the 4th tropical weed science conference (twsc 2013)

Weed Management and Utilization in the Tropics

THE 4th TROPICAL WEED SCIENCECONFERENCE 2013

January 23-25, 2013The Empress Hotel, Chiang Mai, Thailand

PROCEEDING

THE 4th TROPICAL WEED SCIENCE CONFERENCE (TWSC 2013)

ORGANIZED BY

WEED SCIENCE SOCIETY OF THAILAND

AND

DEPARTMENT OF AGRICULTURE

Address : Weed Science Society of Thailand 2nd Floor Weed Science Building Plant Protection Research and Development Office Department of Agriculture Chatuchak , Bangkok 10900 Thailand Tel/ Fax: +66-02-561-1785

CONFERENCE INFORMATION

REGISTRATION AND HOSPITALITY DESK

The registration and hospitality desks will be situated at “1st FLOOR OF CONVENTION Building ” The Empress Hotel, Chiang Mai. The registration will be operated from 14:00 -19:00 p.m. on January 22, 2013 and from 8:00-10:00 a.m. on January 23, 2013. The 4th Tropical Weed Science Conference (TWSC 2013) bag in which the full registered participants will receive upon registration comprising of a copy of program & abstracts, pen, handy drive and name badge.

THE CONFERENCE VENUE

The main Conference activities will take place at the CONVENTION Building, The Empress Hotel, Chiang Mai, THAILAND. This hotel is near Night Bazaar at 10 minute distance walk, 30-40 minutes from the Chiang Mai International Airport.

THE SECRETARIAT OFFICE

The Secretariat office will be at “1st FLOOR OF CONVENTION Building”, set up near the main conference room to facilitate and assist delegates and participants during the Conference. Services will be available from 13.00-18.00 on January 22 and 08:00-16:00 on January 23, 2013. Arrangements for checking PowerPoint presentation by the speakers will also be available at the secretariat office.

POSTERS AND TRADE EXHIBITION

20 Posters, 14 Trade and Professional Exhibition will run concurrently with the Conference display adjacent to the conference room for the duration of the Conference. Presenters are requested to check the program for the board number assigned to them and use the board with the same number. One mounting board measuring 1.8 m high and 1.2 m wide will be available for each poster exhibit. Poster and Trade Exhibition should be mounted from 13:00-19:00 on January 22 and from 08:00-10:00 on January 23, 2013. Presenters are requested to be present at their posters from 08:00-10:00, 16:30-17:30 on January 23-24, 2013.

LANGUAGE

English will be used in all scientific sessions and all Conference communications and publications.

VISUAL AIDS

The following facilities will be available to assist the speakers in their presentation of oral papers.

LCD projectors- Computers to read CDs-

THE HOTEL

The Empress Hotel, the venue of the Conference is situated at The Empress Hotel & Convention Centre 199/42 Chang Klan Road, Chiang Mai 50100 Thailand Tel. (053) 253 199, Fax: (053) 272 467.

Le Méridien Chiang Mai 108 Chang Klan Road, Tambol Chang Klan, Amphur Muang, Chiang Mai, 50100 Thailand Tel. : (66)(53) 253 666

All participants are requested to stay both hotels for convenience in attending the Conference.

TRANSPORTATION

All foreign participants may enter Thailand through either Bangkok or Chiang Mai International Airports. Transfers to domestic flights are available at Bangkok with Chiang Mai. Transportation service ic, airport taxi is available all the time for serving at the taxi counter and hotel limousine need booking prior to the National Organizing Committee. Train travel and air-conditioned first-class sleepers are also convenient for participants. It can also be reached comfortably both day and night by deluxe air-conditioned coaches.

SOCIAL EVENTS

Welcome reception dinner will be organized on Wednesday, January 23, 2013 at 18.30-21.00 p.m. at The Imperial Ballroom, The Empress Hotel.

Farewell dinner will be organized on Friday, January 25, 2013 at 18.30-21.00 p.m. at Imperial Ballroom, The Empress Hotel.

PROFESSIONAL TOUR TO GROWING SITES

A one-day professional excursion is being organized on January 25, 2013 to enable participants to visit Rice Research Center or Mae Kuang Udomthara Dam, Royal Project or Chiang Mai’s Tiger Kingdom. Refreshments and lunch box will be provided. Admission fee of this excursion for an accompanying person is free of charge. Those who wish to join this field trip please sign your name at the registration desk to secure a seat in the bus or van.

MEALS

For vegetarians, please inform the National Organizing Committee for preparing your meals earlier.

ACKNOWLEDGEMENT

The National Organizing Committee wishes to acknowledge the following organizing companies and persons whose generous support has made this conference and the professional tour possible.

Royal Project Foundation- Chiang Mai Rice Research Center - Mae Kuang Udomthara Dam - Office of Agricultural Research and Development Region 1- Chiang Mai Royal Agricultural Research center-

ContentsPage

Organizing Committee............................................................................................................ 7

Allelopathy in Sustainable Agriculture: Rice Allelopathy and Momilactone..................... 8Hisashi Kato-Noguchi

Allelochemicals in Cuscuta campestris Yuncker......................................................... 23BakiHj Bakar, Sow Tein Leong, Muhammad Remy Othman, MohamadSuffianMohamad Annuar and Khalijah Awang

Allelopathic Potential of Jasminum officinale f. var. grandiflorum (Linn.) Kob. and Its Physiological Mechanisms on Bioassay Plants.......................................................... 29Montinee Teerarak, Patchanee Charoenying and Chamroon Laosinwattana

Studies on Natural Herbicide Resistance (HR) among traditional and developed rice (Oryza sativa L.) varieties cultivated in Sri Lanka and inducing HR with Chemicalmutagens, NaN3 and EMS...................................................................................... 26Shyama Ranjani Weerakoon, R. G. Danushka Wijeratne and Seneviratne Somaratne

Rapid bioassay method for herbicide dose-response study and herbicide resistance diagnosis................... ......................................................................................... 37Chuan-Jie Zhang, Soo-Hyun Lim and Do-Soon Kim

Efficacy and Rice Crop Tolerance to Mixtures of Penoxsulam+Cyhalofop as One-Shot Rice Herbicide in ASEAN Countries.......................................................... 41N. Lap, S. Somsak, I.M. Yuli, Le Duy, Lee Leng Choy, Ermita, Bella Victoria, B.V. Niranjan, R.K.Mann

Potential of Organic Herbicide from Aglaia odorata Lour........................................... 48Chamroon Laosinwattana Montinee Teerarak and Patchanee Charoenying

Diversity of Hyphomycetes Fungi from Diseased Weeds............................................. 55Duangporn Suwanagul, Jitra Kokae and Anawat Suwanagul

Impacts of meadowfoam seed meal amendment on weeds and soil microbial activity....... 61Suphannika Intanon, Andrew Hulting, David Myrold, and Carol Mallory-Smith

Imidazolinone tolerance variety for weedy rice control in direct-seeded rice: The Malaysian Experience.......................................................................................69Azmi, M, Yim, K.M. and George, T.V.

Phylogenetic relationships of Echinochloa species based on phenotypic and SSRs markers....................................................................................................... 74Eun-Jeong Lee, Min-Jung Yook, Do-Soon Kim

Eco-efficient weed management approaches for rice in tropical Asia............................. 78A.N. Rao and A. Nagamani

Morphological and physiological responses of Miscanthus spp. to varying temperature and light intensity....................................................... ......................................... 88Jastin Edrian Revilleza, Soo-Hyun Lim, Ji-Hoon Chung, Do-Soon Kim

Flucetosulfuron performance improved by adjuvant................................................... 91Jin Won Kim, Seong-Hyu Shin, Jong-Nam Lee, Se-Eun Lim, Soo-Hyun Lim, Do-Soon Kim

Baseline sensitivity of Echinochloa crus-galli to alternative herbicides selected for managing herbicide resistant Echinochloa species..................................................... 95Ji-Soo Lim, Soo-Hyun Lim, Do-Soon Kim

Weed and Weedy Rice Control by Imidazolinone Herbicides in ClearfieldTM Paddy in Vietnam............................................................................................................. 98Duong Van Chin, Tran Cong Thien, Huynh Hong Bi, Nguyen Thi Nhiem and Tran Thi Ngoc Son

Utilization of weeds in Thailand............................................................................ 103Pensee Nantasomsaran, Komson Nakornsri and Patpitcha Rujirapongchai

A General View of Weeds in Lowland Rice and Up-Land Crops in The South of Vietnam............................................................................................................. 113Ho Van Chien

The 4th Tropical Weed Science Conference, 23-25 January 2013, Chiang Mai, Thailand

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Organizing CommitteeOrganizer: Weed Science Society of Thailand Department of Agriculture, Ministry of Agriculture and Cooperatives

Advisory Board:Mr. Dumrong JirasuthasDr. Manthana MilneProf. Umporn SuwannamekProf. Rungsit Suwanmankha

International Organizing CommitteeProf. Stephen B. Powles (Australia)Prof. Carol Mallory-Smith (USA)Prof. Koichi Yoneyama (Japan)Prof. Nilda R. Burgos (USA)Prof. Do Soon Kim (Korea)Prof. Hisashi Kato-Noguchi (Japan)Prof. Steve Adkins (Australia)Prof. Michael Braverman (USA)Assoc. Prof. Sansanee Jamjod (Thailand)Dr. David Johnson (IRRI)

National Organizing CommitteeChairman: Dr. Chanya ManeechoteVice-Chairman: Dr. Sarawut RungmekaratSecretary: Ms. Nongnuch YokyongsakulProceedings: Dr. Chamroon LaosinwattanaRegistration: Ms. Wanida ThantawinCeremony: Dr. Acharaporn Na Lampamg NoenplubReception: Mr. Virach Chantarasmee Public Relations: Ms. Punnee WitchachooTreasurer: Dr. Sujin JenweerawatVenue: Mr. Sirichai SathuwijarnExcursion: Ms. Jeerawan PetpaisitSponsor: Mr. Komsan NakornsriField trials: Mr. Jeerawat Jitprom


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Allelopathy in Sustainable Agriculture: Rice Allelopathy and Momilactone

Hisashi Kato-NoguchiDepartment of Applied Bioresource Science, Faculty of Agriculture, Kagawa University, Miki,

Kagawa 761-0795, Japan,E-mail address: [email protected]

Abstract

Given the agricultural importance of rice, it has been extensively studied with respect to its allelopathy as part of a strategy for sustainable weed management options. All available information indicates that rice plants possibly release allelochemicals into the neighbouring environments, and a number of compounds, such as phenolic acids, fatty acids, phenylalkanoic acids, hydroxamic acids, terpenes and indoles, have been identified in rice plant extracts, root exudates, and decomposing residues as potential rice allelochemicals. However, the studies demonstrate that the diterpenoid momilactones are the most important rice allelochemicals, with momilactone B playing a particularly critical role. Rice plants secrete momilactone B into the neighboring environments over their entire life cycle at phytotoxic levels, and momilactone B seems to account for the majority of the observed rice allelopathy. Allelochemicals can provide a competitive advantage for host-plants through suppression of soil microorganism and inhibition of the growth of competing plant species because of their antibacterial, antifungal, and growth inhibitory activities. The use of allelopathic rice can definitely reduce the ecological impact in rice cultivation, particularly by reducing the amount of herbicide used. The rice allelopathy may be one of the options in the sustainable weed management strategies.

Keywords: Allelopathy, Allelochemical, Momilactone, Root exudates, Sustainable weed management.

Introduction

Weeds cause reductions in rice yield and quality and remain one of the biggest problems in rice production. The negative impacts of commercial herbicide use on the environment make it desirable to diversity weed management options. Allelopathy is one of the options (Rimando and Duke, 2003; Macías et al., 2007; Kong, 2008; Tesio and Ferrero, 2010). Allelopathy is the direct influence of an organic chemical released from one living plant on the growth and development of other plants (Inderjit and Duke, 2003; Belz, 2007; Macías et al., 2007). Allelochemicals are such organic chemicals involved in the allelopathy (Rice 1984; Putnam and Tang 1986;Inderjit 1996). Allelochemicals can provide a competitive advantage for host-plants through suppression of soil microorganism and inhibition of the growth of competing plant species because of their antibacterial, antifungal, and growth inhibitory activities (McCully, 1999; Hawes et al., 2000; Bais et al., 2004). Rice has also been extensively studied with respect to its allelopathy as part of a strategy for sustainable weed management, such as breeding allelopathic rice strains. A large number of rice varieties were found to inhibit the growth of several plant species when these rice varieties were grown together with these plants under the field or/and laboratory conditions (Dilday et al., 1994; 1998; Kim et al., 1999; Olofsdotter et al., 1999; Azmi et al., 2000; Gealy et al., 2003; Seal et al., 2004a; Kim et al., 2005). These findings suggest that rice may produce and release allelochemicals into neighboring environment, thus encouraging the exploration of allelochemicals in rice. Many secondary compounds, such as phenolic acids, fatty acids, indoles and terpenes were identified in rice root exudates and decomposing rice residues as putative allelochemicals (Takeuchi et al., 2001; Rimando and Duke, 2003; Khanh et al., 2007). However, these compounds are almost ubiquitous in plants and rice allelopathy can not be explained by these compounds (Olofsdotter et al., 2002b; Seal et al., 2004b). Tricyclic diterpen, known as momilactone B, which are unique to rice, have been isolated (Kato-Noguchi et al., 2002). Momilactone B inhibits the growth of typical


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rice weeds like Echinochloa crus-galli and E. colonum at concentrations greater than 1 μM (Kato-Noguchi et al., 2008). Rice plants secrete momilactone B from the roots into the rhizosphere over their entire life cycle (Kato-Noguchi et al., 2003). These observations suggest that rice plants may inhibit the growth of the neighboring plants through the secretion of momilactone B into their rhizosphere. Although considerable progress in our understandings has been made, much work remains to be done before we fully understand this process in rice and can utilize it for more environmentally benign weed management. This chapter summarized the research history of rice allelopathy and putative allelochemicals found and discussed possible involvement of these compounds in rice allelopathy.

The first observation of rice allelopathyThe first observation of allelopathy in rice was made in field examinations in Arkansas, U.S.A.

in which about 191 of 5,000 rice accessions inhibited the growth of Heteranthera limosa (Dilday et al., 1989). This finding led to a large field screening program. More than 16,000 rice accessions from 99 countries in the USDA-ARS germplasm collection have been screened. Of these, 412 accessions inhibited the growth of H. limosa and 145 accessions inhibited the growth of Ammannia coccinea (Dilday et al., 1994, 1998). In Egypt, 1,000 rice varieties were screened for suppressive ability against Echinochloa crus-galli and Cyperus difformis under field conditions, and inhibitory activity was found in more than 40 of them (Hassan et al., 1998). Similar attempts have been conducted in some other countries, and many rice varieties were found to inhibit the growth of several plant species (Kim and Shin, 1998; Olofsdotter et al., 1999; Pheng et al., 1999). It is obscure, however, whether the inhibition was caused by only allelopathic effects. Plant-to-plant interference is a complex combination of competitive interference for resources such as light, nutrients and water, and allelopathic interactions (Qasem and Hill, 1989; Einhellig, 1996; Belz, 2007). Competitive interference and allelopathy cannot be separated under field conditions (Fuerst and Putnam, 1983; Leather and Einhellig, 1998). Considering the allelopathic potential of plants, however, it is essential to distinguish between the effects of competitive interference and allelopathy (Fuerst and Putnam, 1983; Leather and Einhellig, 1986; Inderjit and Olofsdotter, 1998). Thus, bioassays in allelopathy research should be designed to eliminate the effects of competitive interference from their experimental systems. Many scientists have also paid attention to test solution characteristics for bioassays in allelopathy research because the growth of roots and shoots of several plants as well as germination are inhibited by extreme pH and osmotic potential in test solutions (Wardle et al., 1992; Haugland and Brandsaeter, 1996; Hu and Jones, 1997).

Rice allelopathy in controlled environmentsWell-designed bioassays under controlled environments can only evaluate the allelopathic

potential of plants (Leather and Einhellig, 1986; Inderjit and Olofsdotter, 1998). A laboratory whole-plant bioassay for allelopathic rice screening, called “relay-seedling assay”, was developed at the International Rice Research Institute in the Philippines (Navarez and Olofsdotter et al., 1996). This bioassay may eliminate the effects of competitive interference for resources between rice and test plants from the experimental system, and may evaluate the allelopathic potential of rice. By using this bioassay, several rice varieties were found to possess strong growth inhibitory activity. In addition, the 111 rice varieties were tested for their growth inhibitory activity under laboratory and field conditions, but the results were inconsistent (Olofsdotter et al., 1999). Screenings for allelopathic rice have also been undertaken in several other laboratories. These studies shown that there was a marked difference among rice varieties in growth inhibitory activity and that about 3-4% of tested rice varieties had strong allelopathic potential (Fujii, 1992; Hassan et al., 1998; Kim et al., 1999; Olofsdotter et al., 1999; Azmi et al., 2000). These results suggest that some rice varieties may possess allelopathic potential.

The allelopathic potential of rice seedlings of eight cultivars was determined at an early developmental stage in Petri dishes under controlled laboratory conditions (Kato-Noguchi and Ino, 2001). Three plants, alfalfa (Medicago sativa), cress (Lepi dium sativum) and lett uce (Lactuca sativa) were chosen for the bioassay as test plants because of their known germination behaviors. According to the test solution of Weidenhamer et al. (1987), phosphate buffer (pH 6.0) was chosen as the test solution, which did not affect the germination and growth of cress, lettuce, alfalfa or rice, and did not


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cause any significant pH changes during the bioassay. In addition, no effect of osmotic potential of the test solutions in all dishes was detected on the germination and growth of these plant species. The trial indicated that all rice cultivars tested inhibited the growth of roots, shoot and fresh weight of these test plants. However, the effectiveness of cv. Koshihikari on growth inhibition was greatest among these rice cultivars and more than 60% inhibition was recorded by cv. Koshihikari in all bioassays. Test plants could germinate and grow with the rice seedlings without competition for nutrients and water because no nutrients were added in the bioassay and water was supplied regularly (Kato-Noguchi and Ino, 2001). Light is also unnecessary in the developmental stages of these seedlings, since seedlings mostly withdraw nutrients from the reserve of their seeds during early developmental stages (Fuerst and Putnam, 1983). Thus, the inhibitory effects of these rice seedlings may not be due to competitive interference, suggesting that rice seedlings may have allelopathic potential. Allelopathic potential of these rice seedlings against typical rice weeds, Echinochloa crus-galli, were also determined by a “donor-receiver bioassay” (Kato-Noguchi et al., 2010). All rice cultivars inhibited the growth of shoots and roots of E. crus-galli seedlings, but with a different level of inhibitory activity. Koshihikari showed the greatest inhibitory activity on both shoot and root growth of E. crus-galli.

Phenolic acidsPhenolic acids are often mentioned as putative allelochemicals and the most commonly

investigated compounds among potential allelochemicals since they have been found in a wide range of soils (Hartley and Whitehead, 1985; Inderjit, 1996; Dalton, 1999). Hsu et al. (1989) evaluated the inhibitory activities of phenolic acids against germination of lettuce and alfalfa. 4-Hydroxybenzoic acid and salicylic acid were the most active and inhibited the germination at a concentration greater than 0.5-1.5 mM. Olofsdotter et al. (2002b) evaluated whether phenolic acids are responsible for rice allelopathy. They found that allelopathic rice cultivars did not release a significantly greater amount of phenolic acids than non-allelopathic cultivars. The maximum release rate of phenolic acid from rice plants was approximately 10 μg/plant /day. Therefore, at a conventional plant density (100 rice plants/m2), the release rate of phenolic acids would be approximately 1 mg/m2 day. Considering the inhibitory activity of phenolic acids, it was concluded that, even if all phenolic acids were as phytotoxic as 4-hydroxybenzoic acid, the release level of phenolic acids from rice is not sufficient to cause growth inhibition of neighboring plants (Olofsdotter et al., 2002b). Five major phenolic acids in rice root exudates, 4-hydroxybenzoic acid, vanillic acid, syringic acid, p-coumaric acid and caffeic acid, were mixed and their biological activities were determined against Sagittaria monotevidensis (Seal et al., 2004b). The concentration required for 50% growth inhibition (IC50) of the mixture of these five phenolic acids was 502 μM. The concentrations of these phenolic acids detected in rice roots exudates were by far less than 500 μM (Seal et al., 2004b). The inhibitory activity of a mixture of all 15 compounds identified in rice roots exudates, resorcinol, 2-hydroxyphenylacetic acid, 4-hydroxyphenylacetic acid, 4-phenylbutyric acid, 4-hydroxybenzoic acid, vanillic acid, syringic acid, salicylic acid, cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, 5-hydroxyindole-3-acetic acid and indole-5-carboxylic acid and abietic acid, was also determined and IC50 of the mixture was found to be 569 μM (Seal et al., 2004a, 2004b). In addition, it was clarified that synergistic action of phenolic acids on growth inhibition did not work well (Seal et al., 2004b). These studies indicate that any compounds found in rice root exudates including phenolic acids are not responsible for the allelopathy of rice. All information available suggests that phenolic acid concentrations in rice root exudates were much lower than the required threshold of these phytotoxic levels, and phenolic acids seem not to act as rice allelochemicals.

MomilactonesMomilactone A and B were first isolated from rice husks as growth inhibitors (Kato et al., 1973;

Takahashi et al., 1976). Momilactone A and B were later found in rice leaves and straw as phytoalexins (Cartwright et al., 1977; 1981; Kodama et al., 1988; Lee et al., 1999). Thereafter, the function of momilactone A as a phytoalexin has been extensively studied and several lines of evidence indicate that momilactone A has an important role in rice defense system against pathogen attacks (Nojiri et al., 1996;


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Araki and Kurahashi, 1999; Takahashi et al., 1999; Tamogami and Kodama, 2000; Agrawal et al., 2002). Although the growth inhibitory activity of momilactone B was much greater than that of momilactone A (Takahashi et al., 1976; Kato et al., 1977), the efforts to find the function of momilactone B were limited. Momilactone A and B, respectively, inhib it ed the growth of Amaranthus lividus, Digitaria sanginalis and Poa annua at concentrations greater than 20 ppm (ca. 60 µM) and 4 ppm (ca. 12 µM) (Chung et al., 2005). Momilactone A and B were also reported to inhibit the growth of Echinochloa crus-galli and E. colonum, which are the most noxious weeds in rice fields, at concentrations greater than 10 and 1 µM, respectively. Thus, effectiveness of momilatone B on growth inhibition is much greater than that of momilactone A. The growth inhibitory activities of momilactome B are also greater than those of momilactone A under other bioassay systems, (Takahashi et al., 1976; Kato et al., 1977; Fukuta et al., 2007; Toyomasu et al., 2008). Momilactone A and B, respectively, inhibited root and shoot growth of rice seedlings at concentrations greater than 100 and 300 µM. IC50 values of momilactone A and B on rice root and shoot were not obtained because of their weak inhibitory activities against rice. The inhibitory activities of momilactone A and B, respectively, on the root and shoot growth of rice seedlings were 1 - 2 % and 0.6 - 2 % of those on the root and shoot growth of E. crus-galli and E. colonum. Thus, the effectiveness of momilactone A and B on the growth of rice seedlings was much less than that on the growth of E. crus-galli and E. colonum. These results suggest that the toxicities of momilactone A and B to rice seedlings are much less than those to the two weed species (Kato-Noguchi et al., 2008). Momilactone A and B were secreted from rice plants into the rhizopsphere throughout all life cycle stage of rice (Kato-Noguchi et al., 2003; 2008). The secretion level of momilactone A and B increased until flowering initiation, and decreased thereafter. The level of momilactone A and B at day 80 (around flowering) was 1.1 and 2.3 μg/plant/day, which was 55- and 58-fold greater than that at day 30. Although concentration of momilactone A in rice was greater than that of momilactone B, secretion level of momilactone B was greater than that of momilactone A, which suggests that momilactone B may be selectively secreted into the rhizophere than momilactone A. Considering the growth inhibitory activity and concentrations found in the bioassay medium, momilactone A may cause only 0.8 - 2.2% of the observed growth inhibition of E. crus-galli roots and shoots by rice. However, momilactone B in the medium was estimated to cause 59 - 82% of the observed growth inhibition of E. crus-galli roots and shoots by the rice seedlings. In addition, the concentrations of momilactone B in the medium reflected the observed differences in the growth inhibition of E. crus-galli by the eight rice cultivars investigated (Kato-Noguchi et al., 2010). This suggests that the allelopathic activity of rice may be primarily depend on the secretion level of momilactone B. Therefore, momilactone B may play a very important role in rice allelopathy.

Rice allelopathy and allelochemicalsSince the first observation of allelopathy in rice by Dilday et al. (1989), more than 16,000 rice

accessions from 99 countries in the USDA-ARS germplasm collection have been screened. Of these, 412 accessions inhibited the growth of Heteranthera limosa and 145 accessions inhibited the growth of Ammannia coccinea (Dilday et al., 1994; 1998). Similar attempts have been conducted in some other countries, and a large number of rice varieties were found to inhibit the growth of several plant species when these rice varieties were grown together with these plants under field and/or laboratory conditions (Kim et al., 1999; Olofsdotter et al., 1999; Azmi et al., 2000; Gealy et al., 2003; Seal et al., 2004a; Kim et al., 2005). These findings suggest that rice may produce and secrete allelochemicals into its neighboring environments. Although mechanisms of the exudation are not well understood, it is suggested that plants are able to secrete a wide variety of compounds from root cells by plasmalemma-derived exudation, endoplasmic-derived exudation, and proton-pumping mechanisms (Hawes et al., 2000; Bais et al., 2004). Through the root exudation of compounds, plants are able to regulate the soil microbial community in their immediate vicinity, change the chemical and physical properties of the soil, and inhibit the growth of competing plant species (MuCully, 1999; Hawes et al., 2000; Bais et al., 2004). Momilactone B was secreted from rice plants into the rhizopsphere throughout all life cycle stage of rice (Kato-Noguchi et al., 2003; 2008). Considering the inhibitory activity and the secretion level, momilactone B may play a very important role in rice defense mechanism in the rhizosphere as an allelochemical.


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Rice allelopathy for sustainable agricultureSustainable agriculture has now received more attention from agricultural scientists, ecologist

and social economists. Sustainable agriculture requires making efficient use of resources internal to the farm, and relying on a minimum of essential external inputs (Tesio and Ferrero, 2010). Putnam and Duke (1974) have first evaluated the possibility of using allelopathic crops to manage weeds in agricultural sites to minimize serious problems in the present agricultural production, such as environmental pollution, human health concern and depletion of crop diversity. Allelopathy may represent a new frontier for the implementation of the practices applicable in the integrated weed management strategies by using suppressive cover crops, crop rotation and selection of varieties with strong allelopathic potential to biologically reduce the intensity of weeds. Another important approach is the identification of genes with allelopathic activities, and the application of breeding and transgenic techniques to place allelopathic traits into useful crops. Progress made in understanding mechanisms of allelochemicals selectively, physiological modes of action, and genetic regulation of the biosynthesis should represent the basis for manipulation of germplasm resources. In addition, allelochemicals have potential as either herbicides or templates for new herbicide classes because of strong herbicidal potential (Duke, 1986; Dodge, 1987; Putnam, 1988; Gross and Parthier, 1994; Seigler, 1996; Duke et al., 2000; Macías et al., 2007).

The use of allelopathic rice cultivars and allelochemicals can definitely reduce the ecological impact, particularly by reducing the amount of herbicide used. Allelopathic rice cultivars combined with cultural management options is, therefore, an interesting and potential technique, contributing to alternative chemical control of weeds in paddy ecosystems (Weston, 1996; Olofsdotter, 2001; Olofsdotter et al., 2002a). Such an allelopathy-based technique for paddy weed control is the most easily transferable to the low-input management systems prevailing in most Asian rice farming systems (Kong, 2008). Therefore, the rice allelopathy may be one of the options in the sustainable weed management strategies.

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Araki, Y. and Kurahashi, Y. (1999). Enhancement of Phytoalexin Synthesis During Rice Blast Infection of Leaves by Pre-Treatment with Carpropamid.. J. Pesti. Sci. 24: 369-374.

Azmi, M., Abdullah, M.Z. and Fujii, Y. (2000). Exploratory Study on Allelopathic Effect of Selected Malaysian Rice Varieties and Rice Field Weed Species. J. Trop. Agric. Food Sci. 28: 39-54.

Bais, H.P., Park, S.-W., Weir, T.L., Callaway, R.M. and Vivanco, J.M. (2004). How Plants Communicate Using the Underground Information Superhighway. Trends Plant Sci. 9: 26-32.

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Dalton, B.R. (1999). The Occurrence and Behavior of Plant Phenolic Acids in Soil Environments and their Potential Involvement in Allelochemical Interference Interactions: Methodological Limitations in Establishing Conclusive Proof of Allelopathy. In: Inderjit, Dakshini K.M.M. & Foy C.L. (Eds.) Principals and Practices in Plant Ecology: Allelochemical Interactions. Boca Raton, Florida: CRC Press, pp 57-74.


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Allelochemicals in Cuscuta campestris Yuncker

BakiHj Bakar1, Sow Tein Leong2, Muhammad Remy Othman1, MohamadSuffianMohamad Annuar1 and Khalijah Awang2

1Institute of Biological Sciences, University of Malaya, 50603Kuala Lumpur, Malaysia2Department of Chemistry, Faculty of Science, University of Malaya, 50603Kuala Lumpur, Malaysia

Email: [email protected]

Abstract

Golden dodder (Cuscuta campestris Yuncker), commonly known as Rumput Emas in Malaysia is a parasitic weed infesting many crops and weed species alike. A phytochemical study on the chemical constituents of C. campestris was carried out. Six compounds were isolated through chromatographic method and identified as sitosterol1, pinoresinol2, arbutin3, kaempferol4, quercetin5, and astragalin6. The ethanolic extract of C. camprestris displayed inhibitory allelopathic effects at doses 500 ppm and above by inhibiting seeds germination and seedling growth of lettuce, radish and weedy rice as the test plants. The allelopathic potential of three compounds isolated, viz. kaempferol4, sitosterol1 and pinoresinol2 were investigated. All the three compoundsat doses of 1-100μM showed stimulatory effects on plant growth of radish, lettuce and weedy rice seedlings. The response of all assayed species was dose-dependent.

Keywords: Cuscuta campestris, seed germination, seedling growth, flavonoids, sitosterol, pinoresinol, arbutin

Introduction

Synthetic herbicides when used repetitively not only are likely to persist in the environment as bound residues and damage other organisms, but also may lead to the emergence of resistantweed species (Adam et al. 2010).The release of bioactive compounds or allelochemicals from natural sources has the ability to suppress the growth of weeds (Baki et al. 2009). Natural products are a source of compounds that might be used as herbicide directly or as lead structure for herbicide discovery(Duke et al., 2000). Our preliminary screening study showed that the crude aquous extracts of C. campestris as a parasitic weed with special features – the haustoria infesting and growing on different types of weeds and plant crops alike, possess some potent bioactive constituents that are herbicidal in nature, and strongly inhibited the growth of lettuce and radish seedlings (Othman et al. 2012). Cuscuta campestris Yuncker from the family Cuscutaceae (Convolvulaceae) is an annual obligate angiosperm parasite with golden yellow colour (Shen et al. 2005). This parasite twines on other plants and attaches to the above-ground parts of a wide range of host plants. A single plant of C. campestris may attack varieties of host plants at a time and the host can be weeds or crops (Bungard et al., 1999; Baki et al. 2009, Othman et al. 2012). It grows in abandoned area like shrubs and bushes on roadsides and open spaces in Malaysia (Baki et al. 2009).

In this communication we investigated the allelopathic activities of C. campestris and assess its potential as a natural herbicide, primarily focusing on its bioactive chemical constituents and chemical bioassay.

Materials and Methods

Extraction, Isolation and SeparationThe Cuscuta samples were separated from the hosts after collection from Johore, Malaysia in

October–December 2010. All the samples were dried in the oven for 24 - 48 hours at 60°C.About 200g of the dried samples of Cuscuta was dismembered and extracted with ethanol at room temperature


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for three times. The ethanol extract was freed from the solvent by rotary evaporator and freeze dried. About 5g of the crude was subjected to column chromatography (CC) with silica gel as the adsorbent. Gradient elutions with three solvents were employed as the mobile phase; hexane, ethyl acetate (EA), and methanol. Every fraction was collected in 200 mL of eluents. A total of 28 fractions were collected from the first column. Each fraction was tested on TLC to check for purity. Fraction II (Hexane: EA; 100: 0), VI (Hexane: EA; 80: 20), and X (Hexane: EA; 20: 80) were further subjected into CC in order to obtain single spot on TLC. Fraction II (245.7mg) was fractionated in hexane-ethyl acetate. Of 38 fractions that were tested with TLC and NMR, fractions 19-23 gavesitosterol1 (1.3mg).Fraction VI (71.4mg) was subjected to CC using hexane: ethyl acetate solvent system to give two compounds; pinoresinol2(3.5mg, 50:50) and kaempferol4(5.6mg, 0:100). From fraction X, 27 sub fractions were obtained and sub fraction 10 was subjected again to CC with dichloromethane and ethyl acetate as solvent systems. From this fraction, eluted by a mixture of dichloromethane-ethyl acetate (50: 50), was purified quercetin5(0.5mg), while Fraction XII was further purified using HPLC to give arbutin3(2.1mg) and kaempferol-3-O-glucoside 6(1.3mg). All the structures were identified by comparing their NMR and UV spectra with those reported in the literature.

Bioassays Bioassays based on seed germination, shoot and root growths of radish (Raphanus sativus), lettuce (Lactuca sativa) and weedy rice (Oryza sativa) were used to study the allelopathic potential of C. campestris. About 1 ml of ethanol extract of dried C. campestris was dissolved in 200 ml deionized water with 1 % of ethanol. The extract was set as original concentration (5000 ppm) and diluted into 100 ppm, 200 ppm, 500 ppm, and 1,000 ppm. An 8 ml aliquot of the extract was pipette in to petri dish that was lined with filter paper and previously sown with 20 seeds of radish (R. sativus). The control used was deionized water with 1% of ethanol. These petri-dishes were placed in Precision Plant Growth Chamber Model 818 (230 V, 860 watts) for 7 days. Three replicates were prepared for each treatment. The plants were frozen seven days after treatment in order to avoid subsequent growth until measurements were recorded. The seed germination, shoot and root lengths, and dry mass were recorded. Selected constituents, namely, kaempferol, sitosterol and pinoresinol from C. campestris were prepared with 1% of ethanol in deionized water (100μM) and the rest (1 and 10μM) were obtained by dilution. The same bioassay method was used to determine the allelopathic potential of these chemical constituents on lettuce seed germination and root and shoot lengths following exposures as such.

Statistical AnalysisThe statistical analyses were performed on the data using analysis of variance (ANOVA) using

the SPSS Program version 15.0. Any significant difference between treatment means was tested with Tukeys’ test at p<0.05. The percentages of shoot and root growths were calculated according to the following formula:

where Pc and Pt are the shoot or root lengths of the control and the treated samples, respectively.

Results and Discussion

Identification of Chemical Constituents from Cuscuta campestrisSix different chemical constituents were isolated from C. campestris: three were flavonoids,

one sterol, one lignan and one arbutin. The three flavonoids were kaempferol4, kaempferol-3-O-glycoside6 and quercetin5. The concentration of kaempferol4 was relatively high vis-a-vis the six chemical constituents in the parasitic weed. All these constituents are putative allelochemicals which show allelopathic effects on the test plants (see section 2.2). The identifications of these constituents are based on the comparison of the spectral data with those reported. The chemical structures of sitosterol1, pinoresinol2, arbutin3, kaempferol4, quercetin5, and astragalin6 are shown below.


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HO

H

HH

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1

2

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6

7

8

9

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11

12

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21 22

23

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26

27

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7

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6

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7

8

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6

7

8

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Sitosterol (1) (De-Eknamkul and Potduang, 2003; Rawat et al., 1998), C29H50O : 1H NMR (CDCl3) δ3.49 (1H, m, H-3), 5.32 (1H, m, H-6), 0.65 (3H, s, H-18), 0.98 (3H, s, H-19), 0.89 (3H,d, J=6.4Hz, H-21), 0.80 (3H, d, J=1.8Hz, H-26), 0.78 (3H, d, J=, H-27), 0.84 (3H, s, H-29). 13C NMR (CDCl3) δ37.3 (C-1), 31.7 (C-2), 71.9 (C-3), 42.3 (C-4), 140.8 (C-5), 121.8 (C-6), 32.0 (C-7), 32.0 (C-8), 50.2 (C-9), 36.6 (C-10), 21.2 (C-11), 39.8 (C-12), 42.4 (C-13), 56.9 (C-14), 24.4 (C-15), 28.3 (C-16), 56.1 (C-17), 11.9 (C-18), 19.5 (C-19), 36.2 (C-20), 18.9 (C-21), 34.0 (C-22), 26.1 (C-23), 45.9 (C-24), 29.2 (C-25), 19.9 (C-26), 19.1 (C-27), 23.1 (C-28), 12.1 (C-29),.


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Pinoresinol (2) (Do et al., 2009; Li Hui et al., 2004), C20H22O6 : 1H NMR (CDCl3) δ6.86 (1H, s, H-2 and

H-2’), 6.87 (1H, d, J=2.28Hz, H-5 and H-5’), 6.81 (1H, dd, J=1.84, 6.64Hz, H-6 and H-6’), 4.71 (1H, d, J=4.12Hz, H-7 and H-7’), 3.07 (1H, m, H-8 and H-8’), 3.84 (2H, dd, J=4.12, 5.52Hz, H-9 and H-9’), 4.21 (2H, dd, J=6.88, 2.28Hz, H-9 and H-9’), 3.89 (3H, s, 3-OCH3 and 3’-OCH3).

13C NMR (CDCl3) δ132.95 (C-1 and C-1’), 108.67 (C-2 and C-2’), 146.78 (C-3 and C-3’), 145.31 (C-4 and C-4’), 114.35 (C-5 and C-5’), 119.06 (C-6 and C-6’), 85.97 (C-7 and C-7’), 54.23 (C-8 and C-8’), 71.75 (C-9 and C-9’), 56.05 (3-OCH3 and 3’-OCH3).

Kaempferol (4) (Hadizadeh et al., 2003; Rawat et al., 1998), C15H10O6 : 1H NMR (CD3OD) δ6.15 (1H,

d, J=1.84Hz, H-6), 6.37 (1H, d, J=2.28Hz, H-8), 8.05 (1H, d, J=9.16Hz, H-2’ and H-6’), 6.87 (1H, d, J=9.16Hz, H-3’ and H-5’). 13C NMR (CD3OD) δ146.7 (C-2), 135.8 (C-3), 176.0 (C-4), 161.2 (C-5), 97.9 (C-6), 164.2 (C-7), 93.1 (C-8), 156.9 (C-9), 103.2 (C-10), 122.4 (C-1’), 129.3 (C-2’ and C-6’), 115.0 (C-3’ and C-5’), 159.2 (C-4’).

Quercetin (5) (Guvenalp and Demirezer, 2005; Rawat et al., 1998), C15H10O7 : 1H NMR (CD3OD)

δ6.17 (1H, d, J=2.0Hz, H-6), 6.38 (1H, d, J=2.0Hz, H-8), 7.72 (1H, d, J=2.2Hz, H-2’), 6.87 (1H, d, J=8.52Hz, H-5’), 7.61 (1H, dd, J=2.0, 6.6Hz, H-6’). 13C NMR (CD3OD) : δ146.4 (C-2), 136.0 (C-3), 176.1 (C-4), 161.2 (C-5), 97.9 (C-6), 164.2 (C-7), 93.1 (C-8), 156.9 (C-9), 103.0 (C-10), 120.3 (C-1’), 114.6 (C-2’), 144.9 (C-3’), 147.3 (C-4’), 114.9 (C-5’), 122.7 (C-6’).

Kaempferol-3-O-glucoside (6)(Lee et al., 2004), C21H20O11 : 1H NMR (CD3OD) δ6.20 (1H, d, J=1.96Hz,

H-6), 6.39 (1H, d, J=1.96Hz, H-8), 8.05 (1H, d, J=8.8Hz, H-2’ and H-6’), 6.88 (1H, d, J=8.8Hz, H-3’ and H-5’), 5.25 (1H, d, J=7.1Hz, H-1’’), 3.33-3.96 (5H, m, H-2’’, H-3’’, H-4’’, H-5’’, H-6’’). 13C NMR (CD3OD) δ159.2 (C-2), 135.5 (C-3), 179.6 (C-4), 161.7 (C-5), 100.2 (C-6), 166.6 (C-7), 95.0 (C-8), 158.7 (C-9), 105.7 (C-10), 122.9 (C-1’), 132.4 (C-2’ and C-6’), 116.2 (C-3’ and C-5’), 161.7 (C-4’), 104.2 (C-1’’), 75.8 (C-2’’), 78.1 (C-3’’), 71.5 (C-4’’), 78.5 (C-5’’), 72.7 (C-6’’).

Arbutin (3)(Cepanec and Litvi, 2008), C12H16O7 : 1H NMR (CD3OD) δ6.93 (1H, d, J=8.8Hz, H-2 and

H-6), 6.66 (1H, d, J=8.8Hz, H-3 and H-5), 4.70 (1H, d, J=7.2Hz, H-1’), 3.25-3.88 (5H, m, H-2’, H-3’, H-4’, H-5’, H-6’). 13C NMR (CD3OD) : δ152.4 (C-1), 116.6 (C-2 and C-6), 119.4 (C-3 and C-5), 152.4 (C-4), 103.7 (C-1’), 75.0 (C-2’), 78.0 (C-3’), 71.4 (C-4’), 78.0 (C-5’), 62.6 (C-6’).

Bioassay of Ethanol Extract of Cuscuta campestrisTable 1 shows the results on the effect of different concentration of ethanol extract of C.

campestris on the seeds of lettuce, radish and weedy rice. The effect of the extract of C. campestris on seed germination was not significant except lettuce. Interestingly, the growth of shoot and root of all three assayed species were severely affected. The efficacy of the extract was measurably higher on the roots than shoots of the lettuce and weedy rice seedlings. The responses of all assayed species were dose-dependent (Fig. 1 and Fig. 2). At low concentration (100-1000 ppm), there was negligible and non-significant reduction in germination of lettuce. At higher dose (5000 ppm), germination of lettuce seeds was markedly decreasing (45% inhibition). The inhibitory effectsof the extract at 5000 ppm on the growth of lettuce shoots were highly significant at 89% compared with the control. Synergistic effects on shoot growth of lettuce seedlings were observed at other applied doses, explaining perhaps the hormonal role of the extracts at low concentrations. The roots of lettuce displayed higher sensitivity than the shoots to the ethanol extracts of C. campestris. A significant synergistic effect with 14-47% stimulation on root growth obtained when low concentrations of 100 -200 ppm. However, when treated with higher dosage, root growth decreased considerably at 43-95% (Table 1). The ethanol extract of C. campestris did not show any significant effect on radish seed germination. However, the root growth appeared greatly affected. With parallel increase in concentration, root growth was inhibited with values ranging from 27% to 84%, although there was no obvious inhibition at low concentration (100 -200 ppm) (Fig. 2). The effect of C. campestris extract on radish shoot growth was not as great as the effect on roots. In fact, the growth of radish shoot was inhibited only when higher dosage (1000 -5000 ppm) applied (Table 1). There were negligible effects of C. campestris extract on weedy rice germination.


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Yet, the growth of weedy rice seedlings is sensitive to lower concentration of C. campestris extract. The shoot and root growthsof weedy rice seedlings were inhibited by C. campestris ethanol extract at 200ppm, with differential sensitivity of 5-63% reductions of shoot growth and 15-93% reductions of growth compared with the respective controls (Table 1).

Allelopathic Potential of Chemical Constituents of Cuscuta campestris on LettuceThree bioactive compounds, kaempferol, pinoresinol and sitosterol were selected to determine

their allelopathic potentials against the test plants. The results were reported as percentage differences in germination (Table 2), root growth and shoot growths and dry weight (Table 2) compared with the respective controls. Overall, exposures to each of the three chemical constituents failed to have any significant effect on the germination of lettuce seeds. Similar exposures to 1 – 100 μM showed synergistic effects on the growth of shoots and roots of lettuce seedlings vis-à-vis the control. The dose-mediated synergistic effects on the growth of lettuce seedlings were observed when the concentrations were increased. This shows that the responses of lettuce to kaempferol, pinoresinol and sitosterol were dose-dependent with the roots displaying greater sensitivity than the shoots.

Pinoresinol2showedthe greatest stimulatory effect on the growth of rootsand shoots of lettuce. Exposures to 1 – 100 μM of pinoresinol resulted in 60-76% increase in root growth of lettuce, and 21-64% in the shoot growth (Table 2). The parallel figures for exposures to kaempferol4 were 20-60% increase in shoot growth and for the root growth these were 58-67% (Table 2). Sitosterol1 showed the least growth synergistic effects on lettuce growth with only 13-49% in shoot growth the root growth increased up to 46-63% following exposures to 1 – 100 μM (Table 2). However, the beneficial effects of these three constituents on seedling growth of lettuce were reduced with parallel increase in doses in excess of 100 μM suggesting inhibitory effects at higher concentration.

Conclusions

The biological and pharmacological activities of C. campestris are remarkable (Agha et al., 1996; Istudor et al., 1984). Nevertheless, not much research was done on the allelopathic potentials of the weed against other weed species and crops. The preliminary results in this research suggested that application of C. campestris extract at 500 ppm and above has allelopathic potential on other weeds (e.g. weedy rice) and crops (lettuce and radish). However, the pure compounds did not exhibit measurable allelopathic activity against the test plants at concentration of 100 μM and below. This observation on shoot and root growths of lettuce suggest that the allelopathic potential of these constituents may be synergistic at low doses, but inhibitory effects on growth may prevail at higher doses. Further investigation on a broader range of doses and application times of C. campestris in petri-dishes and soils, on different plants is commendable to be carried out to improve their efficacies for weed control.

Table 1. Effects of ethanol extract of Cuscuta campestris on the germination and growth of lettuce, radish, and weedy rice seedlings.

Concentration (ppm)

Germination (%)

Shoot Length (mm) Root Length (mm) Dry Weight (g)

Lettuce

0 100.0b (0.0) 10.0b (0.0) 39.3d (0.0) 0.010a (0.0)

100 100.0b (0.0) 14.0c (-39.0) 57.8f (-47.1) 0.011a (-10.0)

200 100.0b (0.0) 13.0c (-30.0) 44.7e (-13.8) 0.012a (-20.0)

500 98.3b (1.7) 12.4bc (-23.1) 22.4c (43.0) 0.013 a (-30.0)

1000 100.0 b (0.0) 12.5bc (-24.1) 11.2b (71.6) 0.011a (-10.0)

5000 55.0a (45.0) 1.1a (88.9) 1.9a (95.3) 0.017b (-70.0)


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Concentration (ppm)

Germination (%)

Shoot Length (mm) Root Length (mm) Dry Weight (g)

Radish

0 100.0a (0.0) 32.6b (0.0) 71.9c (0.0) 0.15a (0.0)

100 98.3a (1.7) 41.2c (-26.5) 71.0c (1.2) 0.17a (-13.3)

200 98.3a (1.7) 40.4c (-23.9) 62.2bc (13.5) 0.15a (0.0)

500 100.0a (0.0) 29.6b (9.2) 52.2b (27.4) 0.16a(-6.7)

1000 100.0a (0.0) 15.9a (51.1) 16.1a (77.6) 0.16a (-6.7)

5000 95.0a (5.0) 11.4a (65.1) 11.6a (83.8) 0.18a (-20.0)

Weedy rice

0 100.0a (0.0) 52.4d (0.0) 51.2c (0.0) 0.34a (0.0)

100 100.0a (0.0) 59.3e (-13.1) 50.3c (1.8) 0.33a (2.9)

200 100.0a (0.0) 49.8cd (5.0) 43.2b (15.6) 0.35a (-2.9)

500 98.3a (1.7) 45.6bc (12.9) 43.6b (14.8) 0.36ab (-5.9)

1000 98.3a (1.7) 41.7b (20.4) 40.6b (20.8) 0.36ab (-5.9)

5000 100.0a (0.0) 19.4a (63.0) 3.5a (93.1) 0.39b (-14.7)

Values in the column with the same letter are not significantly different at p<0.05.Values in the parentheses are inhibition percentages over control.Values in the parentheses with (-) are promotion percentages over control.

Table 2. Effect of three constituents from Cuscuta campestris on the germination and growth of lettuce (Lactuca sativa) seedlings

Concentration (μM) Germination (%) Shoot Length

(mm) Root Length (mm) Dry Weight (g)

Kaempferol0 96.67a (0.0) 8.52a (0.0) 11.85a (0.0) 0.032a (0.0)1 100.00a(-3.44) 13.66b (-60.27) 19.76b (-66.70) 0.010a (69.29)10 98.33a(-1.72) 12.56b (-47.43) 18.94b (-59.83) 0.010a (68.97)100 96.67a (0.0) 10.25a (-20.31) 18.71b (-57.89) 0.009a (73.04)

Pinoresinol0 96.67a (0.0) 8.52a (0.0) 11.85a (0.0) 0.032a (0.0)1 100.00a (-3.44) 13.99c (-64.18) 20.91b (-76.48) 0.011a (64.58)10 98.33a(-1.72) 12.78c (-49.93) 22.48b (-89.69) 0.010a (67.40)100 100.00a (-3.44) 10.33b (-21.18) 18.97b (-60.06) 0.010a (69.59)

Sitosterol0 96.67a (0.0) 8.52a (0.0) 11.85a (0.0) 0.032a (0.0)1 98.33a (-1.72) 12.74b (-49.48) 19.36b (-63.36) 0.010a (68.97)10 100.00a (-3.44) 12.34b (-44.85) 20.67b (-74.40) 0.010a (70.22)100 96.67a (0.0) 9.62a (-12.83) 17.30b (-45.98) 0.009a (70.85)

Values in the column with the same letter are not significantly different at p<0.05.Values in the parentheses are inhibition percentages over control.Values in the parentheses with (-) are promotion percentages over control


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Acknowledgements

The authors acknowledge the financial support from University of Malaya to the senior author through the UMRG Research Grant No. RG007-09SUS.

References

Agha, A. M., Sattar, E. A., Galal, A., 1996.Pharmacological Study of Cuscuta campestris Yuncker.Phytotherapy Research 10:117-120.

Adam, J., Jeremy, N., Baki, B.B. & Alias, Z. 2010. Resistant Goosegrass (Eleusineindica [L.]Gaertn.)Biotypes to Glufosinate and Glyphosate in Malaysia.Weed Biology and Management 10(4):256-260.

Baki, B. B., Remy, M. O., Aini, H., Khalijah, A., Fujii, Y., Annuar, M. S. M., Zazali, A., 2009. Distribution Patterns, Host Status and Damage Susceptibility of Crop Plants and Weed Species to Cuscuta campestris Yuncker in Malaysia. Korean Journal of Weed Science 29(3):185-193

Bungard, R. A., Ruban, A. V., Hibberd, J. M., Press, M. C., Horton, P., Scholes, J. D., 1999. Unusual carotenoid composition and a new type of xanthophyll cycle in plants. Proc. National Academy of Sciences 96:1135-1139.

Cepanec, I., Litvi, M., 2008.Simple and efficient synthesis of arbutin. Arkivoc 2:19-24.De-Eknamkul, W., Potduang, B., 2003. Biosynthesis of β-sitosterol and stigmasterol in Croton sublyratus

proceeds via a mixed origin of isoprene units. Phytochemistry 62:389-398.Do, K. H., Choi, Y. W., Kim, E. K., Yun, S. J., Kim, M. S., Lee, S. Y., Ha, J. M., Kim, J. H., Kim, C. D.,

Son, B. G., Kang, J. S., Khan, I. A., Bae, S. S., 2009. Pinoresinol-4,4’-di-O-β-D-glucoside from Valeriana officinalis root stimulates calcium mobilization and chemotactic migration of mouse embryo fibroblasts. Phytomedicine 16: 530-537.

Duke, S. O., Romagni, J. G., Dayan, F. E., 2000. Natural products as sources for new mechanisms of herbicidal action. Crop Protection 19:583-589.

Guvenalp, Z., Demirezer, L. O., 2005. Flavonol Glycosides from Asperulaarvensis L. Turkish Journal of Chemistry 29:163-169.

Hadizadeh, F., Khalili, N., Hosseinzadeh, H., Khair-Aldine, R., 2003.Kaempferol from saffron petals. Iranian Journal of Pharmaceutical Research 2:251-252.

Istudor, V., Predescu, I., Popa, E., Badoi, F., Sialvara, S., 1984. Study of polyphenol and saponoside derivatives in Cuscuta campestris var. typica F. orsoviana Buia. Farmacia (Bucharest) 32:173-182.

Lee, J., Ku, C., Baek, N.-l., Kim, S.-H., Park, H., Kim, D., 2004. Phytochemical constituents from Diodiateres. Archives of Pharmacal Research 27:40-43.

Rawat, M. S. M., Pant, G., Prasad, D., Joshi, R. K., Pande, C. B., 1998. Plant growth inhibitors (Proanthocyanidins) from Prunus armeniaca. Biochemical Systematics and Ecology 26:13-23.

Rice, E. L., 1984. Allelopathy.Academic Press, London.Rizvi, S. J. H., Rizvi, V., 1992. Allelopathy: Basic and Applied Aspects. Chapman & Hall, London.Shen, H., Ye, W., Hong, L., Cao, H., Wang, Z., 2005. Influence of the obligate parasite Cuscuta campestris

on growth and biomass allocation of its host Mikania micrantha. Journal of Experimental Botany 56:1277-1284.


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Allelopathic Potential of Jasminum officinale f. var. grandiflorum (Linn.) Kob. and Its Physiological Mechanisms on Bioassay Plants

Montinee Teerarak1, Patchanee Charoenying1 and Chamroon Laosinwattana1

Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand

2 Department of Chemistry, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand

Abstract

Higher plants are a rich source of valuable allelochemicals used for weed control technologies based on natural products. Jasminum officinale f. var. grandiflorum (Linn.) Kob. belongs to the plant family Oleaceae. The main active compound of oleuropein was isolated from methanolic crude extracts of J. officinale dried leaves. The pure active compound of oeuropein exhibited lower growth inhibition compared to methanolic extracts from J. officinale leaves (mixture of allelochemical compound). In cytogenetic bioassay, methanolic crude extract produced lower mitotic index, alternation of phase index and induction of mitotic abnormalities. Allelochemicals found in J. officinale adversely affected seed germination and seedling growth of Echinochloa crus-galli (L.) seeds treated with wettable powder formulation of J. officinale crude extract inhibited seed germination by impeding seed imbibition and induction of α-amylase activity. Taken together, the findings presented in this study strongly suggest that J. officinale may harbor biologically active products the natural properties of which may be exploited to create a successful biorational herbicide.

Keywords: Allelopathy, α-amylase, biorational herbicide, cytogenetics, imbibition, Jasminum officinale,

Introduction

The indiscriminate use of new technology of agrochemicals for success for modern agriculture has made soil sick, caused environmental pollution, development of resistance in pest, toxic residues in our food and quality of life. This indicates that the new technology is not sustainable over long periods. Therefore, the recent emphasis in agriculture has shifted from a primary goal of maximizing yields over the short term, to a sustainable productivity over long periods of time. Sustainability can be achieved in an agriculture that is ecologically sound, resource conserving and not environmentally degrading. Natural products have historically been a valuable source of many pesticides, used either directly as crude preparations, as pure compounds, or as structural leads for the discovery and development of natural product based pesticides. The impact of natural products have historically been greater on the development of fungicides and insecticides than on herbicide (Vyvyan, 2002), but the potential benefits of natural product based herbicide remain underestimated. Higher plants are rich source of valuable allelochemicals used for weed control technologies based on natural products. The initiation of laboratory bioassays of allelochemicals on seed germination efficiency and seedling growth is an important component of modes of action and predict the ability of allelochemicals to field performance potential for improving weed management. The aims of this study were focused on (i) isolation and identification of the main active compound from J. officinale leaves; (ii) examination of the growth inhibitory effect of the main active compound compared with its compound in mixture; and, (iii) determination of modes of action of the compound in mixture.


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Material and methods

Plant materials and methanol extractJ. officinale leaves were harvested, chopped into 1-cm-long pieces and oven-dried at 45°C for

5 days. A hundred grams of dried leaves were used to make methanol extraction by soaking in 1 liter of methanol at 12°C for 48 h to yield a final concentration of 100 g dry leaf per liter (g/L). The resultant extract was filtered through four layers of cheesecloth to remove any fiber debris, followed by a second filtration through Whatman No. 1 filter paper, and set as original concentration (100 g/L). This original stock extract was kept at 5°C until used.

Methanol extract of J. officinale leaves on seed bioassayEchinochloa crus-galli L. Beauv. and Phaseolus lathyroides L. seeds were transferred to Petri-

dishes containing filter paper moistened with 5 mLof distilled water and 25-100 g/L of J. officinale methanol extract. Petri dishes were kept in a growth chamber. The number of germinating seeds was counted and seedling growth was measured as the root and shoot lengths at seven days after treatment.

Prelimary isolation of active compoundCrude extracts were prepared from J. officinale dried leaves by extract with 90% methanol in

water. A 90% methanol crude extract (OR) from J. officinale leaves was partitioned into aqueous (AQ) fraction, acidic (AE) and neutral (NE) fractions. Each fraction of OR, AQ, NE and AE fractions in wettable powder form was prepared to contain four concentrations of each fraction from 1000 to 8000 ppm. Bioassays on seed germination and seedling growth were tested as previously described. The fraction showing the highest inhibition activity was isolated active compound by column chromatography and the active substance was characterized by detailed spectroscopic analysis.

Cytogenetic bioassayBulbs of Allium cepa L. were used for cytogenetic experiment. After outer scales were removed

and basal ends were cut, the bulbs of A. cepa L. were placed in containers, with their basal ends dipped in distilled water, and germinated under standard laboratory conditions. When the newly emerged roots reached 1.50–2.00 cm in length, they were used in the test. The newly emerged roots were treated with a series of concentrations of the J. officinale extracts (12.5, 25, 50, 100, 200 and 400 ppm) for 18 h. Root tips of A. cepa were cut off and fixed in ethanol:acetic acid (3:1). The fixed root tips were macerated in a mixture of hydrolytic enzymes, squashed stained with Giemsa solution for 10 min. Mitotic index, phase index and chromosomal abnormalities were recorded.

Preparation of methanol extract of J. officinale leaves in wettable form (WP)One kilogram of 100 mesh J. officinale leaf powder was extracted (ratio 1 kg: 10 L), with

methanol at 25°C constant temperature. After 24 hours of extraction, the brown supernatants were filtered through four layers of cheesecloth and re-filtered through Whatman no. 1 filter paper (Whatman Inc. Clifton, NI, USA.). Following filtration, the brown supernatants were dried by evaporation of the solvent using a rotary evaporator (BUCHI Rotavapor R255), BUCHI, Lausanne, Switzerland) under a partial vacuum at 45°C until a constant crude extract weight was reached. Wettable powder formulation of crude extract (JWP) was prepared by dissolving sticky crude extract with acetone in a mortar jar and then wettable powder (kaolinite:anionic surfactant; 97:3 (w/w)) was added into the mortar jar in a 3:7 ratio (crude extract:wettable powder). The mixture was slowly pulverized until completely dry. Acetone was added three times and kept in the dark at a low temperature until used.

JWP on seed germination, seed imbibition and α-amylase activityThe JWP was dissolved in distilled water to contain five concentrations of 500, 1000, 2000, 4000

and 8000 ppm. Bioassays on seed germination and seedling growth were tested as previously described. Measurement of seed imbibition was done by following the method of Turk and Tawaha (2002) whereas extraction and measurement of activity of α-amylase was done by following the methods of Bernfield (1955) and Sadasivam and Manickam (1996).


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Statistic analysisEach treatment consisted of four replications in completely randomized design. Analysis of

variance was calculated for all data and comparisons between treatments were made at probability level p ≤ 0.05 using Tukey’s Studentized Range Test.

Results

The results indicated that methanolic extracts of J. officinale at all concentrations markedly reduced the percentage of seed germination of wild pea weed compared with that of the distilled water control, while barnyardgrass had no significant effect at the 25 and 50 g/L concentrations. For initial seedling growth, root length of both weed seedlings was inhibited by a magnitude greater than that of the shoot length (Table 1). The main active compound from J. officinale was isolated and determined by spectral data as a secoiridoid glucoside named oleuropein. The pure active compound of oeuropein exhibited lower growth inhibition compared to methanolic extracts from J. officinale leaves (mixture of allelochemical compound) which may well act synergistically. In cytogenetic bioassay, Allium root treated with J. officinale extract induced a decrease in mitotic index and alternation of phase index (Fig. 1). In addition, the toxic effect of J. officinale extract induced cell division aberrations.

Table 1 Allelopathic effect of the methanolic extract of Jasminum officinale f. var. grandiflorum (Linn.Kob.) on the germination, shoot length and root length of Echinochloa cruss-galli (L.) Beauv. and Phaseolus lathyroides (L.).

Conc. (g/L)

Echinochloa cruss-galli (L.) Beauv Phaseolus lathyroides (L.).-----------------------------% of control-----------------------------

germination Root length shoot length germinationRoot length

Shoot length

0 100a 100a 100a 100a 100a 100a25 97a 87b 98a 85a 50b 75ab50 75a 28c 95a 62b 15c 52b75 33b 5c 63b 10c 2d 20c

100 0c 0d 0c 0d 0d 0d

Different letters within the same column indicate significance differences (p<0.05) between treatments.

Allelochemicals found in J. officinale adversely affected seed germination and seedling growth of E. crus-galli. seeds treated with JWP. The inhibition percentages on E. crus-galli seed germination treated with 500 to 8,000 ppm for 7 days was about 0 to 70%, respectively, whereas shoot length was inhibited 19.04 to 71.82% and root length was 76.31 to 100% inhibition, respectively (Table 2). Further studies were extended to explore the impact of JWP on imbibition and α-amylase activities of E. crus-galli seeds (Table 3). For all treatment concentrations, no significant differences in imbibition after the 12 and 24 h imbibition time were observed. After the 48 h imbibition period, the percentage of imbibition caused marked changes for all concentrations used. Application of 500 ppm JWP had a stimulatory activity of α-amylase on E. crus-galli. An increased concentration of JWP inhibited α-amylase activity. However, the activity of α-amylase was not significantly inhibited at concentrations of 1000 and 2000 ppm crude methanolic extract in wettable powder during whole experiment. It was significantly inhibited when imbibing the seeds in JWP at concentrations of 4000 and 8000 ppm for a period of 12 h, 24 h and 48 h. Generally, the imbibition and α-amylase activities in the treated E. crus-galli seeds were progressively depressed with increasing JWP concentrations (Table 3).


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0102030405060708090

100

0 12.5 25 50 100 200 400

concentration (ppm)

perc

ent

% Prophase % Metaphase

% Anaphase-Telophase Mitotic index

Fig. 1 Mitotic index and mitotic phases of onion root-meristem cells exposed to different concentrations of Jasminum officinale f. var. grandiflorum (Linn.) Kob. crude methanolic extracts for 18 h.

Table 2 Effects of crude metanolic extract from J. officinale in wettable powder form (JWP) on seed germination and seedling growth of E. crus-galli seeds.

Concentration (ppm)

Inhibition (% of control)

Germination Shoot length Root lengthControl 0.00c 0.00cd 0.00c

500 0.00c -8.00d -15.29d1000 7.50c 19.04bc 76.31b2000 5.00c 20.81bc 97.12a4000 40.00b 30.20b 100.00a8000 70.00a 60.18a 100.00a

The values represent the means. Different letters within the same column indicate significance differences (p<0.05) between treatments.

Table 3 Effects of crude methanolic extract from J. officinale in wettable powder form (JWP) on seed imbibition and α-amylase activities of E. crus-galli seeds at different imbibition periods.

Concentration (ppm)

Seed imbibition (%)

α-amylase activities (µmol maltose min-1 g-1(FW))

12h 24h 48h 12h 24h 48hcontrol 32.84a 45.11a 79.54a 3.15a 4.37a 9.49a500 32.99a 45.83a 66.37b 3.15a 4.82a 9.52a1000 34.78a 46.85a 50.62c 2.63ab 4.17ab 8.08ab2000 34.00a 46.61a 49.18c 2.12ab 4.09ab 6.81abc4000 31.97a 40.40a 47.44c 1.69bc 3.48b 5.38bcd8000 29.50a 35.31a 41.45c 1.34c 2.89b 4.39cd

The values represent the means. Different letters within the same column indicate significance differences (p<0.05) between treatments.


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Discussion

Allelochemicals from J. officinale suppressed growth plant bioassays. The main active compound of oleuropein was isolated from methanolic crude extracts of J. officinale dried leaves. Oleuropein was less inhibition effect on germination and seedling growth of bioassay plants than crude extract which may well act synergistically. This finding is congruent with the observation of synergistic of biological compounds reported by Lydon et al., (1997), Inderjit et al., (2002) and Kilani et al., (2008). In cytogenetic bioassay, methanolic extract produced lower mitoitc index, alternation of phase index and induction of mitotic abnormalities. A similar observation of commercial herbicides like pentachlorophenol, 2,4-D and butachlor was reported by Ateeq et al. (2002). In addition, the toxic effect of J. officinale extract induced cell division aberrations. These findings correspond to others in the published literature which have examined the effects of several herbicides, including dimethyl 2,3,5,6-tetrachloro-1,4-benzenedicarboxylate (DCPA), propham, chloropropham (carbamates), dithiopyr and thiazopyr, all of which have shown interference with mitosis (Dyer and Weller, 2005).

Allelochemicals found in J. officinale adversely affected seed germination and seedling growth of E. crus-galli seeds treated with JWP inhibited seed germination by impeding seed imbibition and induction of α-amylase activity. Seed which inhibited imbibition may be limited in specific enzymes required for metabolism of reserved food and hence have poor seed germination. The decrease in α- amylase activity as a result of exposure to JWP could suggest the retardation of substrate production for respiration and consequently limited energy production (Taiz and Zeiger, 2006). For this reason, JWP may adversely affect seed germination. It was shown that the activity of α-amylase was inhibited by the presence of allelochemicals. Kato-Noguchi and Macías (2005) previously reported that lettuce (Lactuca sativa L. cv. Grand Rapids) seeds treated with 6-methoxy-2-benzoxazolinone (MBOA) inhibited seed germination by impeding induction of α-amylase activity.

Conclusion

Allelochemical, oeuropein was isolated from J. officinale. The pure active compound of oeuropein exhibited lower growth inhibition compared to crude form (mixture of allelochemical compound) which may well act synergistically. Crude forms of J. officinale adversely affected germination and growth tested weeds and showed the modes of negative action on physiological and cellular processes.

Acknowledgements

The authors acknowledge The Thailand Research Fund (TRF, Grant number DBG-5080019) for financial support.

References

Ateeq, B., Farah, M.A., Ali, M.N. and Ahmad, W. (2002). Clastogenicity of Pentachlorophenol, 2,4-D and Butachlor Evalualted by Allium Root Tip Test. Mutat. Res. 514: 105-113.

Bernfeld, P. (1955). Amylases α and β. in: Colowick, S.P., Kaplan, N.O. (Eds.), Method in Enzymology. Academic Press, New York, 149-158.

Dyer, W.E. and Weller, S.C. (2005). Plant Response to Herbicides, in: Jenks, M.A., Hasegawa, P.M. (Eds.), Plant Abiotic Stress. Blackwell Publishing, Oxford, pp. 171-214.

Inderjit, Streibig, J.C. and Olofsdotter, M. (2002). Joint of Action of Phenolic Acid Mixtures and Its Significance in Allelopathy Research. Physiol. Plant. 144: 422-428.

Kato-Noguchi, H. and Macías, F.A. (2005). Effects of 6-Methoxy-2-Benzoxazolinone on the Germination and α-Amylase Activity in Lettuce Seeds. J. Plant Physiol. 162: 1304–1307.


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Kilani, S., Sghaier, M.B., Limem, I., Bouhlel, I., Boubaker, J., Bhouri, W., Skandrani, I., Neffatti, A., Ammar, R.B., Dijoux-Franca, M.G., Ghedtra, K. and Chekir-Ghedira, L. (2008). In vitro Evaluation of Antibacterial, Antioxidant, Cytotoxic and Apoptotic Activities of The Tubers Infusion and Extracts of Cyperus rotundus. Bioresour. Technol. 99: 9004-9008.

Lydon, J., Teasdale, J.R. and Chen, P.K. (1997). Allelopathic Activity of Annual Wormwood (Artemisia annua) and The Role of Artemisinin. Weed Sci. 45: 807-811.

Sadasivam, S. and Manickam, A. (1996). Biochemical Methods. New Age International (P) Ltd., New Delhi.

Taiz, L. and Zeiger, E. (2006). Plant Physiology, 4th edn. Sinauer Associates, Massachusetts.Turk, M.A. and Tawaha, A.M. (2002). Inhibitory Effects of Aqueous Extracts of Black Mustard on

Germination and Growth of Lentil. Agron. J. 1: 28-30.Vyvyan, J.R. 2002. Allelochemicals as Leads for New Herbicides and Agrochemicals. Tetrahedron 58:

1631-1646.


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Studies on Natural Herbicide Resistance (HR) among traditional and developed rice (Oryza sativa L.) varieties cultivated in Sri Lanka and

inducing HR with Chemical mutagens, NaN3 and EMS

Shyama Ranjani Weerakoon1*, R. G. Danushka Wijeratne1 and Seneviratne Somaratne1

1Department of Botany, Faculty of Natural Sciences, The Open University of Sri Lanka, P. O. Box 21, Nawala, SRI LANKA.

Abstract

At present, weeds are the major biotic constraint to increased rice production worldwide affecting growth and causing a considerable reduction in yield. Application of high concentrations of pre-emergent-broad-spectrum systemic herbicide, Glyphosate is prevalently used to control rice weeds in South East Asian countries including Sri Lanka and which intern cause severe damages to cultivated rice too. Therefore, inducing herbicide resistance (HR) in cultivated rice is a novel measure used to increase selectivity and enhance crop safety and production. Hitherto, HR in Sri Lankan rice varieties has neither been evaluated nor been attempted to induce. Therefore, varying concentrations of chemical mutagens, Sodium Azide (NaN3) 1.5, 3.0 and 6.0 mmol l-1 and Ethyl Methyl Sulphonate (EMS) 4.5 mmol l-1 concentrations were used to induce HR against Glyphosate at 0.25 and 0.5 g l-1 concentrations. Six traditional and eighteen developed-cultivated rice varieties (Bg, Bw, At and Ld series developed by Rice Research Development Institute, Sri Lanka) were used in the study. Experimental design used was RCBD with five replicates and three blocks in each treatment-combination. Plants with ≥ 40% resistance were considered as resistant to Glyphosate. Observations on time taken-to seed germination, -to flowering; measurements of plant height and number of leaves at 12-weeks after sawing, leaf-length, breadth, panicle-length, number of seeds/panicle of resistant plants and controls were recorded.Ten developed-cultivated varieties (Bg250, Bg94-1, Bg304, Bg359, Bg406, Bg379-2, Bg366, Bg300, Bw364, At362) and three traditional rice varieties (“Kalu Heenati”, “Sudu Heenati”, “Pachchaperumal’) were found to be naturally resistant to 0.25 g l-1 Glyphosate concentration. NaN3-treatment enhanced HR in four varieties (Bg406, Bg379-2, Bg300, Bw364) from 40% to 56% and a susceptible variety (Bg352) developed HR up to 56%. In EMS-treatment, five varieties of developed-cultivated rice (Bg300, Bg359, Bg304, Bg403, Bw364) and two traditional varieties (“Suduru Samba”, “Murungakayan”) were induced HR and four varieties (Bg300, Bg359, Bg304 and “Suduru Samba”) enhanced their HR against Glyphosate at 0.25 g l-1 concentration. Mutagenic chemicals NaN3 or EMS could successfully induce and enhance Glyphosate-resistance in traditional and developed-cultivated rice varieties. Mutant varieties exhibited a considerably higher resistance to Glyphosate. NaN3-mutant and EMS-mutant rice were morphologically different from corresponding parental varieties. EMS showed more effective mutagenic action than NaN3 by showing a decreasing trend and significant difference in agro-morphological characters compared to their respective parental varieties. EMS induced HR in most rice varieties showed a considerable yield penalty associated with growth stunting trends in agro-morphological characters. However, there was no indication of yield penalty in rice varieties with NaN3 induced HR via affecting agro-morphological characters. Increasing concentrations of NaN3 and Glyphosate have shown a negative effect on agro-morphological characters of rice varieties. Mutated rice varieties yielded the higher Glyphosate resistance and they possess higher candidacy to incorporate them in rice breeding programs and to develop HR rice varieties in near future. However, further studies using varying concentrations of NaN3 and EMS with/without gamma irradiation with several traditionally-cultivated and developed-cultivated rice varieties are recommended for better results.

Keywords: Glyphosate, Herbicide Resistance, Oryza sativa, Sodium Azide, Ethyl Methyl Sulphonate, Sri Lanka


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Introduction

Weeds are the major biotic constraint to increased rice production worldwide. Weeds can cause severe yield losses to cultivated rice in relation to the density, type of weeds and cultivated rice varieties (Diarra et al. 1985a; Diarra et al. 1985b; Fisher and Ramirez, 1993; Eleftherohorinos et al. 2002). Therefore, weed control is an essential component of profitable crop production and weeds can be controlled mechanically; chemically or by crop rotation and most farmers rely on a blend of these methods. Application of high concentrations of pre-emergent-broad-spectrum systemic herbicide, Glyphosate is required to control rice weeds in the rice fields of South East Asian countries including Sri Lanka. Glyphosate causes damages to the cultivated rice as well (Labrada, 2007; Davis et al, 2009). It inhibits 5-enolpyruvylshikimate-3-phosphate synthase, an enzyme involved in the shikimic acid pathway of plants (Cioppa et al., 1986). Glyphosate can cause a significant damage to rice yield with a reduction of yield up to 80% (Davis et al., 2009). Inducing herbicide resistance (HR) into rice is a new means to confer selectivity and enhance crop safety and production (Guttieri et al., 1996). Over the last two decades, mutational techniques have become one of the most important tools available to progressive rice-breeding programs. Imidazolinone-resistant rice was developed through chemically induced seed mutagenesis with Ethyl Methyl Sulfonate (EMS) (Gealy et al., 2003) and “Clear field” rice variety was also developed through EMS mutagen against herbicides Imidazolinone (Lang and Buu, 2007). Similarly, Sodium Azide (NaN3) has been used to produce rice mutant for the enhancement of agronomic traits (Nakata et al., 2008; Young Seop et al., 2009). Screening for herbicide resistance is often based on visual evaluation, mortality or growth inhibition compared to untreated plants. There are evidences that HR crops can bring significant benefit to farmers, consumers and the environment. Farmers are benefited from the excellent broad-spectrum weed control provided by such herbicides and from substantially lower costs of growing some HR crops. HR crops provide additional crop choice, enabling implementation of alternate weed management tactics to target specific weeds while maintaining crop sequences. Therefore, inclusion of an HR crop in a cropping program along with a range of weed management tactics can ensure to control hard-to-control weeds (Devine and Buth, 2001).

There is an inadequacy of research efforts on screening of the herbicide resistance among cultivated rice varieties and development of HR rice varieties up to date in Sri Lanka. Therefore, in the present study six traditionally cultivated and eighteen developed-cultivated (Bg, At, Bw, Ld series) rice (Oryza sativa L.) varieties were chosen to screen the herbicide resistance (HR) against pre-emergent-broad-spectrum herbicide, Glyphosate and to induce HR against Glyphosate via mutagenesis using the chemical mutagens, Sodium Azide (NaN3) and Ethyl Methyl Sulphonate (EMS).


Materials - Rice varietiesTwenty four rice varieties with germination percentage of > 85% were selected for the study.

Six traditionally cultivated varieties (“Kalu Heenati”, “Sudu Heenati”, “Suwadal”, “Suduru Samba”, “Pachchaperumal” and “Murungakayan”) and eighteen inbread-developed(cultivated) rice varieties (Bg94-1, Bg250, Bg300, Bg304, Bg305, Bg352, Bg357, Bg358, Bg359, Bg360, Bg366, Bg379-2, Bg403, Bg406, Ld365, At362, At308, Bw364) were collected from Research Centers of Rice Research Development Institute at Batalagoda, Ambalanthota, Bombuwela and Labuduwa, Sri Lanka for the study. These lines were maintained in a greenhouse in the premises of the Open University of Sri Lanka, Nawala, Sri Lanka.

Method 1 - Natural Glyphosate resistance among traditional and developed(cultivated) rice varieties

Seeds of the twenty four rice varieties mentioned above were surface sterilized and placed in moist chambers for germination. The germinated rice seedlings (height about 4 cm) were immersed in


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Glyphosate solutions with two different concentrations, 0.25 g l-1 and 0.5 g l-1 (360 g l-1 Glyphosate) for 4 days. Randomized Complete Block Design (RCBD) was used in the experiment and there were five replicates used for each treatment and three blocks in each treatment combination. Control treatment was carried out without Glyphosate. All seedlings were subsequently transferred to soil medium (sterilized mud) and allowed to grow and observations were taken for ten weeks. Dead plants were considered as susceptible to the herbicide and surviving plants with a substantial growth were considered as resistant to the herbicide. For each rice variety, number of resistant plants and percentage resistance was calculated. The plants with percentage resistance of ≥ 40% were considered as resistant to Glyphosate. The percentage (%) of resistance was calculated using the following equation.

Method 2 - Mutation Studies using NaN3 The seeds of inbred-developed (cultivated) varieties were exposed to the chemical mutagen

Sodium Azide (NaN3) at 1.5 mmol l-1, 3.0 mmol l-1 and 6.0 mmol l-1 concentrations for a day and followed the same steps of the Method 1. Agro-morphological characters were measured or counted at 12 weeks after sawing (WAS). These characters were time-to seed germination, time-to flowering, plant height at, number of leaves, leaf length and width, length of panicle and number of seeds/panicle of the plants with herbicide treatment and the controls without herbicide treatment were also recorded.

Method 3 - Mutation Studies using EMSIn the mutational studies with EMS, seeds of each rice variety were exposed to the chemical

mutagen Ethyl Methyl Sulfonate (EMS) at 4.5 mmol l-1 concentration for a day and followed same steps described in Method 1 and Method 2. Agro-morphological characters of the rice plants at 12 WAS were recorded.

Statistical analysesA descriptive statistics were performed on the data set (mean, standard deviation). The GLM

(General Linear Models) was used to test the effects of factors (rice variety, NaN3 and Glyphosate concentrations) on agro-morphological characters. Since there were no significant interactions among the factors, One-way-analysis of variance (ANOVA) was performed on agro-morphological characters. All statistical analyses were carried out using SAS Version 9.2 (SAS, 2008).

Results

The comparison of natural Glyphosate resistance among traditional and developed (cultivated) rice varieties revealed that thirteen varieties (Bg250, Bg94-1, Bg304, Bg359, Bg406, Bg379-2, Bg 366, Bg300, Bw364, At362, “Kalu Heenati”, “Sudu Heenati”, “Pachchaperumal’) were naturally resistant to 0.25 g l-1 Glyphosate. The higher percentage of resistance was resulted in 0.25 g l-1 Glyphosate concentration, and 0.5 g l-1 concentration caused reduction in the percentages of resistance due to inhibited seed germination. Certain herbicide resistant rice varieties which were mutated with NaN3 (Bg406, Bg379-2, Bg300, Bw364) have enhanced their percentages of resistance from 40% to 56% while a susceptible variety (Bg352) developed HR up to 56% under 0.25 g l-1 Glyphosate concentration. Seven varieties after mutation with EMS (Bg300, Bg359, Bg304, Bg403, Bw364, “Suduru Samba”, “Murungakayan”) were resistant to 0.25 g/l Glyphosate. EMS treatment has enhanced the percentages of resistance in Bg300, Bg359, Bg304 and “Suduru Samba”.


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A combined ANOVA (results not given in the manuscript) revealed that all main effects (rice variety, NaN3 and Glyphosate concentration) were not statistically significant (p ≥ 0.05). Further, in relation to all agro-morphological characters observed, no statistically significant differences between NaN3 mediated-mutated rice plants were observed compared to non-mutated rice plants under treatment of different Glyphosate concentrations (Table 1). However, an increase in number of days taken to seed germination was observed for rice plants mutated with higher concentration of NaN3 (Table 2). Decreasing trends were observed in time-to flowering, number of leaves/plant, leaf length, leaf width and panicle length in the mutated rice plant along the increasing Glyphosate concentrations. Apparently, number of seed/panicle indicated a considerable reduction across increasing Glyphosate concentrations (Table 2). In contrary, there were statistically significant differences (p ≤ 0.05) between the EMS-mediated-mutated rice plants with non-mutated rice plants related to agro-morphological characters such as plant height and number of leaves/ plant at 12 WAS, leaf length and leaf width, under treatment of different Glyphosate concentrations (Table 1). However, time-to flowering, time-to seed germination, panicle length and number of seed/panicle indicated no statistically insignificance when compared mutated rice plants with non-mutated rice plants (Table 1). Mutational study with EMS revealed that mutated rice plants showed a considerable reduction in the agro-morphological characters, plant height and number of leaves/ plant leaf length and leaf width, panicle length and number of seed/panicle across the increasing Glyphosate concentrations (Table 3).

Discussion

There were no previous records on the existence of natural herbicide resistance in traditional and inbred-developed rice varieties in Sri Lanka. The present study revealed that several traditional and developed rice varieties were possessing considerably high HR against Glyphosate. Further, the study showed that NaN3 and EMS could successfully be used to induce HR in Glyphosate susceptible rice varieties as well as to enhance the existing HR. The chemical mutagen EMS has been used to develop HR varieties against the herbicide Imidazolinone (Gealy et al., 2003; Lang and Buu, 2007).

The NaN3-mutant rice varieties were not morphologically different from their parent varieties. Meanwhile, increasing NaN3 concentrations indicated a negative effect on agro-morphological characters of rice varieties. A similar observation has made by Awan et al.,1980 and YoungSeop et al. 2009 with NaN3, reported that there was a decreasing trend in germination rate and seedling height with the increasing NaN3 concentration in Oryza sativa L. ssp. Japonica and few other varieties. However, in the present study no yield penalty was observed in NaN3-mutant rice varieties because yield parameters such as time-to flowering and number of seeds/panicle was not significantly differ compared to parental varieties. EMS seems to be having more effective mutagenic action than NaN3 by showing a decreasing trend and significantly difference in agro-morphological characters such as plant height, number of leaves/plant, leaf length and leaf width compared to their respective non-mutated varieties. Though EMS induced HR in most of the rice varieties studied, there was a considerable yield penalty associated with growth stunting trends in agro-morphological characters. However, more studies with different concentrations of EMS and NaN3 are necessary to make a solid conclusion.


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Table 1. Summary of ANOVA performed on the agro-morphological characters along the different concentrations of EMS, NaN3. S = Significant at p ≤ 0.05; NS = Not significant, p ≥ 0.05

Source Sum of Squares df Mean Square F Significance

EMS treatmentsGerminating time 0.028 2 0.014 0.301 NSHeight 9185.002 2 4592.501 16.673 SNumber of leaves/plant 149.433 2 74.717 15.52 SLeaf width 267.159 2 133.579 8.181 SLeaf length 2220.196 2 1110.098 7.804 SFlowering time 1041.436 2 520.718 1.448 NSPanicle length 2580.56 2 1290.280 39.480 SNumber of seeds/plant 11934.44 2 5967.220 36.887 S

NaN3 treatmentsGerminating time 6.786 2 3.393 3.791 NSHeight 952.84 2 476.42 1.612 NSNumber of leaves/plant 57.371 2 28.686 3.326 NSLeaf width 0.123 2 0.061 1.8 NSLeaf length 701.198 2 350.599 2.697 NSFlowering time 1999 2 999.5 0.571 NSPanicle length 169.633 2 84.817 1.948 NSNumber of seeds/plant 249.018 2 124.509 1.518 NS


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Table 2.Summary of the morphological and yield characters of parental varieties and Glyphosate resistant rice produced by different concentrations of NaN3. Mean values and standard error of mean is given within parenthesis. NA = Not available

Glyphosate

concentration (g/l)

Sodium asideConcentration

(mmol/l)Variety

No. of days taken to

flowering

No. of days taken to

germinateHeight (cm) No. of

leavesLeaf length

(cm)Leaf width

(cm)Panicle

length (cm)No. of seeds/

panicle

Mutated line

0.25 6 Bg 304 NA 5 (0) 56.5 (5.0) 6.0 (1.4) 44.0 (2.8) 1.0 (0.1) NA NA

0.25 3 Bg 352 38.0 (53.7) 3 (0) 32.3 (18.0) 11.0 (1.4) 19.5 (13.4) 0.6 (0.3) 1.3 (1.8) 2.0 (2.8)

0.5 3 Bg 366 NA 4 (0) 19.0 (2.3) 4.0 (0) 13.0 (6.0) 0.6 (0) NA NA

0.25 1.5 Bg 406 NA 3 (0) 19.0 (2.3) 6.0 (1.4) 12.0 (0) 0.6 (0) NA NA

0.25 3 Bg 406 NA) 5 (0) 33.5 (6.4) 13.0 (2.8) 16.0 (1.4) 0.7 (0.1) NA NA

0.25 1.5 Bw 364 74.5 (2.1) 3 (0) 57.0 (12.7) 12.5 (0.7) 19.3 (6.0) 0.8 (0.1) 12.0 (5.7) 14.5 (10.7)

Parental line

control control Bg 304 47.5 (0.7) 2 (0) 59.0 (1.4) 11.0 (1.4) 47.5 (2.1) 1.1 (0.1) 29.5 (3.6) 50.0 (1.4)


control control Bg 366 60.5 (2.1) 3 (0) 66.5 (0.7) 13.0 (1.4) 47.5 (6.4) 1.2 (0.2) 35.0 (1.4) 65.0 (0)


control control Bw 364 57.5 (2.1) 3 (0) 64.5 (5.0) 11.5 (0.7) 55.0 (2.8) 1.4 (0.2) 34.0 (1.4) 58.5 (6.4)


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Table 3. Summary of the morphological and yield characters of parental varieties and Glyphosate resistant rice produced by different concentrations of EMS. Mean values and standard error of mean is given within parenthesis. NA = Not available

Glyphosate concentration (g/l)

Ethyl Methyl Sulphonate

Concentration(mmol/l)

Variety No. of days taken to flowering

No. of days taken to germinate Height (cm) No. of leaves Leaf length

(cm)Leaf breadth

(cm)Panicle length

(cm)No. of seeds/

panicle

Mutated lineControl 4.5 Bg 300 59.0(1.4) 3.0(0) 66.5(2.1) 11. 5(0.7) 1.2(0.1) 43.5(3.5) 22.5(2.1) 35.5(5.0)Control 4.5 Bg 304 54.0(1.4) 3.0(0) 94.0(2.9) 11. 5(0.7) 1.8(0) 48.5(3.5) 32.5(5.0) 81.0(22.6)Control 4.5 Bg 359 60.0(1.4) 3.0(0) 64.5(3.5) 10.5(0.7) 1.4(0.1) 39.5(2.1) 26.0(1.4) 33.0(1.4)Control 4.5 Bg 403 66.5(0.7) 3.0(0) 67.0(2.8) 12.5(0.7) 1.4(0) 42.5(2.1) 26.5(3.5) 52.0(5.7)Control 4.5 Bw 364 57.5(0.7) 3.0(0) 79.0(2.8) 12.5(0.7) 1.3(0.1) 45.0(4.2) 25.5(0.7) 32.0(2.8)Control 4.5 Murungakayan 63.5(0.7) 3.0(0) 91.7(2.4) 10.5(0.7) 1.4(0) 58.5(5.0) 23.0(1.4) 33.0(1.4)Control 4.5 Suduru Samba 70.5(2.1) 3.0(0) 129.0(4.2) 9.5(0.7) 1.7(0.1) 71.1(7.1) 31.5(3.5) 58.5(3.5)

0.25 4.5 Bg 300 62.5(0.7) 3.0(0) 38.0(1.4) 9.0(0) 0.8(0) 20.0(1.4) 11.5(0.7) 12.5(3.5)

0.25 4.5 Bg 304 61.5(0.7) 3.0(0) 54.5(0.7) 8.0(0) 0.9(0) 23.5(3.5) 14.5(2.1) 13.5(5.0)0.25 4.5 Bg 359 62.0(1.4) 3.0(0) 46.5(0.7) 8.0(0) 0.8(0.1) 21.0(2.8) 13.0(2.8) 10.5(7.8)0.25 4.5 Bg 403 74.0(1.4) 3.0(0) 55.5(2.1) 10.5(0.7) 1.0(0) 27.0(2.8) 14.5(0.7) 16.5(0.7)0.25 4.5 Bw 364 62.0(2.8) 3.0(0) 57.5(0.7) 7.5(0.7) 0.9(0) 28.5(3.5) 14.5(2.1) 12.5(3.5)0.25 4.5 Murungakayan 67.5(2.1) 3.0(0) 74.0(1.4) 8.0(0) 1.1(0.1) 43.0(4.2) 13.5(2.1) 12.5(3.5)0.25 4.5 Suduru Samba 75.5(2.1) 3.0(0) 96.5(3.5) 7.5(0.7) 1.4(0) 53.0(8.5) 25.5(0.7) 31.5(3.5)0.5 4.5 Bg 300 61.5(3.5) 3.0(0) 37.5(2.1) 7.0(0) 0.9(0) 20.0(2.8) 10.0(1.4) 8.5(0.7)0.5 4.5 Bg 359 64.5(0.7) 3.0(0) 44.5(2.1) 7.5(0.7) 0.7(0) 23.0(2.8) 13.0(2.8) 10.0(0)0.5 4.5 Bg 403 NA 3.0(0) 45.3(1.8) 7.5(0.7) 0.7(0) 27.3(4.6) NA NA0.5 4.5 Bw 364 NA 3.0(0) 42.5(2.1) 6.5(0.7) 0.8(0.1) 21.5(5.0) NA NA0.5 4.5 Murungakayan NA 3.0(0) 62.5(2.1) 7.5(0.7) 0.7(0) 34.0(7.1) NA NA0.5

4.5 Suduru Samba 77.5(5.0) 3.0(0) 52.0(1.4) 6.5(0.7) 0.8(0) 29.0(2.8) 19.0(2.8) 17.0(2.8)Parental line

Bg 300 52.0(1.4) 2.0(0) 64.0(1.4) 12.5(0.7) 1.2(0.1) 52.0(4.2) 28.5(3.5) 86.5(6.4)

Bg 304 50.0(2.8) 2.0(0) 63.0(4.2) 11.0(1.4) 1.1(1.1) 49.5(5.0) 30.5(2.1) 59.0(11.3

Bg 359 57.0(2.8) 3.0(0) 66.0(2.8) 14.5(2.1) 1.3(0.1) 53.5(3.5) 30.5(2.1) 41.0(2.8)

Bg 403 69.0(2.8) 3.0(0) 70.5(2.1) 12.0(0) 1.4(0.1) 60.0(5.7) 32.0(0) 49.5(9.2)

Bw 364 57.5(2.1) 3.0(0) 64.5(5.0) 12.5(0.7) 1.5(0.1) 57.0(5.7) 36.0(1.4) 72.0(12.7)

Murungakayan 56.5(3.5) 2.0(0) 98.0(8.5) 14.0(0) 1.5(0.1) 70.5(6.4) 34.0(4.2) 63.5(10.6)

Suduru Samba 68.0(1.4) 2.0(0) 149.5(3.5) 10.0(0) 1.5(0.1) 94.0(4.2) 62.5(6.4) 108.5(16.3)


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Conclusion

The results of the present study led to reach the following conclusions: (a) Several traditional and inbred-developed rice varieties possesses HR against Glyphosate (b) The mutant varieties showed considerably higher resistance to the Glyphosate, (c) Chemically mutated varieties using NaN3 was morphologically different from respective parental varieties (d) Increasing NaN3 and Glyphosate concentrations have a negative impact on agro-morphological characters of rice varieties (e) EMS seems to be having more effective mutagenic action than NaN3 (f) Mutated rice varieties with high Glyphosate resistance have higher potential to incorporate in rice breeding programs as well as could lead to develop herbicide resistant rice varieties in future.

Acknowledgements

The research grant provided by the Faculty of Natural Sciences, The Open University of Sri Lanka is greatly appreciated.

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Rapid bioassay method for herbicide dose-response study and herbicide resistance diagnosis

Chuan-Jie Zhang, Soo-Hyun Lim and Do-Soon KimDepartment of Plant Science, Seoul National University, Seoul 151-742, Korea

Corresponding author: E-mail: [email protected]

Abstract

This study was conducted to develop a rapid bioassay method for herbicide dose-response of Echinochloa species and herbicide resistance diagnosis in Echinochloa spp. Germinated seeds of Echinochloa spp. were placed on the paper wick of 18 cm x 16.5 cm growth pouch containing herbicide solution at a range of concentrations. Shoot and root lengths of Echinochloa spp. were then measured after incubation for 6 days. Dose-responses in root length by the growth pouch method were well described by logistic function and confirmed to be similar to those of whole plant assay regardless of herbicide modes of action, suggesting that the growth pouch method can be used for herbicide bioassay. The growth pouch method was then applied to rapid diagnosis of ACCase or ALS inhibitor resistance in Echinochloa spp. Resistant and susceptible biotypes were discriminated at 180 to 300 mg and 80 to 120 mg ai L-1 of cyhalofop-butyl for E. crus-galli and E. oryzicola, respectively, and at 350 to 500 mg and 650 to 1000 mg ai L-1 of penoxsulam for E. crus-galli and E. oryzicola, respectively. Therefore, the growth pouch method can be used for herbicide resistance in Echinochloa spp. with significant time and cost-savings as compared with the conventional whole-plant assay.

Keywords: Diagnosis, Echinochloa species, growth pouch, herbicide resistance, rapid bioassay

Introduction

Echinochloa species is one of the most troublesome weeds in rice cultivations both transplanted and direct-seeded. Reliance upon herbicides and continuous use of herbicides with the same mode of action has led to the development of herbicide resistance in weed populations (Holt et al., 1993). Studies have been confirmed to be resistant to several herbicides in Echinochloa species. Several dose-response methods of weed species have been developed, including whole-plant assay which has been the most widely used for herbicide dose-response and resistance diagnosis. Other methods, such as Petri dish assay (Moss, 1990), trimmed seedling, tiller and stem node tests (Kim et al., 2000), chlorophyll fluorescence measurement (Norsworthy et al., 1998), leaf disc flotation test (Kemp et al., 1990), and pollen germination test (Richter and Powles, 1993) were also developed. However, many of these methods are expensive, limited, difficult to assess, time-consuming, or limited for a certain growth stage. The methods for herbicide dose-response test and resistance diagnosis should be rapid, accurate, cheap, reproducible and readily available and should provide a steady result on herbicide performance. Therefore, this study was conducted to develop a new method by using growth pouch for dose-response test of herbicides with different modes of action. The method was then further applied to herbicide resistance diagnosis in Echinochloa specie.


Whole-plant assay of herbicides with different modes of actionPot experiments were conducted to evaluate dose-responses of whole plants of E. crus-galli

to herbicides with different modes of action in the glasshouse at Experimental Farm Station of Seoul National University, Suwon, Korea. Herbicides, bentazone, cyhalofop-butyl, bispyribac-sodium, penoxulam, glufosinate and glyphosate were applied to E. crus-galli (Suwon biotype) at 5th to 6th leaf


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stage at different dosage ranges. All the treatments were replicated three times and were arranged in a completely randomized design. Above-ground fresh weight was assessed at 30 days after application (DAA), and data were expressed as percentage of the untreated control. GR50 values, dose required to inhibit plant growth by 50% of the untreated control were determined by fitting the data to the standard dose-response model (Streibig, 1980).

Growth pouch test of herbicides with different modes of actionGerminated seeds of E. crus-galli (Suwon biotype) were placed in the growth pouch containing

a range of concentrations of bentazone, cyhalofop-butyl, bispyribac-sodium, penoxsulam, glufosinate, glyphosate and then kept in an incubation room maintained at 35/25oC (day/night) with supplementary light where the roots were being kept in the dark. De-ionized water was added to the growth pouch every day to replace water loss due to evaporation. The root and shoot length were measured at 6 days after treatment (DAT). The test consisted of 3 replications of a completely randomized block design.

Diagnosis of ACCase and ALS inhibitor resistant Echinochloa spp. Origin of Echinochloa species. E. crus-galli, Seosan-5, Seosan-152, and Suwon (reference susceptible) biotypes, previously determined as resistant or susceptible to cyhalofop-butyl (Im et al., 2009) were used for whole plant and growth pouch tests with cyhalofop-butyl. E. oryzicola, Gimje, Iksan, and Suwon (reference susceptible) biotypes, previously determined as resistant or susceptible to penoxulam (Kim, 2010) were also used for whole plant and growth pouch tests with penoxulam.

Whole-plant assay. Pot experiments were conducted to evaluate dose-responses of E. crus-galli and E. oryzicola biotypes to cyhalofop-butyl and penoxsulam at a range of their doses. Fresh weight was assessed at 30 DAA and the data were fitted to the standard dose-response model (Streibig, 1980) to estimate GR50 value of each biotype. The R/S ratio was calculated by dividing GR50 values by the GR50 of the reference susceptible Suwon biotype.

Growth pouch test. Germinated Echinochloa seeds were placed in the growth pouch containing a range of concentrations of cyhalofop-butyl and penoxsulam and then kept in the incubation room maintained at 35/25oC with supplementary light with root being kept in the dark. Germination pouches were topped up with de-ionized water every day to replace water loss due to evaporation. The root length was measured at 6 DAT. The test consisted of 3 replications of a completely randomized block design.


Growth pouch method for herbicide dose-response studyShoot and root growths of E. crus-galli were significantly affected by herbicides in both whole-

plant assay and growth pouch tests. GR50 values were estimated using shoot fresh weights in the whole-plant assay and root length in the growth pouch test.

Based on the GR50 values we can concluded that the values for shoot length was much bigger than those of root length except glufosinate and were not calculable for cyhalofop-butyl and penoxulam, indicating that roots of Echinochloa species are more sensitive to the herbicides tested in this study than shoot except glufosinate. Comparison between the two testing methods thus suggests that the growth pouch test can be used to test herbicide with different modes of action by monitoring root length within a week.

Diagnosis of ACCase inhibitor resistant Echinochloa spp.In E. crus-galli biotypes, the GR50 values by the whole-plant assay of Seosan-5 and Seosan-

152 biotypes were 4.3 and 3.6 times as high as that of Suwon biotype, respectively (Table 2). The GR50 values by the growth pouch test of Seosan-5 and Seosan-152 were 5.4 and 3.6 times greater than that of Suwon biotype, respectively (Table 2). In E. oryzicola biotypes, the GR50 values by the whole-plant assay of Gimje and Iksan were 4.2 and 5.9 times as high as that of Suwon (Table 2). The


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GR50 values by the growth pouch test of Gimje and Iksan were 3.4 and 4.9 times greater than that of Suwon biotype, respectively (Table 2). The optimum concentrations of cyhalofop-butyl to discriminate between resistant and susceptible biotypes are 180-300 mg and 80-120 mg a.i. L-1 for E. crus-galli and E. oryzicola, respectively.

Diagnosis of ALS inhibitor resistant Echinochloa spp.In E. crus-galli biotypes, the GR50 values by the whole-plant assay of Seosan-5 and Seosan-152

biotypes were 7.8 and 4.1 times greater than that of Suwon biotype, respectively (Table 2). The GR50 values by the growth pouch test of Seosan-5 and Seosan-152 were 7.4 and 5.3 times greater than that of Suwon biotype, respectively (Table 2). In E. oryzicola biotypes, the GR50 values by the whole-plant assay of Gimje and Iksan were 7.4 and 11.6 times greater than that of Suwon, respectively (Table 2). The GR50 values by the growth pouch test of Gimje and Iksan were 6.1 and 9.8 times greater than that of Suwon biotype, respectively (Table 2). The optimum concentrations of penoxsulam to discriminate between resistant and susceptible biotypes are 350-500 mg and 650-1000 mg a.i. L-1 for E. crus-galli and E. oryzicola, respectively.

Table 1. GR50 values of herbicides tested by the whole-plant and the growth pouch methods.

Mode of action Herbicide

GR50 valuesWhole-plant assay

(g a.i. ha-1)Growth pouch test

(mg a.i. L-1)

Shoot weight Root length Shoot length

PS II Bentazone 13095.2 2479.0 6066.3ACCase Cyhalofop-butyl 15.7 21.4 NA

ALSBispyribac-sodium 13.6 193.3 1278.0

Penoxsulam 5.5 6148.7 NAGS Glufosinate 113.1 187.2 108.0

EPSPS Glyphosate 303.4 71.7 345.9

Table 2. R/S ratios in the whole-plant assay and the growth pouch tests of resistant (Seosan-5, Seosan-152, Gimje, Iksan) biotypes of Echinochloa species to cyhalofop-butyl and penoxsulam.

Test method HerbicideE. crus-galli E. oryzicola

Seosan-5 Seosan-152 Gimje Iksan

Whole plantCyhalofop-butyl 4.3 3.6 4.2 5.9

Penoxsumal 7.8 4.1 7.4 11.6Growth pouch

Cyhalofop-butyl 5.4 3.6 3.4 4.9Penoxsumal 7.4 5.3 6.1 9.8

In summary, the growth pouch method produced accurate and consistent results with significant time and cost-savings as compared with the whole-plant assay. It is cheap and can be conducted with a minimum facility without spray application equipment. Moreover, the method using germinated seeds requires very little amount of herbicide molecule. Therefore, this method can be used not only for herbicide resistance diagnosis but also for early screening of a new molecule with herbicidal activity in herbicide discovery.


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References

Holt, J.S., Holtum, J. A.M. and Powles, S.B. (1993). Mechanisms and Agronomic Aspects of Herbicide Resistance. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44: 203-229.

Kim, D.S., Caseley, J.C., Brain, P., Riches C.R. and Valverde, B.E. (2000). Rapid detection of propanil and fenoxaprop resistance in junglerice (Echinochloa colona). Weed Science 48: 695-700.

Kim, D.S. (2010). Evolution and Impact of Herbicide Resistant Echinochloa Species. Proceeding of Workshop on Effective Management of Herbicide Resistant Weeds, RDA, Suwon, Korea (in Korean).

Im, S.H., Park, M.W., Yook, M.J. and Kim, D.S. (2009). Resistance to ACCase Inhibitor Cyhalofop-Butyl in Echinochloa crus-galli var. crus-galli Collected in Seosan, Korea. Kor. J. Weed Sci. 29: 178-184.

Moss, S.R. (1990). Herbicide Cross-Resistance in Slender Foxtail (Alopecurus myosuroides). Weed Sci. 38: 492-496.

Norsworthy, J.K., Talbert, R.E. and Hoagland R.E. (1998). Chlorophyll Fluorescence for Rapid Detection of Propanil-Resistant Barnyardgrass (Echinochloa crus-galli). Weed Sci. 46: 163-169.

Richter, J. and Powles, S.B. (1993). Pollen Expression of Herbicide Target Site Resistance Genes in Annual Ryegrass (Lolium rigidum). Plant Physiol. 102: 1037-1041.

Streibig, J.C. (1980). Models for Curve-Fitting Herbicide Dose Response Data. Acta Agric. Scand. 30: 59-63.


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Efficacy and Rice Crop Tolerance to Mixtures of Penoxsulam+Cyhalofop as One-Shot Rice Herbicide in ASEAN Countries.

N. Lap1, S. Somsak2, I.M. Yuli 3, Le Duy 4, Lee Leng Choy 5, Ermita, Bella Victoria 6 ,

B.V. Niranjan 7 , R.K.Mann 8

1&4DowAgroSciences Vietnam, 2DowAgroSciences Thailand, 3DowAgroSciences Indonesia 5 Dow AgroSciences Malaysia, 6Dow AgroSciences Philippines, 7Dow AgroSciences ASEAN & 8Dow AgroSciences LLC USA.

Abtract

Penoxsulam, a triazolopyrimidine sulfonamide rice herbicide, provides good control of Echinochloa spp., annual sedges and many broadleaf weeds. Cyhalofop-butyl, an aryloxy phenoxypropionate rice herbicide, provides good control of many grass weeds such as Echinochloa spp. and Leptochloa chinensis. The pre-mix formulation of 10g ai Penoxsulam + 50g ai Cyhalofop-butyl/liter (Accept60OD/TopShot60OD) and the tank-mix of penoxsulam (Clipper25OD/Rainbow25OD) + Cyhalofop-butyl(Clincher100EC/Cranstan100EC) are broad-spectrum herbicide products that are applied post-emergence and have residual weed control activity to control many grass, broadleaf and sedge weeds with excellent rice tolerance in ASEAN countries. Combination products containing penoxsulam + cyhalofop-butyl can increase rice productivity in a wide diversity of rice production systems in direct-seeded, water-seeded and transplanted rice. Small plot field research trials and on-farm demonstration trials were completed from 1998 to 2011. Trials were conducted in many locations across ASEAN countries over a 13 year period. In large plot on-farm demonstrations from 2003 to 2011, premixes or tank mixes of penoxsulam + cyhalofop-butyl at 10 g ai+50 g ai/ha to12.5 g ai+62.5 g ai/ha, respectively, applied as a foliar post-emergence treatment at 7 to18 days after sowing or transplanting provided >90% control of common weeds in rice. This high level of weed control resulted in a 20 to 50 % yield increase when compared to rice production in untreated areas. Both active ingredients in the mixtures are highly efficacious on Echinochloa spp. Each herbicide has a different mode of action, so these pre-mix products are a good tool for Echinochloa spp. resistance management in rice. Pre-mixes and tank mixes of penoxsulam + cyhalofop-butyl demonstrated excellent rice crop safety. The herbicide mixtures were applied post-emergence at rates up to 5 times the labeled use rate ( 300 g/ha) at 7 to 18 days after sowing or transplanting and did not injure the rice crop or reduce yields.

Keywords: penoxsulam, cyhalofop–butyl, efficacy, yield, direct-seed rice, transplanted rice, grasses, sedges, broadleaf weeds.

Introduction

Rice is a major staple food with a planted area of 29 MM ha in ASEAN countries in 2009, which provides for 600 million people in the area. Rice production should be increased to meet the demand of the increasing population. One of the most important methods to increase rice production is to minimize the loss caused by weed competition in rice fields. These weeds are not only reducing rice production but also affecting rice seed quality. Since the beginning of agriculture, growers have tried to control rice weeds by any means. One of the means is using synthetic herbicides. Many different herbicides have been commercialized; however, farmers still prefer a one shot treatment that will provide broad-spectrum weed control.

The premix of Penoxsulam + Cyhalofop-butyl has been developed by Dow AgroSciences since 2005 and commercialized under the trade name of TopShot60OD in Indonesia, Philippine, Vietnam and Accept60OD in Thailand. Penoxsulam is an ALS inhibitor that belongs to group


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B1 (triazolopyrimidine sulfonamides), and penoxsulam is broad spectrum herbicide that controls Echinochloa spp, broadleaf and annual sedge weeds. Cyhalofop-butyl is an ACCase inhibitor in Group A (Aryloxyphenoxypropionates) which provides very high control of grassy weeds like Echinochloa spp and Leptochloa spp. Both active ingredients are very safe to rice and have broad spectrum control efficacy. This report is a summary of 106 field trials across Vietnam, Philippine, and Thailand since 1998-2011.


Field studies were conducted on field stations of Rice Research Institutes in Malaysia, Philippine, Indonesia and Vietnam and on farmer fields in Thailand. Trials were randomized complete block design (RCBD) with 3 or 4 replications with plot area of 16-25 m2. Target crop was Oryza sativa (Indica) cultivated by direct-seeding or transplanting. In wet-seeded rice, water was partially drained from the field, with post emergence foliar application made to exposed weeds, and the paddy reflooded within 48 hours after application. Other rice cultivation followed local farming practice.

For the tank mixture trials, Penoxsulam 25 OD (25 g/l) and Cyhalofop-butyl 100 EC (100 g/l) were used at early stage of development. The herbicide premix was Topshot 60 OD (10 g/l Penoxsulam + 50 g/l Cyhalofop-butyl); tested rates were 1, 1.25 and 1.5 liters/hectare. Each treatment was diluted in a spray volume of 320-400 liter water per hectare, and applied by knapsack sprayer with fan nozzle.

Individual weed control evaluation was made at 14 DAA (Days After Application), 28 DAA and 42 DAA by visual observation on biomass reduction of weed compared with untreated plot as percentage of control. Phytotoxicity was recorded at 1, 3, 5,7,14 and 28 DAA by visual assessment based on 1-9 injury scale level. Rice yields were harvested in 5m2 frame in each plot then calculated into theoretical rice yield per hectare. Collected data were statistical analyzed by ARM7 (owned by Dow AgroSciences).

Results and Discussions

Rice Crop PhytotoxicityThe tested rates from 5-50 g ai + 25-250 g ai/ha of the tankmix or premix of penoxsulam +

cyhalofop, respectively, did not cause any injury symptom to indica rice when applied at 3-18 days after sowing or transplanting. Table 1 shows that up to 5 times label rate, e.g. 300 g ai/ha applied at 7-18 days after sowing/transplanting did not injure the rice crop.

Efficacy of tank-mixture Penoxsulam + Cyhalofop on Leptochloa chinenses (LEFCH)Penoxsulam at 10-30 g ai/ha does not provide commercial control of LEFCH. However,

Cyhalofop-butyl makes an excellent tank mix partner for control of LEFCH without antagonizing the control of ECHCG and other weeds listed. The result of this mixture as shown in Table 2 demonstrates that 10+50 to 12.5+62.5 g ai/ha of Penoxsulam + Cyhalofop-butyl, respectively, applied at 3-16 DAS provided more than 85% control of LEFCH without antagonizing ECHCG control.

Efficacy of premix Penoxsulam+Cyhalofop_butyl on Leptochloa chinensis (LEFCH)Table 3 shows that at 4-18 DAS (Day After Seeding) application timing, the premix at rates of

0.75-1.5 l/ha provided very high herbicidal activity on LEFCH. In general, the 1 l/ha rate provided equal efficacy on LEFCH compared with rates of 1.25 and 1.5 l/ha, at same application timing, with results similar among ASEAN countries.


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Table 1 Crop Injury of Penoxsulam + Cyhalofop-butyl to rice at 1-28 DAA when applied 3-18 days after sowing across ASEAN countries

Days after

seeding

Rates(g ai/ha)

No. of observation

Crop injury level*

1DAA 3DAA 5DAA 7DAA 14DAA 28DAA

4-9

60 g mixtures N=60 1 1 1 1 1 1

120 g mixtures N=60 1 1 1 1 1 1

300 g mixtures N=60 1 1 1 1 1 1

225 g Butachlor + 225 g Propanil N=52 1 3 2 1 1 1

10-14

60 g mixtures N=54 1 1 1 1 1 1

120 g mixtures N=54 1 1 1 1 1 1

300 g mixtures N=54 1 1 1 1 1 1

36 g Fenoxaprop _ ethyl N=48 3 4 5 3 1 1

* crop injury based on 1-9 scale with 1 = totally safe and 9 = complete kill.

Table 2. Efficacy of Penoxsulam + Cyhalofop on LEFCH at 28 DAA (% Biomass Reduction)

Days after seeding Country

No. of observation

Penoxsulam + Cyhalofop (g ai/ha)10 + 50 12.5 + 62.5 15 + 75

3 -5 Vietnam n = 12 90.9 94.2 99.66 -9 Thailand n = 12 94.4 100.0 100.0

Vietnam n = 12 89.6 94.2 98.010 –12 Philippines n = 12 96.4 99.7 95.0

95.097.5

Thailand n = 8 89.2 91.7Vietnam n = 12 90.7 94.2

13-16 Philippines n = 20 96.2 100.0 98.491.799.1

Thailand n = 8 70.0 85.0Vietnam n = 16 95.3 95.0

From the data of these trials, the premix of Penoxsulam + Cyhalofop-butyl was developed, results of the premix are presented in the next section.

Efficacy of premix Penoxsulam+ Cyhalofop-butyl on Echinochloa crus- galli (ECHCG)Table 4 demonstrates that 1 l/ha of Penoxsulam + Cyhalofop-butyl premix provided >90%

control of ECHCG with an application window of 0-18 DAS across ASEAN countries. Very high results were seen on ECHCH, because both active ingredients can provide effective control of ECHCG. Thus, the premix of Penoxsulam + Cyhalofop-butyl at 1 l/ha could be a good choice for ECHCG resistance management across ASEAN. It not only provides very high control but has a wide application window.


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Table 3. Efficacy of premix Penoxsulam + Cyhalofop-butyl on LEFCH at 28 DAA (Averaged % Biomass Reduction)

Days after seeding Country No. of

observationRate of product (liter/ha)

1 1.25 1.54-9 Thailand N=52 93 95 96

Vietnam N=24 92 99 9610-14 Philippines N=40 94 96 97

Thailand N=60 96 95 97Vietnam N=64 95 99 98

15-18 Philippines N=32 95 95 98Vietnam N=12 - 88 100

Table 4. Efficacy of premix Penoxsulam + Cyhalofop-butyl on ECHCG at 28 DAA (% Biomass Reduction)



1 1.25 1.50-3 Vietnam N=48 98 97 964-9 Thailand N=52 91 94 97


Thailand N=40 98 97 99Vietnam N=64 97 99 99

15-18 Philippines N=24 93 98 91Vietnam N=8 - 90 100

Efficacy of premix Penoxsulam + Cyhalofop-butyl on Echinochloa colona (ECHCO)As shown in Table 5, control efficacy of the premix on ECHCO is similar to ECHCG. At 10-14

DAS, 1 l/ha provided >92% control of ECHCO. In Philippines, 1-1.25 l/ha at 15-18 DAS application timing provided lower control than at 10-14 DAS, possibly due to weeds being larger and having more tolerance at later timing. However, 1.5 l/ha was similar between 2 application timings.

Efficacy of premix Penoxsulam + Cyhalofop-butyl on Cyperus difformis (CYPDI)As presented in Table 6, 1 l/ha of premix penoxsulam + cyhalofop-butyl at 0-14 DAS provide

very good control of CYPDI, higher rate at 1.25-1.5 l/ha did not show significantly better efficacy.

Table 5. Efficacy of premix Penoxsulam + Cyhalofop-butyl on ECHCG at 28 DAA (% Biomass Reduction)

Days afterseeding Country No. of

observationRate of product (liter/ha)1 1.25 1.5

10-14Philippines N=16 96 99 96Thailand N=8 93 90 97

15-18 Philippines N=8 91 95 97


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Table 6. Efficacy of premix Penoxsulam + Cyhalofop_butyl on CYPDI at 28 DAA (% Biomass Reduction

Days after seeding Country No. ofobservation

Rate of product (liter/ha)1 1.25 1.5

0-3 Vietnam N=40 98 96 100

4-9Thailand N=30 91 92 98Vietnam N=8 100 99 99


Efficacy of premix Penoxsulam + Cyhalofop-butyl on Cyperus iria (CYPIR)Similar to CYPDI, result in Table 7 demonstrates that at 4-18 DAS application timing, 1 l/

ha provided >90% control of CYPIR in Thailand and Philippines. In Thailand, control efficacy of the premix on CYPIR was really high across the application window of 4-14 DAS, it implied that the premix of Penoxsulam + Cyhalofop-butyl can provide very good control of CYPIR in Thailand.

Efficacy of premix Penoxsulam + Cyhalofop-butyl on Fimbristylis miliacea (FIMMI)From the data in Table 8, it is clear that 1 l/ha of premix Penoxsulam + Cyhalofop-butyl can

provide effective control of FIMMI at 0-18 DAS in Thailand, Philippines and Vietnam. The differences between 1, 1.25 and 1.5 l/ha is minor, demonstrating that the FIMMI was very susceptible to the premix in those countries. Moreover, according to N.Lap et al (2003) 10 g a.i/ha Penoxsulam at 3-18 DAS provides 87-90 % control of FIMMI in Vietnam, Thailand and Philippine, it showed that there is no antagonism between penoxsulam and cyhalofop-butyl on FIMMI in this area.

Table 7 Efficacy of premix Penoxsulam + Cyhalofop-butyl on CYPIR at 28 DAA (% Biomass Reduction)

Days afterseeding Country No. of


1 1.25 1.54-9 Thailand N=9 97 98 98

10-14Philippines N=12 85 - 90 Thailand N=24 96 97 97

15-18 Philippines N=48 93 95 -

Table 8 Efficacy of premix Penoxsulam + Cyhalofop-butyl on FIMMI at 28 DAA (% Biomass Reduction)



1 1.25 1.50-3 Vietnam N=48 99 97 97


10-14Philippines N=24 97 98 95Thailand N=90 98 98 99Vietnam N=72 95 97 98

15-18 Philippines N=48 94 96 97


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Efficacy of premix Penoxsulam + Cyhalofop-butyl on Sphenoclea zeylanica (SPDZE) Table 9 shows that the premix at 1 l/ha provided very high control of SPDZE with application

window at 0-14 DAS.

Efficacy of premix Penoxsulam+ Cyhalofop-butyl on Monochoria vaginalis (MOOVA)Table 10 showed that premix penoxsulam + cyhalofop at 1 l/ha provided very high control

of MOOVA at 4-18 DAS in Philippine and Vietnam, higher rate at 1.25 and 1.5 l/ha did not increase control efficacy on this weed.

Effect of premix Penoxsulam + Cyhalofop-butyl on rice yieldThe data in Table 11 shows that the premix at a rate of 1 l/ha helps to increase yield dramatically

up to 106 to 121% compared to the untreated plot. Rate of 1.25-1.5 l/ha did not show any difference to the response from the 1 l/ha rate.

Table 9. Efficacy of premix Penoxsulam + Cyhalofop-butyl on SPDZE at 28 DAA (% Biomass Reduction)


observation

Rate of product (liter/ha)

1 1.25 1.5

0-3 Vietnam N=48 97 99 98

4-9Thailand N=52 96 91 96

Vietnam N=24 95 98 96

10-14Philippines N=24 100 100 100

Vietnam N=40 97 100 99

15-18 Philippines N=40 100 100 100

Table 10 Efficacy of premix Penoxsulam + Cyhalofop-butyl on SPDZE at 28 DAA (% Biomass Reduction)

Days after seeding Country No. ofobservation

Rate of product (liter/ha)1 1.25 1.5

4-9 Vietnam N=8 100 98 99

10-14Philippines N=24 100 100 100


Table 11 Average rice yield of Premix Penoxsulam + Cyhalolop-butyl compared with commercial standards and untreated plots across ASEAN countries

Days after seeding

Rates(g ai/ha)

No. of observation

Average yield*(ton/ha)

Yield increased compared to

untreated

4-9

60 g Premix N=60 7.59 121 %75 g Premix N=60 7.61 122 %90 g Premix N=60 7.68 124 %

225 g Butachlor + 225 g Propanil N=52 6.73 96 %

Untreated N=60 3.43 0 %


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Days after seeding

Rates(g ai/ha)

No. of observation

Average yield*(ton/ha)

Yield increased compared to

untreated

10-14

60 g Premix N=54 7.28 106 %75 g Premix N=54 7.54 113 %90 g Premix N=54 7.58 114 %

34.5 g Fenoxaprop_ethyl + 10 g Ethoxysulfuron N=48 6.52 85 %

Untreated N=54 3.64 0 %

* Theoretical yield, calculated from harvested yield in 5m2/ plot.

Acknowledgements

We thank Dr.DuongVan Chin of the Cuu Long Rice Research Institute (Vietnam); Mr. Henry Dupo, Sr. (Philippines); Hj Muhammad Harun and En Hassan Ahmad (Malaysia Agriculture Research and Development institute) and Dr. Hamdan Pane and Dr.Zainal Lamid (Indonesia) for cooperation in carrying out these trials in respective country for this study.

References

Mann, R.K. et al. Penoxsulam and cyhalofop butyl_Technical Bulletin. Dow AgroSciences internal document

N. Lap, S.Pornkulwat, S.N.A Sayomchai, C. Antipas, M.A. Jaafar, S. Djoko,Vasant L.Patil, S. Sonoredjo and R.K. Mann. 2003. Manila weed conference.

Sujitno, S et al. premix Penoxsulam + Cyhalofop_butyl Postemergence Foliar Applications at 3 to 27 Days After Seeding in ASEAN Direct-Seeded Indica Rice in 2000 – 2001. DERBI DowAgroSciences internal report.


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Potential of Organic Herbicide from Aglaia odorata Lour

Chamroon Laosinwattana1 Montinee Teerarak1 and Patchanee Charoenying 1

Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand

2 Department of Chemistry, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand

[email protected]

Abstract

This study was undertaken to explore the potential of Aglaia odorata Lour.) organic herbicide under laboratory, pot culture and natural field conditions. The aqueous extracts of leaf and branches of Aglaia odorata inhibited the germination and seedling growth of Echinochloa crus-galli and Phaseolus lathyroides and the leaf extract was slightly more inhibitory than the branch extract at the same concentration. These results indicated that leaf and branch from A. odorata contained certain allelochemicals inhibitory to E. crus-galli and P. lathyroides germination and growth. Subsequent research had examination of the different A. odorata forms on the growth of E. crus-galli and P. lathyroides, under laboratory and pot culture conditions. The results showed that the degree of toxicity of different A. odorata forms can be classified in order of decreasing inhibition as pellet > dried leaf powder > aqueous extract. Addition, effects of A. odorata granules was study on seedling growth of major maize weeds and the influence of soil type on its residue’s efficacy. Under experimental greenhouse, emergence and seedling growth of two major maize weeds (large crabgrass (Digitaria adscendens) and horse purslane (Trianthema portulacastrum L.)) was inhibited but varied with soil type. The degree of toxicity of different soil types can be classified in order of decreasing inhibition as sand > sandy loam >clay. Under natural field conditions, an organic herbicide produced from A. odorata in the granule form could be used to suppress D. adscendens weed emergence and growth in maize field and had no adverse effect on maize growth and silage yield.

Keywords: Allelopathy, herbicide formulation, inhibition, organic herbicide, weed control

Introduction

The use of allelopathic plants as mulch or soil incorporated has been suggested for alternative weed management in sustainable agriculture (Fujii, 2001; Singh et al., 2003a; Xuan et al., 2005; Batish et al., 2006a; Khanh et al., 2006). For example, hairy vetch (Vicia vilosa L.) is promising cover crop for weed control in fields, grasslands and orchards in Japan (Fujii, 2001). Dried Saururaceae (Houttuynia cordata Thunb.) powder significantly reduces the Echinochloa and Monochoria paddy weeds at 150 g m–2 and increases the grain yield of rice than control (Lin et al., 2006). However, there are many limitations for using plant residues such as mulch or incorporating them due to heavy fieldwork for applying large amount of plant residues, which is often cost prohibitive. Over many years, various types of allelochemicals have been isolated and characterized from hundreds of plants. Some of the allelochemicals products exploited as commercial herbicides are cineole (shell), benzoxazinones (BASF), quinolic acid (BASF) and leptpspermones (Zeneca) (Kohli et al., 1998). Natural plant products may provide clues to new and safe herbicide chemistry (Duke, 1986). However, most of allelochemicals having potential herbicidal activity but not commercially used because of there are extremely expensive to manufacture. Einhellig (1995) suggested that nearly all allelopathic activities are due to the presence of several compounds in a mixture. The concentration of each compound in a mixture might be significantly less than the concentration of individual compounds needed to cause growth inhibition.This illustrates the significance of joint action of allelochemicals in mixtures (Inderjit et al., 2002). Another challenge is that the use of products formulated from crude extract of joint action of allelochemicals in mixtures


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from strong allelopathic plants, might successfully overcome this problem. The objectives of this research were: (i) to determine the allelopathic effects of aqueous extracts and dried powder and pellet of A. odorata on the germination and initial seedling growth of Echinochloa crus-galli and Phaseolus lathyroides under laboratory and pot culture conditions and (ii) to evaluate the herbicidal potential of A. odorata granule applied to three soil types on the emergence and growth of large crabgrass (Digitaria adscendens) and horse purslane (Trianthema portulacastrum) in an experimental greenhouse and (iii) to evaluate the herbicidal potential of A. odorata granule to control weeds and effects on growth and yield of maize under natural field conditions.

Material and methodPlant material

Plant materials of Chinese rice flower plants were collected before flowering and were separated into the leaves and branches. They were dried in hot-air oven at 45 °C for 72 h. and cut into 1 cm pieces, powdered in a blender and sieved through 40 mesh (420 µm) sieve.

Preparation of A. odorata in 3 forms and seed bioassay under laboratory conditionsIn this experiment there were two factors; (i) two test weed species (E. crus-galli and P.

lathyroides) (ii) A. odorata in 3 forms (aqueous extract, dried leaf powder and pellet). The aqueous extracts were prepared from powdered leaves and branches of A. odorata which extracted with distilled water for 72 h at 10 °C, followed by filtration to remove any debris. Dried leaf powder was prepared from dried leaves which were powdered in a blender. The pellets were prepared by mixing 50% A. odorata dried leaf powder, 25% cassava glue and 25% CaCO3 powder were dried in hot-air oven at 45°C for 3 days. Weed seeds were transferred to Petri-dishes containing filter paper moistened with 5 mL of distilled water and 4 application doses of each different form. Petri dishes were kept in a growth chamber. The number of germinating seeds was counted and seedling growth was measured as the root and shoot lengths at seven days after treatment. Aqueous extract form was applied at 6.25, 12.5, 25 and 50 g dried leaf L–1. The dried powder of A. odorata was added at 31.25, 62.5, 125 and 250 mg/Petri dish and pellets were applied at 62.5, 125, 250, and 500 mg per Petri dish which equivalent to 6.25, 12.5, 25 and 50 g dried leaf L–1, respectively.

Soil application bioassayThe influence of A. odorata pellets was explored in pot studies done in experimental

house, on the seedlings emergence and growth of E. crus-galli and P. lathyroides. Number of emerged weeds plants was counted at 14 days after sowing, while plant height and their biomass were determined at 28 days.

Effect of three soil types on the efficacy of the organic herbicide from A. odorata granulesThe effect of the A. odorata granules on the emergence and seedling growth of D. adscendens

and T. portulacastrum was explored under pot conditions in an experimental greenhouse. Clay soil, sandy loam soil and sand were used as the three soil types. The number of plants of each bioassay species that emerged was counted at 14 days after treatment and the plant height was determined at 28 days after initiating the treatments. The biomass was determined separately at 28 days after initiating the treatments.

Field experiment: Effect on growth of weeds and maizeA. odorata granule, a control with the herbicide atrazine and another control without

organic herbicides or atrazine were applied under maize field conditions. After planting 3 weeks was assessed the emergence of weeds and 12 weeks after planting dry weight was assessed in two 1 m2 quadrates placed randomly in each plot at maize harvest. Weeds were counted by species, clipped at ground level within each quadrate. The weed is dried by oven drying to constant weight at 72°C, for comparison their dry weight. Maize silage yields can be obtained by


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harvesting at the maize silage (12 weeks after planting); In each plot was harvested at stage 20 plants were harvested from the center rows and compared of crop yield for all plant treatments.

Statistical analysis In all experiments, Data were subjected to one-way analysis of variance (ANOVA) and comparison is statistically significant between treatments mean with Tukey’s Studentized Range test at p < 0.05.

Results

The A. odorata in 3 forms at all test concentrations markedly reduced the seed germination (%) of both test species compared to control (Table 1).The leaf and branch aqueous extracts at all concentrations inhibited seed germination and seedling growth, and the degree of inhibition increased with the incremental extracts concentration but the leaf extract was slightly more inhibitory than the branch extract. The aqueous extracts of leaf and branches of A. odorata inhibited the germination and seedling growth of E. crus-galli and P. lathyroides. The degree of toxicity of different A. oderata forms can be classified in order of decreasing inhibition as pellet > dried leaf powder > aqueous extract. In pot culture, pellet reduced the seedlings emergence, plant height and dry biomass of both test weeds: E. crus-galli and P. lathyroides (Table 2). The reduction in emergence of E. crus-galli at the lowest dose (0.5 t ha-1) was 67% and was only 2% in P. lathyroides. At the highest dose (2.0 t ha-1 ), the emergence of E. crus-galli was reduced by 92% and that of wild pea only by 30%.With increasing doses of application, the weed emergence as well as the plant height and dry biomass were decreased further. The pellets were more inhibitory to E. crus-galli than to P. lathyroides.

The effects of applying A. odorata granules to the three soil types on emergence, plant height, and dry weight of D. adscendens and T. portulacastrum are shown in Fig. 1 and 2, respectively. The emergence, plant high and dry mass of D. adscendens and T. portulacastrum were affected by soil type and varied with the amount of A. odorata granules applied. The degree of toxicity of different soil types can be classified in order of decreasing inhibition as sand > sandy loam >clay. The emergence, plant height, and dry weight of T. portulacastrum were less affected than those of D. adscendens. These results indicate that A. odorata granules has strong herbicidal potential for controlling D. adscendens. Under maize field conditions, the number of D. adscendens plants was affected significantly at all A. odorata granule doses and with the atrazine application, except for the lowest A. odorata granule dose (0.25 t ai ha-1) that was applied (Fig.3). The decrease in the number and biomass of the emerged plants of D. adscendens plants in the 1 t ai ha-1 A. odorata granule-applied plots was similar to that observed in the plots that received atrazine. Two additional broad-leaved weed species, T. portulacastrum and Amaranthus gracilis, were found in the experimental plots that receive A. odorata granule, however, at the highest dose of application (1 t ai ha-1), the emergence and dry weight of the two weed species were lower than those in the control plot. The A. odorata granules did not provide the same level of broad-leaved weed (T. portulacastrum and A. gracilis) control as the synthetic herbicide (atrazine), but the broad-leaved weeds that remained after applying the A. odorata granules seemed to be unable to compete with the maize. The yield of the maize plants growing in the plots that received the 0.25 t ai ha-1 A. odorata granule treatment was lower than those in the 0.5 and 1 t ai ha-1 treatment plots (Fig.4). The increase in the maize yield in the plots receiving the A. odorata granule treatment of 1 t ai ha-1 was 5.5% greater than that in the plots receiving the herbicidal treatment.

Discussion

Leaf and branch aqueous extracts of A. odorata were assayed for their effects on seed germination and early seedling growth of E. crus-galli and P. lathyroides L. The leaf and branch aqueous extracts at all concentrations inhibited seed germination and seedling growth, and the degree of inhibition increased with the incremental extracts concentration but the leaf extract was slightly more inhibitory than the branch extract. The results of this study suggest that branch and leaf of A. odorata contain water-soluble


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allelochemicals. Further work is needed to specify and verify the allelochemicals produced by this plant at different forms under laboratory conditions. The degree of toxicity of different A. odorata forms can be classified in order of decreasing inhibition as pellet > dried leaf powder > aqueous extract. The inhibitory effects of A. odorata powder were stronger than the effects of the aqueous extracts because in aqueous extract, the allelochemicals were extracted partially from the leaf powder in water, during the soaking at 10 °C for 72 h. However, the allelochemicals in leaf powder might have released continuously during the experimental period. Interestingly, the inhibitory effects of pellets prepared from the dried leaves were stronger than equal amount of leaf powder. This phenomenon might be attributed to the moisture of cassava glue acting as a compatibile medium during the pellets preparation. The A. odorata pellets under pot culture conditions also suppress the growth and yield of E. crus-galli which may be exploited to create a successful biorational herbicide.

The potential use of A. odorata granules as a weed suppressant was determined under experimental greenhouse and field condition. Under experimental greenhouse, the emergence and seedling growth of D. adscendens and T. portulacastrum was inhibited but varied with soil type; the inhibitory effect of the A. odorata granules depended on soil type; the inhibitory effect was extremely low in clay, as compared to sand, indicating that the allelochemical activity of A. odorata granules was greatly influenced by the soil physicochemical properties such as organic mater content. This result agreed with Kobayashi (2004) who reported that the sensitivity of weed species to phytotoxins depends on the physiological and biochemical characteristics of each species and the activity of phytotoxins become weak in the soil (Laosinwattana et al., 2010). Under natural field conditions, A. odorata granules could be used to suppress D. adscendens weed emergence and growth in maize field and had no adverse effect on maize growth and silage yield. The lack of a reduction in maize silage yield by A. odorata granule agrees with the results reported by Dhima et al., (2010) who found that the use of aromatic plants incorporated as green manure for a maize crop did not significantly affect maize development. Table 1 Effect of application of A. odorata in 3 forms on germination and seedling growth of Echinochloa crus-galli and Phaseolus lathyroides in Petri dish.

Conc. (g dried leaf /L)

------------------------------------------- inhibition (%) over control ------------------------------------------Aqueous extract Dried leaf powder Pellet

Seed germination

Root length

Shoot length

Seed germination

Root length

Shootlength

Seed germination

Root length

Shoot length

Echinochloa crus-galli

6.25 2.5 -1.68 -3.3 25 24.04 0 30 16.83 17.09

12.5 21.75 5.05 62.91 50 69.95 62.91 70 74.76 83.49

25 70 34.14 95.53 78 82.21 97.48 88 91.83 98.06

50 100 100 100 100 100 100 100 100 100

Phaseolus lathyroides

6.25 0 -0.81 2.91 0 16.26 -12.62 3 6.02 2.91

12.5 11 4.55 62.14 20 36.42 39.32 25 53.01 62.14

25 47.5 37.72 88.83 54.5 65.37 64.08 69.5 85.85 95.15

50 74.5 80 91.26 100 100 100 100 100 100


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Table 2 Emergence (14 DAT) and growth (28 DAT) of Echinochloa crus-galli and Phaseolus lathyroides in response to the use of A. odorata pellet formulation as soil surface mulch in the pot experiment.

Pellet dose (t/ha)

---------------------------------- inhibition (%) over control ---------------------------------

Echinochloa crus-galli Phaseolus lathyroides

Emergence Plant height Biomass Emergence Plant height Biomass

0.5 67 34.62 82.43 2 4.86 10.34

1 92 62.81 96.74 15 2.43 18.1

2 100 100 100 30 27.32 41.81

0

20

40

60

80

100

120

0 0.25 0.5 1 0 0.25 0.5 1 0 0.25 0.5 1

clay soil Sandy loam soil Sand soil

Dose (ton (ai)/ha)

% o

f con

trol

Emergence Plant height Dry weight

Fig. 1 Emergence, plant height, and dry weight of Digitaria adscendens in clay, sandy loam, and sand amended with different amounts of A. odorata granules in test pots.

0

20

40

60

80

100

120

0 0.25 0.5 1 0 0.25 0.5 1 0 0.25 0.5 1

clay soil Sandy loam soil Sand soil

Dose (ton (ai)/ha)

% o

f con

trol

Emergence Plant height Dry weight

Fig. 2 Emergence, plant height, and dry weight of Trianthema portulacastrum in clay, sandy loam, and sand amended with different amounts of A. odorata granules in test pots.


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0

20

40

60

80

100

Density Dry mass Density Dry mass Density Dry mass

D. adsxendens T. portulacastrum A. gracillis

% o

f con

trol

non-weed control 0.25 ton/ha A.odorata granule0.5 ton/ha A. odorata granule 1 kg/ha Atrazine2 kg/ha Atrazine

Fig. 3 Effect of A. odorata granules applied as a pre-emergent herbicide on weed density and dry biomass under field conditions.

0 20 40 60 80 100 120

0.25 ton/ha A.odoratagranule

0.5 ton/ha A. odoratagranule

1 ton/ha A.odoratagranule

2.5 kg/ha Atrazine

% of control

Dry Mass Grain yield

Fig. 4 Effect of A. odorata granules applied as a pre-emergent herbicide on dry biomass grain yield of maize under field conditions.

Conclusion

Leaf and branch from A. odorata contained certain allelochemicals inhibitory to E. crus-galli and P. lathyroides germination and growth. The degree of toxicity of different A. odorata forms can be classified in order of decreasing inhibition as pellet > dried leaf powder > aqueous extract. J. odorata pellet forms under pot culture condition also suppress the growth of E. crus-galli. Under natural field conditions, an organic herbicide produced from A. odorata in the form of a powder product could be used to suppress D. adscendens weed emergence and growth in maize field and had no adverse effect on maize growth and silage yield.


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Acknowledgements

This research was supported by grants from The Research Foundation of King Mongkut’s Institute of Technology Ladkrabang and Thailand Toray Science Foundation, Bangkok, Thailand.

References

Batish, D.R, Kaur, M., Singh, H.P. and Kohli, R.K. (2006)a. Phytotoxicity of a Medicinal Plant, Anisomeles indica, Against Phalaris minor and Its Potential Use as Natural Herbicide in Wheat Fields, Crop Prot. 26: 948-952.

Dhima, K.V., Vasilakoglou, I.B., Gatsis, Th.D., Panou-Philotheou, E. and Eleftherohorinos, I.G. (2010). Effects of Aromatic Plants Incorporated as Green Manure on Weed and Maize Development. Field Crops Res. 110: 235-241.

Duke, S.O. (1986). Naturally Occurring Chemical Compounds as Herbicides. Rev. Weed Sci. 2: 15-44.

Einhellig, F.A. (1995(. Allelopathy: Current Status and Future Goals. In: Inderjit, Dakshini, K.M.M., Einhellig, F.A. (Eds.). Allelopathy: Organisms, Processes and Applications. American Chemical Society, Washington, DC, pp. 1-24.

Fujii, Y. (2001). Screening and Future Exploitation of Allelopathic Plants as Alternative Herbicides with Special Reference to Hairy Vetch. Crop Prot. 4: 257-275.

Inderjit, Streibig, J.C. and Olofsdotter, M. (2002). Joint of Action of Phenolic Acid Mixtures and Its Significance in Allelopathy Research. Physiol. Plant. 144: 422-428.

Khanh, T.D., Hong, N.H., Nhan, D.Q., Kim, S.L., Chun, I.M. and Xuan, T.D. (2006). Herbicidal Activity of Stylosanthes guianensis and Its Phytotoxic Components. J. Agron. Crop Sci. 192: 427-433.

Kohli, R.K., Batish, D.R. and Singh, H.P. (1998). Allelopathy and Iits Implications in Agroecosystems. J. Crop Prod. 1, 169–202.

Kobayashi, K. (2004). Factors Affecting Phytotoxin Activity of Allelochemicals in Soil. Weed Biol. Manag. 4: 1-7.

Laosinwattana, C., Boonleom, C., Teerarak, M., Thitavasanta, S. and Charoenying, P. (2010). Potential Allelopathic Effects of Suregada multiflorum and The Influence of Soil Type on Its Residue’s Efficacy. Weed Biol. Manag. 10: 153-159.

Lin, D., Sugitomo, Y., Dong, Y., Terao H. and Matsuo, M. (2006). Natural Herbicidal Potential of Saururaceae (Houttuynia cordata Thunb) Dried Powders on Paddy Weeds in Transplanted Rice. Crop Prot. 25: 1126-1129.

Singh, H.P., Batish, D.R., Kaur, S. and Kohli, R. (2003)a. Phytotoxic Interference of Ageratum conyzoides with Wheat (Triticum aestivum). J. Agron. Crop Sci. 189: 341-346.

Xuan, T.D., Tawata, S.T., Khanh, D. and Chung, I.M. (2005). Biological Control of Weeds and Plant Pathogens in Paddy Rice by Exploiting Plant Allelopathy. Crop Prot. 24: 197-206.


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Diversity of Hyphomycetes Fungi from Diseased WeedsDuangporn Suwanagul1, Jitra Kokaew2 and Anawat Suwanagul2

1Faculty of Biotechnology, Rangsit University Pathum Thani 12000, Thailand 2Thailand Institute of Scientific and Technological Research, Pathum Thani 12120, Thailand

E-mail addresses: [email protected], 76 [email protected], [email protected]

Abstract

Diseased weeds showing leaf spot and leaf blight were collected from vegetable plots in Central of Thailand. Seven weed hosts are included Cyperus rotundus (Cyperaceae), Cyperus brevifolius (Cyperaceae), Brachiaria mutica (Poaceae), Eleusine indica (Poaceae), Dactyloctenium aegyptium (Poaceae), Pennisetum polystachyon (Gramineae) and Oryzae sativa f. spontanea (Poaceae). Tissue transplanting and moist chamber methods were used to isolate microfungi. Identification was base on growth rate, colony color and other microscope features as observed on artificial media. Microscopic characteristics were examined under stereo and light microscopes. A total of 20 fungi isolates, comprising of 9 species, were found. These are included Alternaria alternate, Bipolaris bicolor, Curvularia intermedia, Curvularia pallescens, Drechslera holmii, Exserohilum rostratum, Fusarium oxysporum, Myrothecium verrucaria and Nigrospora oryzae. Five different preservation methods, including PDA slant, liquid paraffin, filter paper, grain culture and 15% glycerol method, were employed to maintain pure culture of all fungal for future use.

Keywords: Weeds, Fungi, Htphomycetes, Biopesticides

Introduction

The Hyphomycetes fungi is importance to human, animals and plants (Sivanesan, 1987; De Hoog et al., 2000). Manoch et al. (2004) reported Alternaria alternata, Stemphylium solani, Curvularia lunata, Thielaviopsis basicola and T. thielavioides caused disease in plant. De Hoog et al. (2000) and Moss (2003) reported human and animals diseases caused by Hyphomycetes fungi such as Arthrinium phaeospermum, Exerohilum rostratum, Memnoniella echinata, Pithomyces chartarum and Stachybotrys chartarum. In the other hand, the Hyphomycetes is beneficial fungi because they can produced many secondary metabolites such as nigrosporin A & B from Nigrospora oryzae can inhibited Bacillus subtilis be equal to streptomycin in vitro (Tanaka et al., 1997). Oidiodrendron griseum produced 10-methoxydihydrofuscin, fuscinarin and fuscin that is efficiency to inhibited HIV-1 (Yoganathan et al., 2003 ). Sin et al., 2002 reported Gilmaniella bambusae, Periconia minmibissia and Tetraploa aristata produced enzyme cellulase and xylanase. Nigrospora oryzae produced secondary metabolite to inhibit spore germination of fungal pathogens such as Fusarium avenaceum, F. culmorum, F. equiseti, F. gramminearum, F. oxysporum, F. lateritium and Botrytis cinerea (Szewczuk et al., 1991).

The benefit and importance of Hyphomycetes fungi have been reported by many mycologist. Simmons (2004) reported new species of Dematiaceous Hyphomycetes from many plants. Varma et al. (2006) reported Curvularia lunata caused leaf spot disease on grass this fungi produced 4-epiradicinol inhibit bacterial growth. Nikonov et al. (2007) reported Alternaria alternata and Curvularia geniculata produced enzyme laccase to degrade lignin and humus. The investigations on Hyphomycetous fungi causing weed diseases have been reported by many researchers (Auld and Mc Rae, 1999; Babu et al., 2003). The exploitation of fungal plant pathogens as biological weed control agents has gained considerable importance due to the secondary metabolite they produced (Bonilla et al., 1999; Boyette et al., 2002; Domsch et al., 1993). The purposes of this study were to isolate and identify Hyphomycetes fungal species from diseased weeds.


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Moist chamber method 1. Each excrement sample was placed in a moist chamber consisting of a glass bowl or plastic box lined

with damp cotton or tissue-paper and placed by the window. They were incubated for 2-7 days or longer at 28oC and observation was made under stereomicroscope. Transferred needle was used to transfer spores or fruiting structures on a slide and mount with a drop of distilled water, then covered with cover slip and examined under light microscope with Normaski Interference Contrast. Photomicrographs were taken were used.

Tissue transplanting method 2. Small pieces of diseased weeds were surface sterilized in 10 % clorox for 3-5 min, rinsed with

sterile distilled water, transferred to sterilized tissue paper and placed on potato dextrose agar (PDA) in a petridish. They were incubated at 28°C for a few days. Hyphal tips were transferred on to PDA slants.

Identification of Fungi3. Macroscopic features were studied including colony growth pattern, color, texture on

different agar media. Fungal growth rate was measured on PDA, CMA. CZA and MEA. Microscopic characters were observed on a slide preparation using sterile distilled water and lectophenol as mounting media and examined under a light microscope (Olympus BH-2 with Normaski Interference Contrast). Photomicrographs of fungal structure were taken under stereo, light microscopes.

Preservation of fungi4. Five different preservation methods were used to maintain all fungal pure cultures.

4.1 PDA slant method (Monoch et al., 2005; Smith and Onions, 1994)Pure cultures of fungi were maintained on PDA slants at 28o C. Sub-culturing was carried out

every 6 months.

4.2 Liquid paraffin method (Monoch et al., 2005)Pure cultures were maintained on PDA agar slant in a vial (1 dram). Liquid paraffin was placed

in a vial and autoclaved twice. Covering the pure culture on agar slant with sterile liquid paraffin about 2/3 of a vial and stored at 28o C. in order to prevent dehydration and slows down metabolic activity and growth through reduced oxygen tension.

4.3 Filter paper method (Jeamjitt, 2007)Fifteen pieces (0.5 x 0.5 cm2) of sterile filter paper Whatman no. 1

were placed on PDA in sterile Petri-dishes. The mycelia were transferred on PDA and incubated for 7-14 days depend on the species. The filter papers with mycelium were transferred to new sterile Petri dishes by using sterile forcep and placed in an electric dessicator for 7-10 days. Dried filter papers covered with mycelium and fruiting bodies were kept in the alumnium foil in a sterile plastic bags, labeled and placed in a box and strorage at - 20o C.

4.4 Grain culture Grain of barley were autoclaved at 121°C for 15 minutes and placed on PDA in sterile Petri-

dishes. Mycelial puge of endophytic fungi were transferred to PDA for 7-14 days. The plant tissue with mycelium were transferred to plastic tube by using sterile forcep, labeled and placed in a box and strorage at -20o C.

4.5 15% glycerol15 % glyceral in vial were autoclaved at 121°C for 15 minutes. Mycelial puge of endophytic

fungi were transferred to 15% glycerol and kept in a box and strorage at 4o C.All fungal pure cultures were maintained at Microbiological Resource Centre , Thailand Institute

of Scientific and Technological Research (TISTR), Phathum Thani, Thailand for future use.


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Result and Discussion

Nine species of Hyphomycetes fungi were found on 7 diseased weeds as follows (Table 1,2;Figure1).

Table 1 Weeds species and Locations of Collection.

No. Weeds species Family Location1 Cyperus rotundus Cyperaceae Bangkok2 Cyperus brevifolius Cyperaceae Chanthaburi3 Brachiaria mutica Poaceae Chanthaburi4 Eleusine indica Poaceae Pathum Thani5 Dactyloctenium aegyptium Poaceae Pathum Thani6 Pennisetum polystachyon Poaceae Pathum Thani7 Oryzae sativa f. spontanea Poaceae Bangkok

Figure 1 Disease symptoms of weeds A; Cyperus rotundus, B; Dactyloctenium aegyptium and C; Eleusine indica

Altenaria alternate (Fr.) Keissler Colonies on PDA attaining 9.0 cm in diam. in 7 days at 28°C, cottony, olivaceous black. Conidiophore geniculate, smooth 2.5-4.5 x 50 µm. Conidia formed in branched chain, 10-15 x 21-55 µm. producing beak, 2.5-5 µm long. A. alternate has been studied and evaluated the potential use of an indigenous isolate of as a mycoherbicide to control Lantana camara (Saxena and Pandy, 2002)

Bipolaris bicolor Paul & Parbery Colonies on PDA attaining 9.0 cm in diam. in 7 days at 28°C, hairy, dark brown. Conidiophore geniculate, smooth 2.5-5 x 54 µm. Conidia strain, cylindrical 18-20 x 55-120 µm., with 7-9 pseudoseptate, hilum flat dark. B. bicolor has been reported the application of to control Johnson grass (Sorghum helepense) Bonilla et al. (1999).

Curvularia intermedia Boedijn Colonies on PDA attaining 8.5 cm in diam. in 7 days at 28°Cbrown, cottony. Conidiophore straight, smooth 4.7-9.0 x 25-754 µm. Conidia strain or curve, 3 distoseptate, 13-20 x 25-35 µm. C. intermedia isolated from diseased crabgrass (Digitaria sp.) has been reported the potential use as microherbicide to control large crabgrass (D. sanguinalis) (Tilley and Walker, 2002).

Curvularia pallescens BoedijnColonies on PDA attaining 8.0 cm in diam. in 7 days at 28°C grey. Conidiophore geniculate,

5.5-6.0-9.0 x 85-180 µm. Conidia strain or curve, 3 distoseptate, slightly curve, ellipsoidal, pale to brown, smooth, 7.2-11.5 x 17-33 µm. C. pallescens isolated from disease leaves of Cyperus rotundus, Dactylotenium aegyptium, Echinochloa colona, Eleusine indica, Digitaria ciliaris, Brachairia reptans, Rottboellia cochinchinensis and Pennisetum polystachyon has showen the symptom very similar to our study. This fungus has been reported to produce Endo-1,4D-glucanase (Kokaew 2005).

BA


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Drechslera holmii (Luttrell) Subran. & JainColonies on PDA attaining 9.0 cm in diam. in 7 days at 28°C velvety. Conidiophore geniculate,

dark brown, 5.5-10.0 x 185-250 µm. Conidia obclavate and rotrate, with 6-11 pseudosepta, end cell pall and cut off by dark thick septa, intermediate cell, golden brown, smooth, 20-31 x 60-125µm, hilum protuberant, dark. Meanwhile, a study of Ellis (1971) reported that D. holmii causing reddish-brown spot and strips on leaves of Dactyloctenium sp. in USA.

Exserohilum rostratum (Drechsler) Leonard & SuggsColonies on PDA attaining 9.0 cm in diam. in 7 days at 28°C velvety. Conidiophore geniculate,

brown, 5.5-7.2 x 125-200 µm. Conidia slightly cuve, obclavate, rotrate, with 4-12 pseudosepta, smooth, 14-22 x 40-180 µm, hilum distinctly protuberant, bipolar germination. This fungus was successfully applied as foliar spay to control seven weedy grasses in citrus orchard (Chandramohan and Charudattan 2001).

Fusarium oxysporum SchlechtColonies on PDA attaining 9.0 cm in diam. in 7 days at 28°C cottony. Conidia of two types,

microconidia 1- celled, hyaline, 2.5-3.0 x 5.5-12.0 lightly cuve, obclavate, rotrate, with 4-12 µm and macroconidia 3 to many septate, fusiform to falcate, hyaline, 3.2-5.5 x 21.0-60.0 µm. F. oxysporum has been reporeted as mycoherbicide to control Striga hermonthica in Wastern Africa (Clotala et al. 1995). Table 2 Micro fungi isolated from weed diseases at different locations.

Fungi Class Host plantAlternaria alternate Hyphomycetes 1,2Bipolaris bicolor Hyphomycetes 1,3Curvularia intermedia Hyphomycetes 1,3,4Curvularia pallescens Hyphomycetes 1,2,4,6Drechslera holmii Hyphomycetes 5Exserohilum rostratum Hyphomycetes 1,2,5,6Fusarium oxysporum Hyphomycetes 2,3,6Myrothecium verrucaria Hyphomycetes 2,3,4Nigrospora oryzae Hyphomycetes 1,2,3

* 1= Cyperus rotundus, 2 = Cyperus brevifolius, 3= Brachiaria mutica, 4= Eleusine indica, 5=Dactyloctenium aegyptium, 6= Pennisetum polystachyon Myrothecium vurrucaria (Alb. & Schw.) Ditm. Ex fr.

Colonies on PDA attaining 9.0 cm in diam. in 7 days at 28°C Sporodochia, green to black surrounded by a zone of white. Conidia in dark green slimy masses, cylindrical, navicular to ellipsoidal, hyaline, smooth, 2.0-4.5 x 6-10 µm.

Boyett et al. (2002) reported the application of M. verrucaria to control Kudzu (Pueraria lobata). Secondary metabolites produced by this fungus such as Verrucaria A,B,C,D,E,F,G,J; Roridins A,B,D,E,H; Muconomycin; Coprogen B; Gliotoxin and Antifungal

Nigrospora oryzae HudsonColonies on PDA attaining 9.0 cm in diam. in 7 days at 28°C at first white, turned to grey. Conidia

broadly ellipsoidal, black, 10-15 µm in diam. A report of Kokaew (2005) that isolated C. pallescens from disease leaves of Cyperus rotundus, Dactylotenium aegyptium, Echinochloa colona, Eleusine indica, Digitaria ciliaris, Brachairia reptans, Rottboellia cochinchinensis and Pennisetum polystachyon which was very similar to the present study. This fungus produced Verrucaria A,B,C,D,E,F,G,J; Muconomycin; Coprogen B; Gliotoxin


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Conclusion

A total of 20 isolates of Hyphomycetes fungi found on diseased weed collected from vegetable plots in Bangkok, Pathum Thani and Chanthaburi provinces were common leaf spot and leaf blight diseases. There were Alternaria alternate, Bipolaris bicolor, Curvularia intermedoia, Curvularia pallescens, Drechslera holmii, Exserohilum rostratum, Fusarium oxysporum, Myrothecium verrucaria, Nigrospora oryzae.

Acknowledgment

We would like to thank the authorities of Faculty of Biotechnology, Rangsit University and Thailand Institute of Scientific and Technological Research for extending facilities on this research.

Reference

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Impacts of meadowfoam seed meal amendment on weeds and soil microbial activity

Suphannika Intanon1, Andrew Hulting1, David Myrold1, and Carol Mallory-Smith1

1 Department of Crop and Soil Science, Oregon State University.109 Crop Science Building, Corvallis, Oregon 97331, USA.

[email protected]

Abstract

Meadowfoam (LimnanthusalbaHartw. exBenth) seed meal, a by-product of meadowfoam oil extraction, has glucosinolate degradation compounds that are similar to those from Brassicaceae. The compounds are reported to be herbicidal. Two field studies were conducted to evaluate the application of meadowfoam seed meal for weed control in lettuce and the effect of meadowfoam seed meal on soil microbial activity. Meadowfoam seed meal was applied either as 2.86 kg m-2 on day 0 or as 1.43 kg m-2 on day 0 followed by 1.43 kg m-2 on day 7. To account for the fertilizer effect of the seed meal, urea was used as a nitrogen source and applied eitheras 16.8 g m-2 on day 0 or as 8.4 g m-2 on day 0 followed by 8.4 g m-2 on day 7. Meadowfoam seed meal treatment suppressed weed emergence and growth. A split application of meadowfoam seed meal provided the best control of spiny sowthistle. Lettuce aboveground biomass was similar between urea and meadowfoam seed meal amended treatments. Soil microbial activity was greater in meadowfoam seed meal treatmentscompared to either urea or non-amended plots. Both fertilizer and bioherbicide effects were found with the use of meadowfoam seed meal.

Keywords: meadowfoam seed meal, LimnanthusalbaHartw. exBenth,glucosinolate, soil amendment

Introduction

Meadowfoam (LimnanthusalbaHartw. ex Benth.) is known as an industrial oil seed crop. It is a winter annual crop in the Limnanthaceae family, which is native to southern Oregon and northern California. It is used as a winter rotation crop in grass seed production systems in the Pacific Northwest. The oil extracted from meadowfoam seed possesses a unique oxidative stability that makes it useful in a wide range of cosmetic and personal care formulations. About 70% of the extracted seed remains after oil extraction. At present, this by-product, known as meadowfoam seed meal (MSM), has little value. Interestingly, MSM has potentially herbicidal glucosinolate degradation compounds that are similar to those from Brassicaceae species. Previous research suggests that the effectiveness of MSM as a soil amendment depends on the concentration applied. Low levels of MSM may be a growth stimulant for vegetable crops (Vaughn et al. 2008). At greater concentrations, MSM has been found to inhibit seed germination and possess herbicidal properties (Lindermanet al. 2007; Machado, 2007; Stevens et al. 2009). A greenhouse study confirmed the herbicidal effect of MSM on seeding emergence and growth compared to the control (Intanonet al. 2011). A field study suggested that the meadowfoam seed meal has both fertilizer and bioherbicide effects (Intanonet al. 2012). However, bioherbicide effects were much less than those observed in the greenhouse at the same concentration.The fertilizer effect was observed when MSM was applied at 1.22 kg m-2 and at 2.04 kg m-2, whereas there was a bioherbicide effect observed at 2.86 kg m-2. Although concentration seems to be a key factor in the application of MSM for weed control,with respect to developing MSM as a bioherbicide, the application of an effective concentration and the method of the meal used under field condition need to be investigated. In addition to the investigation of the bioherbicide effect, theimpact of MSM amendment on microbial activity was included in this study. Soil microorganisms, mainly bacteria and fungi, play important roles in nutrients availabilityfor plants (Wardle and Ghani, 1995). The variability in a microbial


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community can be used to indicate the change in soil quality (Breure, 2005).However, there are no data on soil microbial activity changes related to MSM application so the effects of soil amendment with MSM on microbial activity need to be identified. The goal of this study was to evaluate the application of MSM for weed control in transplanted lettuce. The specific objectives were:1) to determine the crop yield and additional nutrients from MSM application, 2)to investigate the effect of MSM application on weed emergence and growth, 3) to determinethe effect of meadowfoam seed meal on soil microbial activity via basal respiration, and 4) to evaluate the relationship between methods of MSM application, plant growth, and soil microbial activity.

Material and methodsSoil A field study was conductedon a Lewis-Brown Horticulture Research Farm, Oregon State University, Oregon, USA. The soil was a mollisol classified as a Malabonsilty clay loam.

Field study In the summer of 2012, two field experiments were conducted from July 9 to September 12 for the first experiment and from August 1 to October 5 for the second experiment using a randomized complete block design with four replications. There were five treatments which consisted of two amendment materials (urea and activated MSM) with two application methods (either full or split rate application) and one control treatment (non-amended treatment). Meadowfoam seed meal was passed through a 1 mm-sieve before use. Activated MSM consisted of 1% ground meadowfoam seed and 99% seed meal by weight. Activated MSM was applied either as 2.86 kg m-2 on day 0 or as 1.43 kg m-2 on day 0 followed by 1.43 kg m-2 on day 7. To account for the fertilizer effect of the seed meal, urea was used as a inorganic nitrogen source and applied eitheras 16.8 g m-2 on day 0 or as 8.4 g m-2 on day 0 followed by 8.4 g m-2 on day 7.The treatment plot size was 1.6 m2 with 1.07 m border between plots and 1.07 m border around the entire site. All plots were cleared of weeds before starting the experiments. A hundred seeds of Sonchusasper (spiny sowthistle) and of Echinochloafrumentacea (Japanese millet) were sown in an evenly spaced rowin each plot right after meal incorporation. Plots were irrigated using sprinkler irrigation system at an average application rate of 254 mm hr-1.Nine lettuce seedlings (18 days old) were transplanted in the middle row of the plot at7 days after incorporation (DAI). Lettuce was transplanted at 15 cm in-row-spacing. On 35 DAI, seven lettuce plantswere harvestedfor aboveground biomass. Seedlings of S. asper and E.frumentaceawere counted and then harvested for aboveground biomass. Weed sample area was 1.14 m2 in each plot. Other emerged weeds wereseparated by species, counted,and harvested for aboveground biomass on35 DAI and re-harvested on 65 DAI. Lettuce and weeds were dried at 60 C for 72 hr, and weighed.Dry lettuce tissue was used to measuretotal nitrogen (N)and sulfur(S)by CNS-Analyzer (Leco CNS-2000).

Soil measurement Soil samples were taken in each plot to 5-cm depth within an areaof 0.46 m2to studysoil microbial activity. The samples were conducted on 0, 7, 14, and 28 DAI. Three soil cores were sampled in each plot and composited. The moist soil was passed through a 3-mm sieve and incubatedin the dark for 48 hr at 25 C before monitoring the total CO2basal respiration by using Isotopic CO2Analyser(PicarroG2101).

Data analysis The aboveground biomass, emergence, total NS, and basal respiration data for each treatment were analyzed using ANOVA. Means were separated by a least significant difference (LSD) test using PROC GLM in SAS v. 9.2.


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Results

Effect of soil amended treatments on lettuce growth and plant nutrient availability Total lettuce biomass in Experiment 1 was greater than Experiment 2 (Table 1). The total plant sulfur in MSM-amended treatments was 24% and 38% greater in MSM amended treatments compared to non-amended and urea-amended treatments in Experiment 1 and 2, respectively. In Experiment 1, total plant nitrogen in amended plots with urea and MSM was 49% greater than non-amended plots. In Experiment 2, total plant nitrogen in urea-amended treatmentswas not different from non-amended treatment andthe MSM amended treatments had 34% greater plant-available nitrogen compared to non-amended and urea-amended treatments.

Table 1. Drybiomass of seven lettuce plants and chemical analysis per gram of lettuce shoot biomass grown in the middle row of sample plots for 18 days in the greenhouse and 35 days in the field with or without amended materials.Data are represented as means with SE within parentheses. Different letters within a column indicate significant differences at the 0.05 levelwithin an experiment.

Expt Trta Lettuce biomassb (g) Total sulfur (g) Total nitrogen (g)1 NC 33.6 (2.74) nsc 0.068 (0.0043) b+ 1.0 (0.11) b

CU 47.2 (4.44) 0.092 (0.0101) ab 2.1 (0.18) aCR 41.8 (3.06) 0.093 (0.0073) ab 2.0 (0.13) aMS 37.2 (3.89) 0.112 (0.0092) a 1.9 (0.15) aMR 36.3 (5.16) 0.110 (0.0164) a 1.8 (0.27) a

2 NC 25.5 (1.92) a 0.061 (0.0046) bc 1.0 (0.09) bCU 24.6 (2.55) a 0.058 (0.0074) c 1.3 (0.03) abCR 25.9 (0.77) a 0.056 (0.0043) c 1.1 (0.05) bMS 36.9 (5.21) b 0.099 (0.0159) ab 1.7 (0.28) aMR 32.4 (2.40) ab 0.091 (0.0072) a 1.7 (0.12) a

atreatments: NC, non-amended; CU, full rate of urea amendment; CR, split rate of urea amendment; MS, full rate ofmeadowfoam seed meal amendment and MR, split rate of meadowfoam amendmentbaverage dry biomass for seven lettuce plants (n=4)cnot significant +significant difference at 0.01 level

Effect of soil amended treatments on weed emergence and growth The effect of treatments on the total emergence and dry biomass of natural occurring weeds, S. asper, and E.frumentaceadiffered across treatment (Table 2 and 3; Fig. 1). At 35 DAI, total dry biomass of naturally occurring weeds in MSM-amended treatments was reduced more than 85% for Experiment 1 and more than 74% for Experiment 2 (Table 2). There was no difference in total dry biomass of naturally occurring weeds harvested at 65 DAI. However, the biomass from Experiment 1 (on average 0.91 g m-2) was much less than those from Experiment 2 (on average 26.72 g m-2). In both experiments, dry weight per plant of E.frumentacea was not different across treatments (on average of 0.82 g plant-1 for Experiment 1 and 0.66 g plant-1 for Experiment 2). In Experiment 1, a full rate application of MSM treatment prevented S. asper germination, whereas in Experiment 2, MSM treatments of both full and split rate applications preventedS. asper germination. In general, the emergence of naturally occurring weedson 35 and 65 DAI for Experiment 1 was less than Experiment 2 and the emergence of naturally occurring weeds, S. asper , and E. frumentaceawas reduced in both MSM treatments (Table 3). On 35 DAI, MSM treatments provided 89% and 85% suppression of weed emergence when compared to urea-amended and non-amended treatments for


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Table 2. Aboveground biomass of naturally occurring weeds, Sonchusasper and Echinochloa frumentacea harvested on different days after meal or urea incorporation (DAI). Data are represented as means with SE within parentheses. Different letters within a column indicate significant differences at the 0.05 levelwithin an experiment.

Expt TrtaDW naturally

occurring weeds(g m-2)

DW naturallyoccurring weeds

(g m-2)

S. asper (g plant-1)

E. frumentacea(g plant-1)

1 35 DAI 65 DAI 35 DAI 35 DAINC 41.0 (10.79) b 0.44 (0.079) nsb 0.13 (0.007) ab 0.48 (0.052) nsCU 83.1 (9.45) a 2.20 (0.933) 0.14 (0.012) ab 0.98 (0.066)CR 55.9 (13.21) b 0.64 (0.238) 0.15 (0.017) a 0.97 (0.077)MS 6.3 (0.21) c 0.17 (0.087) 0.08 (0.046) b 0.60 (0.209)MR 4.8 (0.32) c 1.08 (0.986) 0 c 1.06 (0.715)

2 35 DAI 65 DAI 35 DAI 35 DAINC 66.5 (13.74) a 29.6 (1.33) ns 0.07 (0.011) a 0.54 (0.195) nsCU 60.6 (6.66) ab 30.7 (6.28) 0.07 (0.010) a 0.36 (0.023)CR 40.1 (2.96) b 27.7 (3.80) 0.07 (0.018) a 0.54 (0.087)MS 9.8 (2.02) c 20.0 (7.88) 0 b 0.91 (0.174)MR 10.3 (0.87) c 25.6 (3.93) 0 b 0.97 (0.253)

a treatments: NC=non-amended, CU=full rate of urea amendment, CR=split rate of urea amendment, MS=full rate of meadowfoam seed meal amendment, MR=split rate of meadowfoam amendment

b not significant

Table 3.Total emergence of naturally occurring weeds, Sonchusasper and Echinochloa frumentaceaharvested at different days after meal or urea incorporation (DAI). Data are represented as means with SE within parentheses. Different letters within a column indicate significant differences at the 0.05 levelwithin an experiment.

Expt TrtaTotal emergence

naturally occurringweeds (seedlings m-2)

Total emergence naturally occurring

weeds (seedlings m-2)

S. asper emergence (% of sown seeds)b

E. frumentaceaemergence (% of sown seeds)b

1 35 DAI 65 DAI 35 DAI 35 DAINC 150.9 (29.6) a 4.8 (0.6) ab+ 25.8 (2.84) a 11.8 (2.14) aCU 173.9 (22.1) a 8.8 (2.3) a 24.0 (2.12) ab 12.3 (1.11) aCR 182.7 (39.3) a 7.2 (1.1) ab 19.5 (3.01) b 9.5 (1.55) aMS 23.2 (5.0) b 2.0 (0.9) b 1.3 (0.95) c 3.0 (1.08) bMR 13.2 (1.4) b 2.9 (2.3) b 0 c 1.3 (0.48) b

2 35 DAI 65 DAI 35 DAI 35 DAINC 308.8 (23.5) a 229.2 (32.4) a 29.0 (3.29) a 10.3 (2.36) abCU 276.3 (38.5) a 212.1 (13.4) a 26.8 (3.64) a 10.8 (1.89) aCR 166.4 (6.6) b 160.5 (6.3) b 20.8 (3.61) a 8.5 (2.63) abMS 38.6 (7.2) c 49.8 (10.4) c 0 b 3.8 (1.89) bcMR 37.9 (3.1) c 58.1 (5.2) c 0 b 1.8 (0.48) c

a treatments: NC, non-amended; CU, full rate of urea amendment; CR, split rate of urea amendment; MS, full rate of meadowfoam seed meal amendment and MR, split rate of meadowfoam amendment

baverage percentage of emerged seeds of the total100-sown seeds+significant difference at 0.01 level


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A)

B)

Fig. 1. Lettuce growth and weed community across soil amended with different materials on 35 DAI in A) Experiment 1and B) Experiment 2. NC, non-amended; CU, full rate of urea amendment; CR, split rate of urea amendment; MS, full rate of meadowfoam seed meal amendment and MR, split rate of meadowfoam amendment.

Effect of soil amended treatments on total CO2 basal respiration In general, there were greater total CO2basal respiration in MSM-amended treatments compared to non-amended and urea-amended treatments (Table 4). Within 28 days, the change of the basal respiration slightly decreased over time in non-amended and urea-amended treatments but steeply decreased in MSM-amended treatments. No difference of total CO2basal respiration was detected across non-amended and urea amended treatments for both experiments. In Experiment 1, the total CO2of MSM-amended treatments were 98%, 96.4%, 95.6%, and 92.6% greater than those of non-amended and urea amended treatments on initial, 7, 14, and 28 days, respectively. In Experiment 2, total CO2of MSM-amended treatments were 97.8%, 97.3%, 96.5%, and 92% greater than those of non-amended and urea amended treatments on initial, 7, 14, and 28 days, respectively.

Effect of the methods of MSM application on plant growth and soil microbial activity Based on the results of lettuce growth, weed emergence, and soil microbial activity, the effects of either a full or a split rate of MSM application treatments on plant growth were not obviously differentiated. The split MSM-amended treatment provided a better control of S. asper in early summer than a full rate application (Table 2 and 3).The differences between a full or split rate of MSM application can be observed in basal respiration across the experiments. In Experiment 1, the soil microbial activity from a full MSM application was greater than a split MSM application on initial and 7 DAI but less on 14 and 28 DAI. While in Experiment 2, the soil microbial activity from a full MSM application was less than a split MSM application for all measurement days (Table 4).

Table 4. Total CO2of basal respiration after incubation in the dark for 48 hr at 25 C. Soil sample were collected at initial, 7, 14, and 28 days after meal or urea incorporation (DAI). Data are represented as means with SE within parentheses. Different letters within a column indicate significant differences at the 0.05 levelwithin an experiment.

MRMSCRCUNC

MRMSCRCUNC


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Expt Trta Basal respiration (μgCO2-C g-1 soil hr-1)

1 0 DAI 7 DAI 14 DAI 28 DAINC 3.7 (0.42) b 2.8 (0.30) c 2.5 (0.13) b 1.4 (0.32) bCU 3.8 (1.05) b 2.6 (0.30) c 2.6 (0.16) b 2.0 (0.07) bCR 7.2 (2.02) b 3.6 (0.82) c 2.1 (0.29) b 1.3 (0.19) bMS 376.2 (92.37) a 96.9 (6.40) a 44.0 (6.44) a 17.0 (1.51) aMR 118.0 (52.27) b 68.8 (17.43) b 63.1 (18.42) a 25.7 (6.73) a

2 0 DAI 7 DAI 14 DAI 28 DAINC 4.6 (0.31) b 2.5 (0.20) c 1.9 (0.26) c 1.7 (0.04) bcCU 6.3 (1.35) b 2.7 (0.33) c 1.8 (0.30) c 0.8 (0.29) cCR 5.2 (1.39) b 4.0 (1.02) c 1.8 (0.27) c 1.3 (0.06) cMS 238.3 (57.97) a 82.4 (12.96) b 37.7 (11.30) b 9.8 (1.39) bMR 255.2 (44.54) a 142.6 (10.22) a 66.3 (6.90) a 22.2 (5.64) a

a treatments: NC, non-amended; CU, full rate of urea amendment; CR, split rate of urea amendment; MS, full rate of meadowfoam seed meal amendment and MR, split rate of meadowfoam amendment

Experiment 1 and 2, respectively. On 65 DAI, MSM treatments inhibited 65% and 73% of total weed emergence for Experiment 1 and 2, respectively. However, there was no difference of total weed emergence between a full rate and a split rate of the MSM application treatments. When considering new emerged weedswithin the same MSM-amended treatment, the amount of new weed emergence on 65 DAI was greater than those on 35 DAI. S. asper emergence was inhibited more than 94% by MSM-amended treatments, whereas E. frumentaceaemergence was suppressed 80.8% for Experiment 1 and 71.7% for Experiment 2. S. asperwas found in situin Lewis-Brown Horticulture Research Farm, whereasE.frumentaceawas not. The dry biomass and emergence data of S. asper in Table 2 and 3 did not include the in situ S. asper in the statistical analyses.

Discussion

The growth of transplanted lettuce plants responded positively to MSM-amended treatments (Table 1; Fig. 1). It is possible that MSMprovided the essential nutrients to support lettuce growth, especially plant-available nitrogen and sulfur. MSM treatment appeared to be an organic source of nitrogen and sulfur. The nitrogen and sulfur were reported as structural elements of MSM based on the chemical analysis of an allelochemical compound (Stevens et al. 2009). The organic nitrogen supplied from MSM was available for lettuce onamount similar and/or greatercompared to an inorganic fertilizer, urea. Similar results were found in conifer seedlings which grew faster in amendment of potting medium with MSM compared to non-amended medium (Lindermanet al. 2007). The conifer seedlings in MSM treatment had high total nitrogen and lacked nutrient deficiency symptoms, especially phosphorus. Lettuce growth in early summer (Experiment 1) was greater than late summer (Experiment 2; Table 1), probably because in early summer, the weather was still cool enough to support lettuce growth and development. In the split MSM treatment of Experiment 2, 44% of lettuce seedlings were replaced after a week of transplanting. The crop injury would imply to the less benefit of a split MSM application compared to a full rate application. The greater activity of MSM amendment in a split MSM application of Experiment 2 compared to those in Experiment 1 was also confirmed in the basal respiration, especially on the initial and 7 DAI (Table 4).Therefore, crop safety date should be longer than a week after MSM application when other environment factors influence crop growth and development. There was less weed pressure in early summer compared to late summer. In Experiment 2, there was more late summer grass and E.arvense infestation (Fig. 1B). The emergence and growth of weeds were inhibitedin MSM-amended treatments (Table 2 and 3). The inhibition was better observed in weed


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emergence compared to weed biomass data. It is possible because both fertilizer and bioherbicide effects were observed with the use of MSM as a soil amendment. The bioherbicide property was detected mainly at the first harvest (35 DAI). At the second harvest (65 DAI), 30 days after the first harvest with regular irrigation, a fertilizer effect was found, especially in Experiment 2 (Table 2). Brassicaceae seed meal as a soil amendment material for weed control also had similar results of co-occurrence of bioherbicide and fertilizer effects (Jonhson-Maynard et al. 2005). Emergence ofnaturally occurring weedsfrom MSM-amended treatments in Experiment 2 wasgreater than those from the first harvest (Table 3). The non-consistent results across the experiments may be due to greater weed pressure in late summer. According to the planted species, MSM had high level of suppression on emergence and growth ofS. asperbut not on the emergence and growth of E.frumentacea (Table 2 and 3; Fig. 1). Emerged E.frumentaceaseedlings from MSM-amended treatments were fewer but larger compared to non-amended and urea-amended treatments because they may take advantage of the addition of plant-available nutrientsfrom MSM. Although, MSM application did not have obvious selectivity for a specific weed species, it can generally suppress annual dicotsand monocots butwas less effective on some perennial plants such as E. arvense (data not shown). In this study, the effect of MSM on soil microbial activity focused only on total CO2 production from basal respiration. Basal respiration reflects the availability of carbon for microbial growth and maintenance and is a measure of the basic turnover rates in soil (Insamet al. 1991). Greater basal respiration ratein MSM-amended treatments compared to non-amended and urea-amended treatments was due to organic inputs from MSM (Table 4). The carbon inputs from MSM increased the gross metabolic activity of mixed microbial population. However, basal respiration aloneis just one measure of soil microbialactivity. Further investigations on the effects of MSM on soil microbial activity will involve multi-parameter approaches including microbial enzyme activity and specific carbon substrates for soil microbial community. The effect of MSM did not last longer more than a week. The repeat application after one week was assumed to increase the bioherbicide effect. Jonhson-Maynardet al. (2005) suggested that the reapplication of seed meal may provide adequate control weeds throughout the growing season but this can also increase late-season weed biomass due to the increase in plant-available nitrogen. However, based on the results of this study, there was no clear evidence of the differences between a full and a split rate of MSMapplication treatments on weed suppression and soil microbial activity.

Conclusions

MSM treatments applied as soil incorporation at 2.86 kg m-2 can inhibit weed emergence and growth. The split rate application of MSM provided a significant benefit for weed control similar to the full rate application. Application time of MSM showed the different results on plant and microbial activity. The early summer application provided better weed control than the late summer application which waspossibly due to less weed pressure in the early summer. However, the other environmental conditions (e.g. temperature and soil moisture) may possibly be involved. The effects of MSM treatment on soil microbial activity and soil quality still need to be determined using multi-parameter approaches and longer time span. In summary, a single MSM application as a pre-emergence soil amendment benefits crop yield, weed suppression, and soil carbon inputs.

Acknowledgements

I would like to thank Dr. John Hart for providing helpful suggestionsand assistance on plant nutrient analysis. I would also like to thank Megan McGinnisfor her advice and support on basal CO2respiration.


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References

Breure, A. M. 2005. Ecological soil monitoring and quality assessment, in:P. Doelman and H. J. P. Eijsackers (Eds.). Vital soil, function value and properties. Elsevier, Amsterdam. pp. 281-305.

Insam, H., C. C. Mitchell, and J. F. Dormaar. 1991. Relationship of soil microbial biomass and activity withfertilization practice and crop yield of three ultisols. Soil Biol.Biochem. 23: 459-464.

Intanon, S., A. Hulting, F. Stevens, J. Kling, R. Reed, and C. Mallory-Smith. 2011. The use of meadowfoam seed meal as a soil amendment to suppress seedling emergence. Proceedings of Western Society of Weed Science, 63rd annual meeting. Spokane, WA, March 7-10, 2011.p 29.

Intanon, S., A. Hulting, J. Kling, and C. Mallory-Smith. 2012. Evaluation of Meadowfoam Seed Meal as a Potential Bioherbicide. Proceedings of the Weed Science Society of America, 52nd annual meeting. Waikoloa, HI, February 6-9, 2012.

http://wssaabstracts.com/public/9/proceedings.html. Accessed: November 2, 2012.Johnson-Maynard, J. M.,Morra, L. Dandurand, C. William, and M. J. Butters. 2005. Brassicaceae seed

meal application for weed reduction and improved nitrogen management in organic farming systems. Project report, University of Idaho, Moscow, ID.

Linderman, R. G., E. A. Davis, and C. J. Masters. 2007. Response of conifer seedlings to potting medium amendment with meadowfoam seed meal, in:J. Janick and A. Whipkey (Eds.). Issues in New Crops and New Use. Alexandria, VA: ASHS Press.pp. 138-142.

Machado, S. 2007. Allelopathic potential of various plant species on downy brome: implications for weed. Agron. J. 99:127–132.

Stevens, J. F., R. L., Reed, S. Alber, L. Pritchett, and S. Machado. 2009. Herbicidal activity of glucosinolate degradation products in fermented meadowfoam (Limnanthesalba) seed meal. J. Agric. Food Chem. 57: 1821-1826.

Vaughn, S. F., M. A. Berhow, and B. Tisserat. 2008. Stimulation of plant growth by (3-methoxyphenyl) acetonitrile applied as a foliar spray in vivo or as a medium amendment in vitro. HortSci. 43: 372-375.

Wardle, D. A. and A. Ghani. 1995. A critique of the microbial metabolic quotient (qCO2) as a bioindicator of disturbance and ecosystem development. Soil Biol. Biochem. 27: 1601-1610.


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Imidazolinone tolerance variety for weedy rice control in direct-seeded rice: The Malaysian Experience

Azmi, M1., Yim, K. M2 and George, T. V2.1 Malaysian Agricultural Research and Development Institute (MARDI), P. O. Box 12301, General Post

Office,50774 Kuala Lumpur, Malaysia. E-mail: [email protected] (Malaysia) Sdn Bhd, 2 Jalan U8/87, Bukit Jelutong, 40706 Shah Alam, Selangor, Malaysia.

E-mail:[email protected], [email protected]

Abstract

Weedy rice (Oryza sativa complex) poses the greatest threat to direct-seeded rice (DSR) because of its taxonomic and physiological similarities to cultivated rice. Farmers in Malaysia have considered weedy rice as of significant and major importance since there are no selective herbicides available for weedy rice control prior to the advent of Herbicide Tolerant Rice (HTR). The development of local imidazolinone tolerant rice (IMI-TR) was through a collaborative project between MARDI and BASF which started in 2003 at MARDI Experimental Station in Seberang Perai, Penang. IMI-TR Line No. 1770 from United States was crossed with popular local rice cultivar of high yielding variety, MR 220. The goal of the project is to offer farmers a new innovative and effective solution to the weedy rice problem. After undergoing many trials and breeding selection, by 2008 two potential varieties, MR 220CL1 and MR 220CL2, were identified. The introduction of these cultivars is justified by the need to offer a new efficient and innovative alternative approach and method to manage weedy rice in DSR. The combination of IMI-TR variety with imidazolinone herbicides is known as the Clearfield® Production System (CPS) and this was launched in Malaysia on 8th July 2010. This was also the first launch of Clearfield® Production System for rice in the Asia Pacific region. This new technology is able to effectively control weedy rice that no other herbicides can control in DSR. The use of the CPS has benefited the rice industry in Malaysia by providing an effective management of weedy rice alongside with other noxious weeds in rice cultivation. Clearfield® Production System for rice offers many advantages and benefits namely it helps to simplify the process of weed management and control the weedy rice with a single OnDutyTM (imazapic/imazapyr) herbicide application. Most of all, it is an innovative and effective agronomic solution that delivers significant value and benefits to rice farmers through effective weed control using farmers normal cultural practices, increased yield potential and improved crop harvest quality. Studies have shown Clearfield® plots yielded 2.5 t/ha more than conventional plots with correspondingly net income being higher by more than USD900/ha. The CPS is non-GMO and signifies the beginning of a paradigm shift in modern agriculture for effective weedy rice and weed control. In 2012 season, about 30% of rice granaries in Malaysia had adopted this technology. The demand for this technology is expected to be increased from season to season in the future as shown from farmers acceptance and adoption since the second season of 2010 till now. In short, the CPS is tested and proven to be technically feasible, economically viable and culturally acceptable in the Malaysian experience.

Introduction

A major factor that contributes to a higher production cost for rice is weed control. Currently, rice farmers throughout the world face a unique weed problem. A weedy relative of cultivated rice known as weedy rice has invaded and severely infested direct-seeded rice (DSR) fields. Weedy rice has long been a major threat in the DSR culture in Asia, especially in Malaysia. Weedy rice matures earlier than cultivated rice, shatters and lodges easily. And if they lodge, they also cause the neighbouring cultivated rice plants to lodge along side. Under moderate weedy rice infestation (15-20 panicles/m2),


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yield loss is approximately 12 to 15%; under high infestation (21 to 30 panicles/m2) yield loss is 15 to 22%; while under heavy infestation (more than 50 panicles/m2) lodging of weedy rice plants may occur and can cause total yield loss under tropical climatic conditions (Azmi and Karim 2008).

Since there are no selective weedy rice herbicides in the pipeline, an alternative is to explore herbicide tolerant rice technology. Rice tolerant to imidazolinone herbicides was developed from a single plant that survived a chemically induced mutation trial in 1993 at Louisiana State University Agricultural Center (Sanders et al., 1998). The method used for obtaining herbicide-tolerant rice was chemical mutagenesis with ethyl methane sulfonate. The acetohydroxy acid synthase (AHAS) enzyme catalyses the first step in the biosynthesis of the branched chain amino acids leucine, isoleucine and valine in plants. The mutation in AHAS enzyme results in alteration to the binding site for the imidazolinone class herbicide, therefore increasing the plant’s tolerance to those herbicides. The other properties of the AHAS enzyme are unaffected. This rice line is considered non transgenic because it was developed through seed mutagenesis and not through gene transfer. Thus it is a non-Genetically Modified Organism (GMO) variety. Genetic engineering involves adding a gene from another organism. The resulting plant is termed GMO, and any subsequent progenies developed from this plant that possess the introduced traits are GMOs as well. Clearfield rice is not a GMO since it did not contain any inserted gene from another organism, and maintains pure DNA from rice in its genome structure. As such, it has been accepted in the world rice market. Clearfield rice (tolerance to imadozolinones) was registered in 2001 and fully released by BASF in 2002 in USA.

The imidazolinone herbicides were developed in the late 1970s and 1980s for use in a range of crops including oil palms, rubber, sugarcane, forestry, soy bean, cereals, sunflower, and lentils. Imidazolinone herbicides include imazapyr, imazapic, imazethapyr, imazamox, imazamethabenz, and imazaquin. They have a broad spectrum of weed control, controlling both monocotyledonous and dicotyledonous weeds. These chemicals can be absorbed through both roots and leaves; hence weeds can be controlled through both the soil and foliar application (Shaner, 1991). They are translocated to the growing points of plants where they inhibit the enzyme AHAS thereby disrupting protein synthesis and cell growth.

Breeding and SelectionThe research to develop imidazolinone-tolerant rice (IMI-TR) lines started in the main season

2003/04 at the MARDI Seberang Perai Experimental Station. Clearfield line No.1770 (from Louisiana State University, USA) was crossed with local popular variety, MR 220. The cross was made to incorporate the PW-16 gene (a dominant gene tolerant to imidazolinone herbicides) from the donor line 1770 to these recurrent parents. The crossed seeds were planted in the off-season 2004. Screening for herbicide tolerance was not done on the F1 plants. The F1 plants were backcrossed to the recurrent parents. The seeds harvested were later planted in the main season 2004/05. Following the BASF Protocol R-50, imazamox was applied at the rate of 70g a.i./ha at 10 days after sowing the B1F1 plants. The B1F1 plants were screened for imidazolinone tolerance together with the donor and recurrent parents as checks. The homozygous susceptible plants (about 50% of the population) and the recurrent parents died at about 15 days after application. The heterozygous tolerant B1F1 plants showed very slight crop injury while the homozygous donor parent showed no crop injury. The surviving B1F1 seedlings were later transplanted in a trough at about 30 days after application. At flowering stage, the B1F1 plants were backcrossed again to the recurrent parent, MR 220. The backcrosses to the recurrent parent was repeated in the off season 2005. The treatment with imazamox was carried out as in the main season 2004/05. No backcrosses were done in the main season 2005/06. The B3F1 plants were allowed to grow for seed multiplication. Screening with imazamox proceeded as before.

Individual plant selection on the tolerant plants started on the B3F2 bulk population in the off season 2006. At this stage, treatment with imazamox at 200 g a.i./ha was applied at 8 days after sowing. This was done to eliminate both the homozygous susceptible and heterozygous tolerant plants; leaving behind the homozygous tolerant plants to grow. At maturation stage, 18 plants were selected. The line selection was carried out until the off season 2007, where 2 lines were selected in the main season 2006/07 and five lines were selected in the off season 2007.


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By the off-season 2007, two potential imidazolinone tolerant lines were selected for purification and evaluation for yield, agronomic performance, resistance against major pests and diseases as well as physical and chemical properties of the grain. The lines were further evaluated in main season 2007/08, from which two lines, MR 220CL1 and MR 220CL2, were identified as potential lines. They were promoted in the off season 2008 for further evaluation under large plot testing in Felcra Padi Estate, Seberang Perak and Sungai Limau Dalam, MADA District IV, Kedah. The introduction of these cultivars is justified by the need to offer a new efficient and innovative alternative method to manage weedy rice in DSR culture. The combination of IMI-TR with imidazolinone herbicides were known as Clearfield Production System (CPS). Clearfield rice production system was launched in Malaysia in October 2010 specifically for control of weedy rice in DSR.

Comparisons between Clearfield Production System and major crop establishment methodsThe CPS is basically the same as wet seeding technique (Azmi et al. 2011). The field is

thoroughly puddle and leveled before sowing. Sowing can be carried out using motor-blower or line seeders such as drum seeder or knapsack row seeder. Under water seeding culture, water is retained in the field for seeding. On the other hand, transplanting requires saturated soil without standing water for good crop establishment. Other comparisons are in Table 1.

Table 1. Comparisons between Clearfield Production System, mechanical transplanting, water seeding and wet seeding technique

Clearfield Production System

Mechanical transplanting Water seeding Wet seeding

Optimal field conditions

Saturated Saturated 5-10 cm water depth

Saturated

Crop establishment

Low incidence of golden apple snail

Low incidence of weedy rice and golden apple snail

High incidence of golden apple snail attack

High infestation of weedy rice and low incidence of golden apple snail

Cost of establishment

Low High Low Low

Cost of weed control

Moderate - High Low Low Moderate - High

Optimal season Off season Main season Main season Off seasonEquipment Motor-blower,

drum seeders, knapsack row seeder

Transplanter Motor-blower Motor-blower, drum seeders, knapsack row seeder

Weeds associated with planting method

A wider range of weeds i.e. grasses, broadleaves and sedges.

Broadleaves but other weeds i.e. grasses and sedges depend on time of flooding

Broadleaves are the dominant weeds

A wider range of weeds i.e. grasses, broadleaves and sedges

Weed control Total weed control with imidazolinone herbicides with minimum roguing of weedy rice.

2,4-D, Sulfonyl ureas, pre-emergence herbicide and roguing of weedy rice.

2,4-D, Sulfonyl ureas and roguing of weedy rice.

Graminicide, 2,4-D and sulfonyl urea product followed by roguing of weedy rice.


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Large scale evaluationA large scale evaluation of CPS cultivation (47.62 ha) was carried out in the off season 2010 in

fields seriously affected by weedy rice infestation (>30% infestation level) in the previous season (main season 2009) in FELCRA Seberang Perak rice estate (Azmi et al. 2012). Weedy rice populations were effectively controlled in the fields (from >30% to <1% level) resulting in an average net yield increment of 0.76 ton/ha from 4.93 ton/ha (main season 2009) to 5.69 ton/ha (off season 2010) giving a better B/C ratio from 2.55 to 3.42. Further analysis of its economic viability was done with sensitivity analysis which showed that with a corresponding decrease of 10% in revenue, the B/C ratio is 3.08 while a corresponding increase of 10% in costs, the B/C ratio is 3.11. Thus this showed the CPS technology is not sensitive or affected by a 10% decrease in revenue or a 10% increase in costs (Table 2).

Table 2. Financial Analysis of Clearfield Production System in Felcra Seberang Perak Rice Estate, off season 2009

Block (47.62 ha)

Area (ha)

MainSeason*2009/10

OffSeason2010** Yield Increment

(ton/ha)Increase In Income

(RM**/ha)Yield (ton/ha)

T 5A 10.68 4.11 5.43 1.32 659.16

T 5B 10.88 3.84 4.61 0.77 384.51

T 6A 14.76 5.61 6.51 0.90 449.43

T 6B 11.30 6.14 6.20 0.06 29.96

Av/ha 4.93 5.69 0.76 380.76

B/C ratio 2.55 3.42

B/C ratio* 2.29 3.08 * Sensivity analysis - return 10%

B/C ratio** 2.32 3.11 ** Sensitivity analysis- cost increase

10%* Felcra management, ** Clearfield Production System; ***1 USD = RM3.00

Comparison between Clearfield Production System versus Conventional SystemA preliminary economic sample survey was carried out on farmers planting non-CPS for season

1/2012 versus CPS for season 2/2012 on their same plots over two seasons. Each respondent or farmer is asked in questionnaire form to compare of before CPS adoption (conventional system) and after CPS adoption on the same rice plot of cultivation. The result showed that the costs of weeds management of conventional system per ha was RM 857 which was about 10% higher than CPS of RM 782 mainly due to the more usage and cost of herbicides applied namely for grasses, broadleaf and sedges control with generally two rounds of application. On the other hand, for CPS only one single application of OnDutyTM without any other herbicides was sufficient to control most of the weed species. The notable and important point here is that the conventional system cannot control weedy rice infestation causing it to increase its infestation rate over each subsequent season of planting rice but with CPS, the weedy rice is effectively contained and controlled. Further analysis showed that for half of the respondents, the costs and frequency of pests and diseases control is lower for CPS as compared to conventional system i.e. three rounds of application averaging RM 140 as compared to six rounds of application of RM 520. This invariably showed that CPS is more tolerant to pests and diseases in the fields resulting in lower costs and number of chemical applications while boosting farmers’ confidence in the product. Next when roguing costs is factored in, the CPS clearly showed a mark and significant reduction in weedy


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rice costs control. In conventional plots, all the farmers’ results indicated that roguing need to be carried out two rounds totalling RM 500 per ha plot while for CPS plots, minimum roguing is required. The bottom line is of course the yield comparison with conventional plots averaging 6.9 ton/ha and when CPS was adopted, the yield increase was 7.9 ton/ha which is about 15% higher. This directly gives a net yield increase of one ton/ha amounting to an additional RM 1,250 to the farmers’ income per ha. The survey was conducted in two sample granary areas with but nevertheless many farmers in more severe weedy rice infested areas do get yield increases of more than two to five ton per ha.

Conclusion

The use of the CPS has benefited the rice industry in Malaysia by providing an effective management of weedy rice alongside with other noxious weeds in rice cultivation. Clearfield® Production System for rice offers many advantages and benefits namely it helps to substantially reduce the cost of weed management and control the weedy rice with a single OnDutyTM (imazapic/imazapyr) herbicide application. Most of all, it is an innovative and effective agronomic solution that delivers significant value and benefits to rice farmers through effective weed control, reduced cost of production, increased yield potential and improved crop harvest quality. Studies have shown Clearfield® plots yielded 2.5 ton/ha more than conventional plots with correspondingly net income being higher by more than USD900/ha. In 2012 season, about 30% of rice granaries in Malaysia adopted this technology. The demand for this technology is expected to be increased from season to season. In short, the CPS is tested and proven to be technically feasible, economically viable and culturally acceptable in the Malaysian experience.

References

Azmi, M., Azlan, S., Chew, S. E., George, T. V., Lim, F. W., Hadzim, K. dan Yim, K. M. (2012). Clearfield Production System for Weed Rice Control in Direct-Seeded Rice (in Malay), MARDI Report N0. 214 (2012): 15 pp.

Azmi, M, Azlan, A. George, T.V., Chew, S. E. and Yim, K. (2011). Control of weedy rice in direct-seeded rice using the Clearfield Production System. Proceedings of Asian-Pacific Weed Science Society Conference, 26-29 Sept. 2011, Cairns, Queensland, Australia. Pp. 50-54.

Azmi, M. and Rezaul, K. (2008). Weedy Rice - Biology, Ecology and Management. MARDI Publication. p. 56.

Sanders, D. E., Strahan, R. E. Linscombe, S. D. And Croughan, T. P. (1998). Control of red rice (Oryza sativa) in imidazolinone tolerant rice. Proc. South. Weed Sci. Soc. 51, 36-37.

Shaner, D. L. (1991). Imidazolinone herbicides. Pesticide Outlook. 2 (4): 21-24.


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Phylogenetic relationships of Echinochloa speciesbased on phenotypic and SSRs markers

Eun-Jeong Lee, Min-Jung Yook, Do-Soon Kim*

Department of Plant Science, Seoul National University, Seoul 151-742, Korea*Corresponding author: [email protected]

Abstract

Echinochloa species are one of the most important weeds, causing many troubles in rice cultivation, and difficult to distinguish among species due to their morphological similarity. Therefore, this study was conducted to find SSR markers and morphological traits that can be used for identification and classification of Echinochloa species. The relationships among 77 different Echinochloa accessions including 57 genotypes from Korea and 5 reference species were studied by applying 23 SSR markers derived from allied species and assessing the 14 morphological traits. E. oryzicola accessions were clearly clustered as a distinctive group from E. crus-galli and other Echinochloa species in both SSRs analysis and morphological trait analysis. All E.crus-galli varieties were clustered in same position together, and we could not find the differences among these varieties. Among 23 SSR markers, we also found 5 SSR makers that could exactly discriminate E. oryzicola from E. crus-galli and other Echinochloa species. Although no clear consensus between the results from SSR marker and morphological trait analyses was founded in this study, our results indicate that both SSR markers and morphological traits can be useful tools to distinguish among Echinochloa species.

Keywords: Echinochloa, Morphological analysis, SSRs analysis

Introduction

The genus Echinochloa (Poaceae) has about 20-50 annual and perennial species that can be found throughout the world and some of them are listed most troublesome weeds in the world (Holm et al, 1977). As rice (Oryza sativa L.) is the main food crop, E. crus-galli (L.) Beauv. (2n=6x=54) and E. oryzicola Vasing. (2n=4x=36), which spread widely in both dry or water flooded soil, are the greatest yield limiting and economical loss making weeds in Korea (Chung, 2001). From a taxonomical perspective, there are many limitations in classification of Echinochloa species because these two species are hard to distinguish as they show morphologic variation and some morphological trait frequently overlap among and within species (Yabuno, 1966). Meanwhile, different morphological trait can reflect different competition ability to crop and response to herbicide. So, it is critical to determination of the level of variation within species for effective control strategies. In this paper, SSR analysis and morphological analysis were conducted to assess the polymorphism of intraspecific variation and provide phylogenic relation of interspecies. In addition, we also expect to make and find genetic and morphological markers that related each other and can distinguish interspecies clearly from this study.


77 Echinochloa accessions were collected in different site (mostly in Korea region) or supplied by Herbiseed (www.herbiseed.com). Seeds were germinated in round Petri dishes and seedlings were transplanted in pots located in the glasshouse. Genomic DNA was extracted from young fresh leaves of individual plants according to serial steps of Sneller et al (1997). DNA were amplified with 23 SSR markers previous designed for its relative species such as pearl millet, maize, sugar cane, sorghum, foxtail millet, Echinochloa and Miscanthus. Data cluster was conducted for SSRs analysis with


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NTSYS-pc 2.1 software (Rohlf, 2000) using Jaccard’s coefficients in order to make an unweighted pair-group (UPGMA) dendrograms. Fourteen morphological characteristics were examined for each population. The characters used on leaf, stem, spikelet, awn and panicle. Moreover, 10 agronomic traits also examined in this study such as growth traits and growth date. Data cluster was conducted for morphological trait analysis with NTSYS-pc 2.1 software (Rohlf, 2000) using correlation coefficients in order to make an unweighted pair-group (UPGMA) dendrograms.


SSRs analysisAmong 79 scorable alleles, 68 had a polymorphism with 86% of polymorphism percentage. The

polymorphism information content (PIC) was obtained for each marker. It varied from a minimum of 0 for CORN05 to a maximum of 0.81 for PSMP2235. Especially, SSR markers designed from sugarcane, pearl millet and Echinochloa species had relatively high PIC value than others. Therefore, these markers might be good candidates for getting polymorphism information.

The cluster analysis using UPGMA methods revealed mainly divided 4 groups (I, II, III, and IV) as marked in Figure 1. First of all, reference species were clearly separated from Echinochloa accessions by developing group IV. Group I contained most of E. oryzicola significantly distinguished from E. crus-galli Group (II and III). Group II mainly composed E. crus-galli accessions. Other E. crus-galli and Echinochloa species created Group III together. In this study, all E. crus-galli varieties were clustered in same position, Group III, and we could not find the differences among these varieties. In addition, unclassified Echinochloa accessions (from Taiwan, Vietnam and Sri Lanka) might be estimated to be classified in E. crus-galli.

As compared electrophoresis gel image and phylogenic tree from SSRs analysis, we found 5 SSR markers that could discriminate E. oryzicola against E. crus-galli and other Echinochloa species. It was considered that these 5 SSR markers mainly contributed to making such dendrogram of Figure 1.

Morphological trait analysisIn morphological traits analysis, only 77 Echinochloa accessions were evaluated besides

reference species. The cluster analysis using UPGMA methods showed mainly divided 3 groups (I, II and III) with similar pattern to SSR analysis. In Group I, most of E. oryzicola and 1 E. crus-galli var. crus-galli were included in it. Group II composed 25 of E. crus-galli var. crus-galli, two of E. crus-galli var. practicola, one of E. crus-galli var. formosensis, 4 of unclassified Echinochloa accessions and 3 of E.oryzicola. One remained E. crus-galli var. crus-galli and other Echinochloa species were together in Group III. E. crus-galli varieties and unclassified Echinochloa has the same tendency with those of SSRs analysis.

The most influential morphological traits were only in seed parts; spikelet size, awn length, hairs on empty glume and first empty glume area. When analyzing Echinochloa accessions with these 4 seeds traits again, we got the results which were significantly parted as 2 groups (E. oryzicola and E. crus-galli). Therefore, we can suggest that everyone easily distinguish E.oryzicola and E.crus-galli with using only these 4 traits.

Through SSRs analysis and morphological trait analysis, we could get the 2 phylogenic trees that were closely alike each other in overall clustering. E. oryzicola accessions were clearly clustered as a distinctive group from E. crus-galli and other Echinochloa species with the lowest phylogenetic relationship among Echinochloa species in both SSRs analysis and morphological trait analysis. However, no clear consensus between the results from these two analyses was founded in this study. This might be because SSR markers were non genetic related markers whereas morphological traits were generally related with gene. Nevertheless, our results indicate that both SSR markers and morphological traits can be useful tools to distinguish among Echinochloa species. In addition, these two analyses will be necessary with complementary view point for enhancing the reliance of each marker. Some individual SSR markers, especially derived from sugarcane, pearl millet and Echinochloa, could discriminate E. oryzicola from E. crus-galli. For uncertain or misclassified Echinochloa accessions, further study using more SSR markers and morphological traits will be needed for more clear classification.


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Fig. 1. Phylogenic tree for Echinochloa accessions and relative species based on SSR marker analysis.


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References

Holm, L.G., Plucknett, D.L., Pancho, J.V., and Herberger, J.R., (1977). The World’s Worst Weeds: Distribution and Biology, East-West Center Press, USA.

Chung, I.M., Ahn, J.K., and Yun, S.J. (2001). Assessment of Allelopathic Potential on Barnyard grass (Echinochloa crus-galli) of Rice Cultivars. Crop Prot. 20: 921-928.

Yabuno, T. (1966). Biosystematic Study of The Genus Echinochloa. Jpn. J. Bot. 19: 277-323.Sneller, C.H., Miles, J.W. and Hoyt, J.M., (1997). Agronomic Performance of Soybean Plant Introductions

and Their Genetic Similarity to Elite Lines. Crop Science. 37: 1595.Rohlf, F.J. (2000). NTSYS-pc: Numerical Taxonomy and Multivariate Analysis System, 2.1 ed. Applied

Biostatistics, New York.


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Eco-efficient weed management approaches for rice in tropical Asia

A.N. Rao*. and A. Nagamani*** Former Agronomist (Weed Scientist),

International Rice Research Institute (IRRI) and President, Society for Participatory Development and Research, Plot: 1294A; Road: 63A; Jubilee Hills;

Hyderabad – 500033, Andhra Pradesh, India. E mail: [email protected]

** Professor, Department of Botany, University College of Science, Osmania University, Saifabad, Hyderabad – 500004.

E mail: [email protected]

Abstract

Weeds continue to be the major impediments of rice production in Asia, in spite of research efforts made so far. The yield losses due to weeds range from 12 to 80 percent depending on the location, associated environment, weed community, rice establishment method, weed management practices used by farmers. Weeds may cause complete rice crop failure, if appropriate weed management measures are not taken up. None of the available weed management options, alone, could suppress the weeds effectively in rice. Weeds are dynamic and in recent years weeds such as weedy rices are posing severe threat to the efforts to enhance rice productivity to meet the demands of increasing population in Asia. Innovative approaches need to be evolved for effectively combating the weed menace in rice in Asia. Eco-efficient weed management in rice is concerned with the efficient and sustainable use of resources in rice production to shift the crop weed balance in favor of rice. It encompasses both ecological and economic dimensions of sustainable rice production. Key components of eco-efficient weed management include: regular weed monitoring to identify shifts in weed populations and identify problematic weeds from time to time, weed seed depleting soil and other management practices, weed competitive rice based cropping systems and rice establishment methods, weed smothering rice varieties, water and nutrient management options that are detrimental to weeds in rice, exploring the feasibility of weeds usage, use of eco-friendly and economic herbicides in combination with cultural practices, and adoption of appropriate weed dissemination prevention strategies. Available knowledge on each of the components of eco-efficient weed management in rice in Asia is synthesized and future research needs are enlisted.

Introduction

Rice is the major staple crop of tropical Asia. Tropical Asia, which extends over 80 degrees of longitude (from 70°E to 150°E) and 40 degrees of latitude (from 30°N to 10°S). The 16 countries that make up the region (Table 1). The region is physically diverse and ecologically rich in natural and crop-weed related biodiversity. The vast variation in environmental conditions in tropical Asia make wide variety of weeds (Moody, 1989; Caton et al., 2004) competing with rice and causing severe yield losses ranging from 12 to 80 percent depending on the location, associated environment, weed community, rice establishment method, weed management practices used by farmers (Rao et al., 2007). Weeds may cause complete rice crop failure, if appropriate weed management measures are not taken up.

Weeds continue to be the major impediments of rice production in Asia, in spite of research efforts made so far. Global climate changes are occurring and will result in further increases in atmospheric greenhouse gases and temperature (> 0.2°C per decade), soil degradation and competing claims for land and water (IPCC, 2007). Agricultural productivity in Asia is likely to suffer severe losses because of high temperature, severe drought, flood conditions, and soil degradation; food security of many developing countries in the region would be under tremendous threat. Climate change is projected to compound the pressures on natural resources and the environment associated with


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rapid urbanisation, industrialisation and economic development (IPPC, 2007). The rapidly changing environmental conditions will affect the future rice production and weed management problem. Weed species’ distribution and their competitiveness within a weed population and within a rice crop will change with changes in atmospheric CO2 levels, rainfall, temperature and other growing conditions (Mahajan et al., 2012*). This may necessitate adaptations in crop management practices, which in turn will affect weed growth or proliferation of certain weed species.

The present total population in tropical Asia is about 1.6 billion, and the population is projected to increase to 2.4 billion by 2025. It is estimated that demand for food and non-food commodities is likely to increase by 75–100% globally between 2010 and 2050 (Keating et al., 2010; Tilman et al., 2011). The increase in demand in tropical Asia is expected to be at least as much. The demand can be met by bridging the yield gap through alleviation of impediments such as weed menace. Depending on the magnitude of environmental changes within the Asian tropical region, environmental conditions also have a large impact on the effectiveness of cultural, physical, mechanical and chemical weed management practices.

In addition to impact of climate change, the continuing losses due to weeds in rice based agro-ecosystems in Asian tropics (Rao et al., 2007), the evolution of herbicide resistance (Valverde and Itho, 2001), shifts in the weed flora in response to weed management (Ramanjaneyulu et al., 2006), the chemical contamination of water sources (Srivastava et al., 2010) and soil erosion through excessive cultivation (Garrity, 1993) all attest to the need to develop systems of weed management, that are sustainable and eco-efficient. Thus alternative weed management strategies are needed for enhancing rice productivity to meet the increasing demand of population in tropical Asia through effective and economical management of weeds in rice. The eco-efficient weed management is one of such innovative approach.

What is eco-efficiency and Eco-efficient weed management ?Eco-efficiency is concerned with the efficient and sustainable use of resources in farm

production and land management (Balasubramanian et al., 2012). Eco-efficiency was debated earlier (British Crop Protection Council (BCPC) Forum (2004; Atkinson and Wilkins 2004), without a single accepted definition of eco-efficiency. However there was an indication that eco-efficiency is related to both ‘ecology’ and ‘economy’ and is concerned with the efficient and sustainable use of resources in farm production and land management. Even though there are no absolute standards which a system needs to satisfy in order to be classed as being eco-efficient, eco-efficiency will be increased when a given level of production is achieved using less resources, with less losses to the environment and without sacrifice to the productive potential of the land or economic performance. The efficient use of plant nutrients, pesticides and energy and the minimization of greenhouse gas emissions are all key concerns that affect eco-efficiency.

The BCPC Forum (2004) concluded that eco-efficient farming should satisfy the identified five key attributes. Eco-efficient weed management is the approach of managing weeds which satisfy the five key attributes identified by BCPC forum (2004), Viz. (i) it makes the maximum use of renewable inputs by utilising resources efficiently and economically, (ii) it is locally non-polluting and does not transfer pollution to elsewhere, (iii) it provides a predictable output, (iv) it conserves functional biodiversity in relation to strengthening ecological processes, reducing greenhouse gas emission and pollution generally and limiting soil erosion, and (v) it is capable of responding rapidly to changes in the social, economic and physical environment. It is also crucial that eco-efficient weed management must satisfy economic criteria in relation to farm profitability.

The eco-efficient weed management must thus form an integrated component of the rice farming system, in order to realise the ultimate goal of eco-efficient rice production. Approaches to enhance eco-efficiency in weed management in rice must include: (i) adoption of the method of rice establishment and crop management practices that are eco-efficient, (ii) Increased ability to predict interactions between rice and weed populations through better knowledge of weeds and their ecology (ii) identifying and integrating weed management practices that make substantial reduction in external inputs, manage weeds with higher resource use efficiency, with minimal adverse impact on environment and result in higher rice productivity in an economic manner.


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In this review, the possible eco-efficient weed management practices that may profitably used are reviewed.

A. Adoption of the eco-efficient method of rice establishment and crop management practices.In tropical Asia, transplanting seedlings into puddled soil (land prepared by wet tillage) is the

major method of rice crop establishment with 74% of rice is established by transplanting (Table 1). Due to bigger size of rice seedling at the time of transplanting, puddle transplanted (CT-TPR) rice competes better with emerging weeds and hence preferred by farmers as major method of rice establishment (Rao et al., 2007). Reducing water percolation losses, easy seedling establishment, and creating anaerobic conditions to enhance nutrient availability are the other benefits derived from puddling soil prior to rice transplanting (Sanchez, 1973). However, puddling and transplanting require large amounts of water and labor, which are becoming scarce and expensive. Higher labor wages of scarce labor are making rice production less profitable in addition to the drudgery involved in transplanting by women. Thus, rice cultivation in Asia is severely constrained by multiple stresses, especially water and labor shortages and adverse effects of puddling on soil physical properties (Sharma et al., 2003) and a succeeding non-rice crop (Hobbs and Gupta, 2003). Flooded rice culture with puddling and transplanting is considered one of the major source of CH4 emission because of prolong flooding resulting in an anaerobic soil condition. It accounts for 10-20% (50-100 Tg yr-1) of total CH4 emission globally on annual basis (Houghton et al., 1996; Reiner and Milkha, 2000). Direct-seeding method of rice (DSR) establishment (both wet and dry) have shown potential to reduce CH4 emissions compared to puddled transplanted rice (Wassmann et al., 2004). Wassmann et al. (2004) suggested that CH4 mitigation effects can be further enhanced if Wet or Dry-direct-seeded rice (DSR) is combined with mid-season drainage. Compared to CT-TPR, emissions of CH4 decreased and of N2O increased in Dry-DSR. Therefore, DSR especially in dry conditions with zero-tillage may be an effective mitigation option in Asia. All these factors demand a major shift from CT-TPR to DSR in irrigated areas. Depending on water and labor scarcity, farmers are changing either their rice establishment methods only [from transplanting to direct seeding in puddled soil (Wet-DSR)] or both tillage and rice establishment methods [puddled transplanting to dry direct-seeding (Dry-DSR)] in combination with resource-conserving technologies (RCTs) following the principles of conservation agriculture (CA) which have been shown to increase the productivity and eco-efficiency of agriculture at the farm level (Hobbs and Gupta, 2003; Sharma et al., 2005; Gupta and Seth, 2007; Ladha et al., 2009). However, weeds menace is much severe in these alternative methods of rice establishment and effective eco-efficient weed management strategies are the prerequisite for attainment of optimal rice productivity in an eco-efficient manner.

B. Increased ability to predict interactions between rice and weed populations through better knowledge of weeds and their ecology

Both rice and weeds need same resources for growth and establishment resulting in crop-weed competition which necessitated the management of weeds to realise higher rice productivity. Understanding the interactions between rice and weeds is essential to create microenvironment favourable to rice and detrimental to weeds. Such an understanding would thus help in enhancing eco-efficiency of weed management strategies. A few examples to illustrate the role of weed ecology in managing weeds eco-efficiently are cited below.

Light plays a key role in the germination and growth of weeds and the germination response of different weed species to light and darkness varies. A few rice weeds (eg. Cyperus iria, Eclipta prostrate) require light for germination and do not germinate in darkness, some weeds do not require light for germination (eg. Melochia corchorifolia, Mimosa invisa) and some weeds do not need light but light stimulates their germination (Echinochloa colona, Digitaria ciliaris, Amaranthus viridis) (Chauhan and Johnson, 2010). Under eco-efficient zero-tillage systems, the weeds species that require light for germination or light simulates germination have the potentiality to become severe as they are retained on soil surface where light is readily available. Such species can be managed by having stale seed bed technique as component of eco-efficient weed management.

Light in the crop canopy is known to influence the growth of weed species. Possibility exists to manage weeds that are susceptible to shading by selecting a rice variety that covers the ground fast


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and allows less light penetration below its canopy. More research efforts are needed to identify the weed species susceptible shade and adopted to shade in rice agro-ecosystems to enhance light resource efficiency.

Adoption of eco-efficient CA practices such as zero-till systems are likely to leave a large proportion of the weed seed bank on or near the soil surface after rice sowing. Higher loss in seed viability and greater seed mortality occurs for weed seeds present on or close to the soil surface than the buried weed seeds (Mohler, 1993). Weed seeds are most vulnerable to surface-dwelling seed predators when on the soil surface and a tropical Asia study, showed 87% post dispersal weed seed predation was from the soil surface (Chauhan et al. 2010).

Much more research needs to be conducted on weed seed bank dynamics, weed shifts with time, identifying and recognising the biological and ecological factors leading to the long term persistence of species in weed communities, and overall understanding of the ecology of weeds to manage weeds eco-efficient manner in the age of changing climate.

C. Components of integrated eco-efficient weed management in rice in tropical AsiaEco-efficiency in the simplest of terms is about achieving more with less-more agricultural

outputs, in terms of quantity and quality, for less input of land, water, nutrients, energy, labor, or capital (Keating, et al., 2010). The eco-efficient conservation agriculture (CA) is characterised by minimum soil disturbance, retention of residue for soil cover and rotation of major crops. Those components could be used for managing weeds in rice. Eco-efficient weed management could not be achieved by any of the single weed management practice and integration of available weed management practices is needed to attain that objective. Some of the components that may be integrated include:

i).Avoiding weed seed contamination in rice: In recent years, weedy rice is becoming major problem in Asian rice ecosystems.. Sowing seed contaminated by weedy rice is likely the primary cause of invasiveness of weedy rice in rice fields (Rao et al., 2007). Rice seed soaking in herbicide solution for controlling rice seed contaminants (Rao and Moody, 1996) or the use of certified seed have proved to be an essential component in weed management (Rao et al., 2007).

ii) Sanitation: Sanitation is one means of minimizing the likelihood of weed introductions and dispersal of existing weeds throughout a farm, especially herbicide-resistant weeds. All farm machinery should be washed well to remove weed seeds and propagules of perennials in attached-soils from the neighbouring weed infested fields before being moved into clean paddy fields.

iii) Soil solarisation: Soil solarization is a preventive method that exploits solar heating to kill weed seeds and therefore reduce weed emergence. In this method, heating of the soil’s surface is done by using transparent low-density polyethylene (LDPE film) sheets placed on the soil’s surface to trap solar radiation (Khan et al., 2003). The use of transparent and black LDPE sheets reduces weed growth and increases rice yield (Khan et al., 2003).

iv) Water management: Variation in soil moisture conditions can affect weed seed emergence and viability differently (Mercado, 1979). Ismail et al. (1995) indicated that emergence of Echinochloa crus-galli, E. colona, Cyperus iria, Ludwigia hyssopifolia and Rhyncospora corymbosa was lower in soils flooded up to 4 cm water depth compared to seeds sown at all sowing depths in saturated soil. However, some aquatic weeds germinate under water and this behavior has been used as a guide in their control (Mercado, 1979). In fields known to be heavily infested with ‘aquatic’ weeds, germination of the seeds can be stimulated by flooding the field and, after seedlings emerged, control measures can be applied to these weed (Mercado, 1979). Even though water management got the potentiality to use as a component of weed management strategy, water availability is becoming scarce in many of the rice growing Asian countries.

v) Crop rotation: Differentiation of crops grown over time on the same field is a well-known primary means of preventive weed control. Different crops obviously bring about different cultural practices,


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which act as a factor in disrupting the growing cycle of weeds and, as such, preventing selection of the flora towards increased abundance of problem species (Karlen, 1994). In contrast, continuous cropping selects the weed flora by favouring those species that are more similar to the crop and tolerant to the direct weed control methods used (e.g. herbicides) via repeated application of the same cultural practices year after year.

vi) Mulching: Major component of CA is retention of residue cover on the soil surface. The spreading of mulch on the soil surface reduces evaporation, saves water, protects from wind and water erosion, and suppresses weed growth (Singh et al., 2007). Residue as mulch can suppress emergence and growth of many weed species depending on the amount and type of crop residue. The rice straw mulch (4t/ha) was effective in weed management under wet-seeded rice, but not in dry-seeded rice in Sri Lanka (Devasinghe, et al., 2011). Mulching + dry land weeder at 20 DAS proved more effective in dry-seeded rice grown without herbicide use (Hussain and Gogoi, 1996). In Nepal, straw mulch + bispyribac-sodium reduced the population of Alternanthera philoxeroides, Ammannia sp., Commelina diffusa, Cyperus difformis, Cyperus iria, and D. junceum 8 weeks after seeding dry-seeded rice (Ranjit and Suwanketnikom, 2005).

vii) Brown manuring: Brown manuring is a no-till‘ version of green manuring, using a herbicide to desiccate the intercrop (and weeds) at flowering instead of using cultivation. The plant residues are left standing. In ‘Brown Manuring‘ practice both rice and Sesbania crops are seeded together and allowed to grow for 30 days. Subsequently Sesbania intercrop is knocked down with 2, 4-D at 500 g ha-1 (Singh et al., 2007). This technology reduces weed population by nearly half without any adverse effect on rice yield. Sesbania surface mulch decomposes very fast to supply N and other recycled nutrients.

viii) Stale seedbed technique: Weed density could be considerably reduced in the eco-efficient CA adopted systems by using the stale seedbed technique. Weeds population during the crop growing season could be reduced by inducing weed seeds to germinate by giving light irrigations before land preparation followed by non-selective herbicides or tillage operations for killing the emerged weed seedlings. Some of the weed species species sensitive to the stale seedbed technique are Cyperus iria, Digitaria ciliaris, Echinochloa colona, Eclipta prostrata, Leptochloa chinensis, Ludwigia hyssopifolia and Portulaca oleracea (Chauhan and Mahajan, 2011). Practising stale seedbed for 14 days gave the highest benefit:cost ratio (Sindhu et al., 2011). In rice-wheat cropping system, the energy utilization for weed management (which accounted for 1.47 to 3.40 per cent of total input energy) was found slightly higher in traditional seedbed (925 to 1788 MJ/ha) than stale seedbed (768 to 1364 MJ/ha) (Chaudhary et al., 2006).

ix) Enhancing crop competitiveness: The detrimental effects of weeds on the crop may be reduced by making the crop more competitive. Improving rice competitiveness against weeds would provide a low-cost and safe tool for the eco-efficient weed management strategy. Any cultural practice that facilitates rapid rice growth and results in rice canopy covering soil surface, and shade out weeds, increase crop competitiveness. Practices that contribute rice competitiveness include: early sowing, selection of varieties with early growth (Zhao et al. 2006; Namuco et al. 2009). optimal seed rates (Zhao et al. 2007); close spacing (Chauhan and Johnson 2010a); adequate plant population and fertilization (Rao et al., 2007) intercropping (Musthafa and Potty, 2001, Duary et al., 2005), and deferred sowing of black gram in rice (30 cm) after one weeding (Midya et al., 2005).

x) Bioherbicides: Micro-organisms also are used as tools for weed management and have a range of properties that make them desirable for ecological weed management in direct-seeded rice. COLLEGO, a powder formulation of Colletotrichum gloeosporioides (Penz.) Sacc. f. sp. aeschynomene, was registered in 1982 for the control of northern jointvetch (Aeschynomene virginica (L.) B.S.P.) in rice. The successful mycoherbicide, Rhynchosporium alismatis had synergistic controlling effect on Damasonium minus (R.Br.) Buch. when bensulfuron-methyl was applied before fungal inoculation (Jahromi et al., 2001). In Asia, the bioherbicide research is yet to reach the practical usage stage.


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xi)Weeds use: In several countries of Asia, weeds are normally used in villages, as food, medicine and other purposes. In the long run, with proper planning and support, the growing and marketing of medicinal herbs which are currently categorized as weeds could become a very good source of income to farmers, while providing an additional, easily available source of natural medicines to the rural population (Ediriweera, 2007). However, practical feasibility and economics of such usage on large scale needs to be determined before it becomes a practical proposition.

xii) Mechanical weeding: Weeding by mechanical devices reduces the cost of labor and also saves time (Subudhi, 2004). The labor involved was least with the Phulbani weeder (57 person-days ha-1), saving nearly 57% labor compared with hand weeding (127 person-days ha-1). It also had better weed control efficiency (Subudhi, 2004). Dry-seeded rice row seeding with interrow weeding using hoes and without any herbicide achieved higher grain yield (Satoshi et al., 2009). Mnguu (2010) opined that rice farmers can use rotary weeding instead of herbicide in controlling weeds and achieve the same grain yield of wet-seeded rice.

xiii) Manual method: Where ever labor is available, manual method could be used as a component of eco-efficient weed management. However, in tropical Asia the labour availability is decreasing and the cost is increasing. Even in regions where labour is available, availability of labour in time is a constraint which is resulting in damage due to weeds even before the manual weeding is done late on their availability.

xiv) Herbicides: In the eco-efficient CA systems, herbicide use is an important component of weed management and choosing an appropriate herbicide and timing is critical. A variety of herbicides have been screened and found effective for burn down, pre‐emergence and post‐emergence weed control in dry‐seeded systems (Kumar and Ladha, 2011). Virtually all rice farmers who practice direct-seeding adopt chemical herbicides because they reduce weed control time in dry‐seeded crops by 500 hours per hectare in comparison to hand‐weeded transplanted rice (Mazid et al., 2006, Ho, 1996). Herbicide resistant rice cultivars may become an important component of eco-efficient weed management in future, especially for managing recently emerging problematic weeds such as weedy rice. However, educating farmers in Asia is needed to effectively use that technological component.

Integration of above components for managing weeds must ultimately lead to enhancement of the eco-efficiency of agro-ecosystems of the tropical Asia.

Can we quantify the eco-efficiency of the components of eco-efficient weed management:Most of the components of the eco-efficient weed management are enlisted here are based on

their qualitative characteristics pertaining to their eco-efficiency as reported in the literature. However, a method to quantify the eco-efficiency is needed to select and integrate the components of eco-efficient weed management. Eco-efficiency was used as a tool to compare herbicide resistant and conventional cropping systems (Cobourn and Kniss, 2012). The formulae used by Cobourn and Kniss, (2012) for calculating eco-efficiency may be used for quantifying the eco-efficiency of different components of eco-efficient weed management. To compare the eco-efficiency of different herbicides, the Environmental Impact co-efficient (EIQ) suggested by Kovach et al., (2010) may be used. Thus it is possible to quantify the eco-efficiency of the components of eco-efficient weed management.

Future research efforts to develop weed management technologies must consider eco-efficiency as the criteria to attain optimal rice yields in an eco-efficient manner in tropical Asia.


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Acknowledgement

Thanks are due to Ms. Jaya Adusumilli for her help.

Table 1. Area, production, productivity of rice and the percentage of area under direct seeding and transplanting in tropical Asia (2010) (Compiled based data from IRRI and Rao et al., 2007)

Rank Area Area( 000 ha)

Productivity (t/ha)

Production (000 MT)

Direct-seeded Rice area* (%)

Transplantedrice area (%)

1 India* 36950 3.26 120620 28 722 Indonesia* 13244.2 5.01 66411.5 18 823 Bangladesh* 11800 4.18 49355 19 814 Viet Nam* 7513.70 5.32 39988.9 39-47 53-615 Myanmar* 8051.70 4.12 33204.5 9 916 Thailand* 10990.1 2.88 31597.20 34 667 Philippines* 4354.16 3.62 15771.7 42 588 Cambodia* 2776.51 2.97 8245.32 10 909 Sri Lanka* 1060.36 4.06 4300.62 77 2310 Nepal* 1481.29 2.72 4023.82 N**11 Laos 870 3.46 3006 33 6712 Malaysia 673.75 3.78 2548 71 2913 Bhutan 21.80 2.83 61.70 N-n N14 Brunei

Darussalam 1.1 1.27 1.4 N N

Tropical Asia 99788.67 3.53 379135.66 26 74Asia 136550 4.45 607320 20.2 79.8

World 153650 4.37 672016 22.7 77.3

*From Rao et al., 2007N = Information missing, N-n = negligible DSR – mainly transplanting)** Manual transplanting is the dominant crop establishment method in lowland ecologies, while direct seeding of seeds to dry soils is dominant in upland ecologies (FAO, 2002).

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Ramanjaneyulu, A.V., Sharma, R. and Giri, G. 2006. Weed shift in rice based cropping systems - a review. Agric. Rev., 27, 73 – 78.

Ranjit, J. D. and Suwanketnikom, R. 2005. Response of weeds and yield of dry direct seeded rice to tillage and weed management. Kasetsart Journal, Natural Sciences; 39, 165-173.

Rao, A. N. and Moody, K. 1996. Seed soaking in herbicide solution for controlling Ischaemum rugosum, a rice seed contaminant. In: Proc. of the 15th Asian Pacific Weed Science Society Conf. 24th–28th July 1995, Tsukuba, Japan.

Rao, A. N., Mortimer, A. M.., Johnson, D. E., Sivaprasad, B. and Ladha, J. K. 2007. Weed management in direct-seeded rice. Advances in Agronomy. 93, 155-257.

Reiner, W., and Milkha, S. A. 2000. The role of rice plants in regulating mechanisms of methane emissions. Biol. Fertil. Soils 31, 20–29

Sanchez, P.A. 1973. Puddling tropical rice soils. 2. Effect of water losses. Soil Sci. 115, 303–308.Satoshi, H., Anuchart, K., Boonrat, B., Akihiko, K. and Junko, Y. 2009. Spatial variability in the growth

of direct-seeded rainfed lowland rice (Oryza sativa L.) in northeast Thailand. Field crops research. 111, 251-261.

Sharma, P. K., Ladha, J. K., and Bhushan, L. 2003. Soil physical effects of puddling in rice-wheat cropping systems. In “Improving the Productivity and Sustainability of Rice-Wheat Systems: Issues and Impacts” (J. K. Ladha, J. E. Hill, J. M. Duxbury, R. K. Gupta, and R. J. Buresh, Eds.), pp. 97–113. ASA, CSSA, SSSA, Madison, WI.

Sindhu, P. V., Thomas, C. G. and Abraham, C. T. 2011. Stale seedbed and green manuring as an effective weed management strategy in dry-seeded rice (Oryza sativa). Indian Journal of Agronomy. 56, 109-115.

Singh, S., Ladha, J. K., Gupta, R. K., Bhushan, L., Rao, A. N., Shiva Prasad, B. and Singh, P. P. 2007. Evaluation of mulching, intercropping with Sesbania and herbicide use for weed management in dry-seeded rice (Oryza sativa). Crop Protection. 26, 518-524.


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Srivastava, S.; Goyal, P. & Mohan Srivastava, M. 2010. Pesticides: Past, present, and future. In: Handbook of Pesticides: Methods of Pesticide Residues Analysis, Nollet, L.M.L. & Rathore, H.S. (Eds.), pp. 47-65, Taylor & Francis, Boca Raton, Florida

Subudhi, C. R. 2004. Evaluation of weeding devices for upland rice in the Eastern Ghats of Orissa, India. IRRN. 29: 79-80.

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Zhao, D.L., Altin, G.N., Bastiaans, L. and Spiertz, J.H.J. 2006. Cultivar- weeds Competitiveness in aerobic rice: Heritability, correlated traits, and the potential for indirect selection in weed- Free environments.CropSci46, 372-380.

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Morphological and physiological responses of Miscanthus spp. to varying temperature and light intensity

Jastin Edrian Revilleza1, Soo-Hyun Lim1, Ji-Hoon Chung1, Do-Soon Kim1

1Department of Plant Science, Seoul National University, Seoul 151-742, KoreaCorresponding author: E-mail: [email protected]

Abstract

C4 plants are known to have a higher yield compared to C3 plants. Generally, C4 species are best suited to tropical and subtropical climates. Miscanthus, commonly known as a rhizomatous grass species and an important bioenergy weed crop, is a highly exceptional C4 species due to its high productivity in temperate climates. However, recent reports were focused only in the comparison between Maize and Miscanthus response to such unfavorable conditions. Therefore, this study was conducted to investigate photosynthetic responses of different Miscanthus genotypes to low temperature conditions and sub-optimal light intensity. Various Miscanthus genotypes collected from different locations were grown under three different temperature levels, 25/20oC, 20/15oC and 15/10oC during a 35-day period and at five different light intensity regimes namely 100%, 75%, 55%, 25% and 5% during a 70-day period. Necessary morphological assessments include plant height, number of leaves and leaf area and essential physiological assessments include photosynthetic rate, chlorophyll content and chlorophyll fluorescence. Results show that different Miscanthus genotypes showed the same trend within the different environmental conditions. However, biomass yield were not affected by these conditions, suggesting that it is independent of the following physiological response. This report demonstrates an overview of Miscanthus tolerance to low temperature and sub-optimal light conditions.

Keywords: Bioenergy crop, biomass, Miscanthus, photosynthesis

Introduction

Miscanthus x giganteus is exceptional among C4 species for its high productivity in temperate climates (Dohleman and Long, 2009) and has been cultivated for biomass production in Europe and USA. Miscanthus genusis composed of 14 major species, of which M. sinensis, M. sacchariflorus and M. floridulus are native to the Eastern Asian region including Korea, China and Japan. Miscanthus x giganteus is the only Miscanthus species being commercially cultivated for biomass production due to its high biomass yield (Chung and Kim, 2012). However, the study of C4 photosynthesis on different environmental conditions are limited and is focused mainly on the difference between Maize and Miscanthus spp. (Naidu et al., 2003; Wu and Wedding, 1987) and focused mainly on Miscanthus spp. grown in Europe and USA with little emphasis on the eastern parts of Asia (Beale et al, 1996). Therefore, the objectives of this study were to investigate photosynthetic responses of different Miscanthus genotypes to low temperature conditions and sub-optimal light intensity and to compare the biomass yield among the different genotypes with relation to its photosynthesis ability.


Various genotypes belonging to Miscanthus x giganteus (reference genotype), M. lutarioriparius, M. sinensis and M. sacchariflorus were selected from 300 genotypes collected from various locations in Korea and neighboring East Asian countries such as China, Japan and Russia (Table 1).

To give the different low temperature treatments, the Miscanthus spp. plants were grown for at least 25 cm tall and were transferred to a growing chamber (Hanbaek, Korea) with varying day and


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night temperatures of 25/20°C, 20/15°C, and 15/10°C,. Assessments were made on a 7-day interval for 35 days. To give the different light intensity treatments, the Miscanthus spp. plants were grown for 60 days and were transferred to different light intensity conditions: 100%, 75%, 45%, 25%, and 5%. Assessments were made on a 7-day interval for 70 days. To assess the photosynthesis rate various Miscanthus spp. genotypes on field conditions, three-year stands of 53 genotypes of Miscanthus spp. planted on the germplasm field in the experimental farm station in Suwon, South Korea. Three leaves from different tillers of the same genotype were used in measuring the different parameters during the summer months of 2012; June, August and September. For the morphological assessments, plant height, leaf number, tiller number and leaf area were measured. For the physiological assessments, LI-6400XT (LI-COR, USA) was used to measure the photosynthesis rate, SPAD (Konica Minolta, Japan) was used to measure leaf chlorophyll content and Handy PEA (Hansatech Instruments, UK) was used to measure the leaf fluorescence. The biomass yield was measured at the end of the observation period.


Plant growth and increase in tiller number was halted at lower temperature and lower light intensity. However, leaf number was not significantly different among the different temperature range and light intensity. Photosynthesis rate, chlorophyll content (SPAD) and leaf fluorescence (Pi) was significantly affected at low temperature and low light intensity at 28 and 56 days after transferring (DAT), respectively. The various Miscanthus spp. genotypes have different rates of responses on these various temperature and light intensity regimes. There was also no clear correlation of photosynthesis to chlorophyll content and leaf fluorescence. Moreover, there was no clear correlation of photosynthesis rate to biomass yield at different temperature ranges and suboptimal light intensities (Figure 1). Therefore, there is diversity with response of different Miscanthus spp. to various environmental conditions such as temperature and light intensity.

A

B

Figure 1. Relationship between biomass yields and photosynthetic abilities of M. x giganteus (●), M. lutarioriparius (■), M. sinensis (▲) and M. sacchariflorus (▼) for varying temperature (A) and varying light intensity (C).

Photosynthetic abilities of 56 Miscanthus genotypes grown in the field condition showed very diverse depending on species, genotypes and months when the measurement was made. Some genotypes have a higher biomass yield even with low photosynthesis rate, while some have a lower biomass yield even with high photosynthesis rate (Table 1). Nonetheless we selected 4 genotypes, two M. sinensis genotypes, one M. lutarioriparius genotype and M. x giganteus (reference) having high biomass yield potential with high photosynthetic ability. Therefore, these three genotypes except M. x giganteus will be further studied for developing a new Miscanthus variety for biomass production.


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Table 1. Miscanthus spp genotypes with high photosynthesis rate (and high biomass yield.

Miscanthus spp. GenotypeCarbon dioxide assimilation (µCO2 m

-2s-1)Biomass yield (g)

June August SeptemberM x giganteus M-57 12.71 10.55 9.75 63.05

M lutarioriparius M-271 9.18 8.59 7.83 66.21M sacchariflorus M-07 8.58 11.23 8.60 9.38M sacchariflorus M-46 8.80 7.70 11.79 17.91M sacchariflorus M-90 12.42 10.23 8.12 20.77M sacchariflorus M-93 8.76 11.01 11.52 10.28M sacchariflorus M-157 8.34 10.12 8.26 9.14M sacchariflorus M-180 7.75 7.60 7.59 19.75M sacchariflorus M-243 11.97 9.25 8.02 26.86M sacchariflorus M-289 9.93 11.20 11.71 18.27

M sinensis M-37 8.74 7.38 12.66 54.03M sinensis M-104 8.19 9.10 8.63 28.64M sinensis M-257 8.02 10.90 8.32 42.28

References

Beale, C.V., Blint, D.A. and Long. S.P. (1996). Leaf Photosynthesis in the C4-grass Miscanthus x giganteus, Growing in the Cool Temperate Climate of Southern England. J. Exp. Bot. 47: 267-273.

Chung, J.H. and Kim, D.S. (2012). Miscanthus as a Potential Bioenergy Crop in East Asia. J. Crop Sci. Biotech.. 15(2): 65-77.

Dohleman, F.G. and Long, S.P. (2009). More Productive Than Maize in the Midwest: How Does Miscanthus Do it?. Plant Physiol. 150: 2104-2115.

Naidu, S.L., Moose, S.P., Al-Shoaibi, A.K., Raines, C.A. and Long, S.P. (2003). Cold Tolerance of C4 Photosynthesis in Miscanthus x giganteus: Adaptation in Amounts and Sequence of C4 Photosynthetic Enzymes. Plant Physiol. 132: 1688-1697.

Wedding R.T. and Wu, M.-X. (1987). “Temperature Effects on Phosphoenolpyruvate Carboxylase from a CAM and a C4 Plant.” Plant Physiol. 85: 497-501.


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Flucetosulfuron performance improved by adjuvant

Jin Won Kim1, Seong-Hyu Shin2, Jong-Nam Lee3, Se-Eun Lim3, Soo-Hyun Lim1, Do-Soon Kim1,*1Department of Plant Science, Seoul National University, Seoul, Korea

2National Research Institute of Crop Science, RDA, Suwon, Korea3Division of Specialty Chemicals, LG Life Science Ltd., Seoul, Korea

* Corresponding author: E-mail: [email protected]

Abstract

This study was conducted to investigate the effects of adjuvant on herbicidal performance of flucetosulfuron (FTS, Fluxo®, LG Life Science Ltd.) against Echinochloa crus-galli and Aeschynomene indica. Adjuvant which reduced contact angles of herbicide droplet generally increased herbicidal performance of FTS. HLB (hydrophilicity and lipophilicity balance) of adjuvant tested are also related with herbicidal performance of FTS. The GR50 values of FTS dramatically decreased with increasing adjuvant concentration in the same manner as the herbicide dose-response. Leaf excision test revealed that FTS uptake and translocation was also enhanced by adjuvant, depending on adjuvant concentration. As a result, we selected several adjuvant showing much more improved performance in FTS activity against E. crus-galli and A. indica and produced new FTS formulation for foliar application. In this presentation, we will also introduce new FTS formulations improved by adding a new adjuvant, which may help to manage paddy weeds in tropical rice cultivation.

Keywords: adjuvant, Aeschynomene indica, contact angle, flucetosulfuron, HLB

Introduction

There are significant differences in foliar uptake of herbicide formulations among plant species (Chamel et al., 1992; Santier and Chamel, 1996; Gouret et al., 1993; Price and Anderson, 1985; Baker et al., 1992; Knoche and Bukovac, 1993). In general, increasing amounts of surfactants will increase agrochemical uptake, although in some cases, such as with glyphosate, if this markedly increases the contact area of the droplet with the leaf surface, it may reduce the concentration per unit area and reduce the uptake of the herbicide (Liu and Zabkiewicz, 1998).

Contact angle was more closely correlated with diuron efficacy than surface tension, and the coefficient of determination between the contact angle and the fresh weight or control percent of barnyardgrass was 0.41. Kinetic, an organosilicones, applied before or in combination with diuron significantly enhanced diuron efficacy. However, Kinetic sprayed after diuron did not affect the efficacy. Kinetic influenced the retention and absorption of diuron rather than its deposition and translocation (Singh et al., 2002).

Surfactants having a high hydrophilic-lipophilic balance (HLB) are absorbed into the cuticle and enhance the water-holding capacity (hydration state) of the cuticle. With increased cuticle hydration, the permeance of hydrophilic herbicides into the cuticle is increased, which increases the herbicide diffusion rate at a constant concentration gradient. Surfactants having a low HLB are absorbed into the cuticle and increase the fluidity of waxes, as measured by a small reduction in melting point. This increased fluidity increases the permeance of lipophilic herbicides in the cuticle, which, in turn, increases their diffusion rate at a set concentration gradient (Hess and Foy, 2000). Therefore, this study was conducted to investigate the effects of adjuvant on the herbicidal performance of flucetosulfuron and to select a suitable adjuvant for tank mix or pre-mix with flucetosulfuron.


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Whole plant response to adjuvantsIndian joint vetch (Aeschynomene indica L.) was seeded on the pot containing sandy loam soil

and was grown up to 40 cm tall. Forty seven adjuvants were prepared at two concentrations, 0.05 and 0.1% (v/v), and then flucetosulfuron was mixed with the prepared adjuvants. The mixtures were applied to A. indica at 15 g flucetosulfuron a.i. ha-1 and 300 L ha-1 (30 psi) of spray volume with a laboratory track sprayer. Herbicidal efficacy was evaluated with visual-rating from 0 to 10 at 14 days after application. All treatments were performed three times and arranged by randomized block design.

Contact angle measurementA 10 μL of 0.05 and 0.1% adjuvant solutions was dropped on a slide glass and its contact angle

was measured three or four times by Drop Shape Analysis System (DSA 10-MK2, Krüss, Germany) at 23oC. Contact angle of distilled water was 30.5o.

Concentration effect of adjuvants on flucetosulfuron performanceOA14 (HLB 13.6) was prepared at 0.025, 0.05, and 0.1%, and then flucetosulfuron was mixed

with the prepared OA14 solutions. The mixture was applied to A. indica at 3.75, 7.5, 15, and 30 g flucetosulfuron a.i. ha-1 under same conditions as whole plant assay. All treatments were performed three times and arranged by randomized block design. Herbicidal efficacy was evaluated with visual-rating from 0 to 10 at 14 days after application.

Results

Relationship between contact angle and FTS activityContact angles of solutions containing tested adjuvants at 0.1% were correlated with visual

efficacy of tested adjuvants in controlling A. indica. Contact angle showed significantly negative correlation with visual efficacy with -0.79 (Figure 1). However, several adjuvants e.g. Silwet L-77, SN10, SN20, TN20, and LN20 were away from such a relationship. Silwet L-77 is an organosilicone adjuvant and showed relatively poor efficacy of flucetosulfuron in spite of less than 1 degree of contact angle. SN10, SN20, TN20, and LN20 belong to plyoxyethylene alkyl amine series, which showed excellent efficacy of flucetosulfuron in spite of large contact angle, ranged from 22.4 through 28.0 degrees. Nevertheless, it is clear that decrease in contact angle by adding adjuvant significantly improves herbicidal performance of flucetosulfuron. Therefore, contact angle can be also a decisive consideration for choosing adjuvant for flucetosulfuron.

Concentration effect of adjuvants on flucetosulfuron performanceApplications of 15 and 30 g a.i. ha-1 of flucetosulfuron with 0.1% adjuvant had six and four

times herbicidal efficacy as good as those without an adjuvant, respectively, regardless of adjuvant type. Regardless of the applied flucetosulfuron dose, there was concentration effect of OA14 on efficacy of flucetosulfuron, showing the more the adjuvant concentration the better the flucetosulfuron efficacy (Figure 2) is. Particularly, without OA14, the herbicidal efficacy of flucetosulfuron was very poor, unable to estimate GR50 value (Figure 2). The GR50 of flucetosulfuron was 29.9, 13.4, 5.39 g a.i. ha-1 at 0.025, 0.05, and 0.1% of OA14, respectively, demonstrating flucetosulfuron performance improved with increasing adjuvant concentration (Figure 2).


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Contact angle (o)0 5 10 15 20 25

Visual efficacy

0

2

4

6

8

10

12r = -0.79

Figure 1. Correlation between contact angle of adjuvant solutions and visual efficacy against Aeschynomene indica of flucetosulfuron sprayed with adjuvants at 0.1% (v/v).

Flucetosulfuron (g a.i. ha-1)0 5 10 15 20 25 30

Visual efficacy

0

2

4

6

8

10No Adjuvant0.025%0.05%0.1%

GR50 (g a.i. ha-1)No adjuvant: Not calcu-lable0.025%: 29.90.05%: 13.40.1%: 5.39

Figure 2. Visual efficacy against A. indica of flucetosulfuron sprayed with OA14 at different concentrations. The continuous lines are fitted values by using the log-logistic model. GR50 is the dose of flucetosulfuron to cause the 50%-growth retardation of A. indica.

References

Baker, E.A., Hayes, A.L. and Butler, R.C. (1992). Physiochemcial Properties of Agrochemicals: Their Effects on Foliar Penetration. Pesticide Science. 34: 167-182.

Chamel, A., Gambonnet, B. and Coret, J. (1992). Effects of Two Ethoxylated Nonylphenols on Sorption and Penetration of [14C]Isoproturon through Isolated Plant Cuticles. Plant Physiology and Biochemistry. 30: 713-721.

Gaskin, R.E., Steele, K.D. and Forster, W.A. (2005). Characterising Plant Surfaces for Spray Adhesion and Retention. New Zealand Plant Protection, 58 : pp. 179-183.

Gouret, E., Rohr, R. and Chamel, A., (1993). Ultrastructure and Chemical Composition of Some Isolated Plant Cuticles in Relation to their Permeability to the Herbicide, Diuron. New Phytologists. 124: 423-431.

Griffin, W.C. (1949). Classification of Surface-Active Agents by HLB. Journal of the Society of Cosmetic Chemists. 1: 311.


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Hess, F.D. and Foy, C.L. (2000). Interaction of Surfactants with Plant Cuticles. Weed Technology. 14: 807-813.

Knoche, M. and Bukovac, M. (1993). Interaction of Surfactants and Leaf Surface in Glyphosate Absorption. Weed Science. 41: 87-93.

Liu, Z.Q. and Zabkiewicz, J.A. (1998). Organosilicone Surfactant Mediated Cuticular Uptake of Glyphosate into Grasses. In: Proceedings of 5th International Symposium on Adjuvants for Agrochemicals, Memphis, USA. pp. 119-124

Price, C.E. and Anderson, N.H. (1985). Uptake of Chemicals from Foliar Deposits: Effects of Plant Species and Molecular Structure. Pesticide Science. 16: 369-377.

Santier, S. and Chamel, A. (1996). Penetration of Triolein and Methyl Oleate through Isolated Plant Cuticles and their Effect on Penetration of [14C]Quizalofop-Ethyl and [14C]Fenoxaprop-Ethyl. Weed Research. 36: 174-176.

Sanyal, D., Reddy, K.N. and Bhowmik, P.C. (2007). Surfactants Enhance Primisulfuron Activity in Common Lambsquarters (Chenopodium album L.). Proceedings of 21st Asian Pacific Weed Science Society (APWSS) Conference. pp. 432-436.

Seefeldt, S.S., Jensen, J.E. and Fuerst, E.P. (1995). Log-Logistic Analysis of Herbicide Dose-Response Relationships. Weed Technology. 9: 218-227.

Singh, M., Tan, S. and Sharma, S.D. 2002. Adjuvants Enhance Weed Control Efficacy of Foliar-Applied Diuron. Weed Technology. 16: 74-78.

Sun, J. (1996). Characterization of Organosilicone Surfactants and their Effects on Sulfonylurea Herbicide Activity. Ph.D. Thesis, Virginia Polytechnic Institute and State University, pp. 133.


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Baseline sensitivity of Echinochloa crus-galli to alternative herbicides selected for managing herbicide resistant Echinochloa species

Ji-Soo Lim1, Soo-Hyun Lim1, Do-Soon Kim1

1 Department of Plant Science, CALS, Seoul National University, Seoul 151-742, KoreaCorresponding author: Email:[email protected]

Abstract

Acetolactate (ALS) and acetyl CoA carboxylase (ACCase) inhibitor resistant Echinochloa species now become problematic in Korean rice cultivation. Alternative herbicides with different modes of action can be used to control these herbicide resistant Echinochloa species. However, continuous uses of these alternative herbicides will eventually make the Echinochloa species become resistant to these herbicides as well. Therefore, this study was conducted to evaluate baseline sensitivity of Echinochloa crus-galli to alternative herbicides selected for managing herbicide resistant Echinochloa species. 63 accessions of Echinochloa crus-galli collected across Korea were tested by whole plant assay. Six early post-emergence herbicides such as mefenacet, pretilachlor, fentrazamide, cafenstrole, oxadiargyl, and oxaziclomefone at a range of their doses were directly treated to the flooded soil when Echinochloa reached the 2 leaf stage. The sensitivity indices estimated by comparing the maximum GR80 value and the minimum GR80 value was lowest for cafenstrole, 2.05, and greatest for mefenacet, 46.8. These sensitivity indices thus suggest that continuous use of these herbicides may be eventually resulted in herbicide resistance in Echinochloa species.

Keywords: Baseline sensitivity, Echinochloa crus-galli, herbicide resistance

Introduction

Herbicide resistance is a serious worry to worldwide farming system (Holt and Lebaron, 1990; Warwick, 1991). For management of herbicide resistance problem, EPPO suggested changing weed management system, and suggested to investigate baseline sensitivity of about dose response(EPPO,1999). Nowadays, in Korea, resistance herbicide weeds are also reported, about ALS inhibitor resistance herbicide in paddy fields (Hwang, Lee, et al.,2001) and cross-resistance is also detected (Kuk, Kim, et al.,2004). So, Korea farmer need alternative herbicides to manage herbicide resistant weeds. The previous study suggested that some alternative herbicides could control Echinochloa spp. including ACCase inhibitor herbicide resistant species. However, these herbicides also have resistance risk if they are continuously used for Echinochloa control. Our understanding of baseline sensitivity of E. crus-galli to these herbicides may help us pre-estimate potential risk of herbicide resistance development to these herbicides but no study has been done in Korea. Therefore, this study was conducted to quantify baseline sensitivity of Korean E. crus-galli accessions to the alternative herbicides and to assess potential resistance risk of Echinochloa crus-galli in Korean paddy fields.


Collecting sufficient number of accessions representing a region or a country is an essential prerequisite for the baseline sensitivity study. More than three hundred Echinochloa spp. were collected across Korea in 2009 and 2011. Among them, 61 E. crus-galli accessions were selected based on their regional distribution for this study. ACCase inhibitor resistant and susceptible accessions (Im, et al.,2009), and one accession collected in Japan in 2010 were also included as a reference.


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Pre-germinated seeds of each accession were transplanted in a well of open multiple-well acryl plate, specially designed for this study, placed on paddy soil in a 0.185 m2 rectangle tray. Seedlings were then thinned to 4 plants per well. The trays were placed in the 30/20 oC glasshouse at the Experimental Farm Station of Seoul National University, Suwon, Korea. All experiments were consisted with three replications of a completely randomized block design.

Herbicides were chosen based on our previous study to select alternative herbicides to control herbicide resistant Echinochloa spp. in Korea (Bae et al. 2009, not published). Six herbicides, mefenacet, oxaziclomefone, cafenstrole, oxadiargy, pretilachlor and fentrazamide were selected as they well controlled Echinochloa spp. even resistant accessions. Echinochloa crus-galli accessions were treated with mefenacet at 65.63 ~ 525 g ha-1, oxaziclomefone at 7.5 ~ 60 g ha-1, cafenstrole at 30 ~ 240 g ha-1, oxadiargyl at 8.5 ~ 68 g ha-1, pretilachlor at 69.4 ~ 555 g ha-1, and fentrazamide at 11.9 ~ 95 g ha-1 at 7 days after sowing after flooding the tray with water at 4 to 5 cm water depth.

Visual assessments were made at 10 days and 20 days after treatment (DAT). The visual efficacy data were fitted to the standard dose-response model (Streibig,1980) by using Genstat 5 (Genstat Committee 1997) to estimate GR50 and GR80 values.


A total of the 63 accessions of E. cruss-galli were assessed for their sensitivity to mefenacet, pretilachlor, fentrazamide, cafenstrole, oxadiargyl, and oxaziclomefone. GR80 values of mefenacet, pretilachlor, fentrazamide, cafenstrole, oxadiargyl, and oxaziclomefone ranged 12.67 - 3544.84 g a.i. ha-1, 12.20 - 372.98 g a.i. ha-1, 2.68 - 58.16 g a.i. ha-1, 34.35 - 95.21 g a.i. ha-1, 4.68 - 461.50 g a.i. ha-1, and 0.34 - 25.81 g a.i. ha-1, respectively. Their mean values were 525.06, 122.36, 24.86, 53.31, 90.66, and 7.25, respectively.

To analyze distribution of sensitivity, GR80 values were fitted to cumulative distribution function by using Genstat 5 (Genstat Committee 1997). All distributions of sensitivity by each herbicide were confirmed normal distribution. Skewness on sensitivity of E. crus-galli to mefenacet, pretilachlor, fentrazamide, cafenstrole, oxadiargyl, and oxaziclomefone were 4.34, 1.11, 0.63, 0.82, 1.72, and 1.46, with kurtosis of 24.44, 2.50, 1.93, -0.12, 2.32, and 2.54, respectively. All distributions of sensitivity were right-skewed and sensitivity of mefenacet and oxadiargyl were more skewed than the others. According to previous study, skewness could be a clue of creeping resistance. Therefore, skewed distributions of mefenacet and oxadiargyl could suggest some processing of resistance development (Espeby et al.,2011). On the other hand, the distributions of cafenstrole and fentrazamide indicate no or little processing in resistance development.

Accessions

Pretilachlor ( g a.i. ha

-1)

0

100

200

300

400

Mefenacet (g a.i. ha

-1)

0

1000

2000

3000

4000

Figure 1. Sensitivity of the 63 accessions of E. cruss-galli collected in Korea from 2009 to 2010. Scatter graph showed herbicide dose for the 80 % growth reduction (GR80 value) in sensitivity test. Each spot represents GR80 value of each accession. The GR80 value was calculated by non-linear regression analysis against log herbicide dose.


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Pretilachlor (g a.i. ha-1) 0 30 60 90 120 150 180 210 240 270 300 330 360

% of accessions

0

5

10

15

20

25

Mefenacet (g a.i. ha-1) 0 200 400 600 800 1000 1200 ~

% of accessions

0

10

20

30

40

Figure 2. Frequency distribution of E. cruss-galli accessions for each herbicide dose for the 80 % growth reduction (GR80 value) in sensitivity test with mefenact and pretilachlor. The GR80 value was calculated by non-linear regression analysis against log herbicide dose.

References

EPPO. 1999. Resistance risk. EPPO Bulletin 29: 325-347. doi:10.1111/j.1365-2338. 1999.tb00838.x.Espeby, L.Å., H. Fogelfors and P. Milberg. 2011. Susceptibility variation to new and established

herbicides: Examples of inter-population sensitivity of grass weeds. Crop Protection 30: 429-435. doi:http://dx.doi.org/10.1016/j.cropro.2010.12.022.

Holt, J.S. and H.M. Lebaron. 1990. Significance and distribution of herbicide resistance. Weed Technology 4: 141-149.

Hwang, I.T., K.H. Lee, S.H. Park, B.H. Lee, K.S. Hong, S.S. Han, et al. 2001. Resistance to acetolactate synthase inhibitors in a biotype of Monochoria vaginalis discovered in Korea. Pesticide Biochemistry and Physiology 71: 69-76. doi:10.1006/pest.2001.2565.

Im, S.H., M.W. Park, M.J. Yook and D.S. Kim. 2009. Resistance to ACCase inhibitor cyhalofop-butyl in Echinochloa crus-galli var. crus-galli collected in Seosan, Korea. Korean Journal of Weed Science 29: 7.

Kuk, Y.I., K.H. Kim, O.D. Kwon, D.J. Lee, N.R. Burgos, S. Jung, et al. 2004. Cross-resistance pattern and alternative herbicides for Cyperus difformis resistant to sulfonylurea herbicides in Korea. Pest Management Science 60: 85-94. doi:10.1002/ps.786.

Streibig, J.C. 1980. Models for curve-fitting herbicide dose response data. Acta Agriculturae Scandinavica 30: 59-64. doi:10.1080/00015128009435696.

Warwick, S.I. 1991. Herbicide resistance in weedy plants: Physiology and population biology. Annual Review of Ecology and Systematics 22: 95-114.


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Weed and Weedy Rice Control by Imidazolinone Herbicides in ClearfieldTM Paddy in Vietnam

Duong Van Chin1, Tran Cong Thien2, Huynh Hong Bi1, Nguyen Thi Nhiem1 and Tran Thi Ngoc Son1

1Cuu Long Delta Rice Research Institute, Can Tho, Vietnam.2BASF Representative Office in Ho Chi Minh City, Vietnam

[email protected] ; [email protected]

Abstract

Two experiments with two types of rice culture , dry- seeded and wet- seeded rice, were carried out at the Experimental Farm of the Cuulong Delta Rice Research Institute (CLRRI) in Vietnam during Spring-Summer and Summer- Autumn seasons of 2006. The tested variety was OM5749-5, an indica rice genotype developed by plant breeders at CLRRI by crossing between promising Vietnamese indica rice with IMI-tolerance japonica rice from Louisiana State University, USA. The tested herbicides are: imazapic, imazapyr, imazapic+imazapyr and imazethapyr+imazapyr. Results revealed that common weeds observed in the experimental field including Echinochloa crus- galli, Leptochloa chinensis, Cyperus iria, Cyperus difformis, Ludwigia octovalvis and especially weedy rice (Oryza sativa) were controlled successfully by the herbicides. The density and dry weight of weeds were brought down significantly as compared to those under untreated check. In dry seeded rice, the average rice grain yield of six imidazolinone treatments is 1.83 t/ha, 101.1% higher than check (0.91 t/ha). The corresponding data in wet-seeded rice are 2.15 t/ha and 0.88 t/ha with 143.9% increment. Rice quality is also improved by the treatments. The number of contaminated weedy rice seeds in rice product, seeds dropped in the soil surface, the percentage of red grains in milled rice is also reduced under herbicide treatments as compared to that of check statistically.

Keywords: ClearfieldTM paddy, herbicide tolerant variety, imidazolinone, weedy rice control

Introduction

Weedy rice, commonly considered as ecotypes of Oryza sativa, is a new pest in rice growing countries in the world including Vietnam. In tropical areas, weedy rice is progenies of crosses between wild rice and cultivated rice or come from degradation of cultivated rice varieties. The major characteristic of weedy rice is easy shattering. Other characteristics are observed as taller plants, fewer tillers, and high percentage of red rice in milled rice (Chin et al. 2000). Weedy rice competes with cultivated rice for sunlight, water and nutrients resulting in reduction in rice yield. The quality of milled rice is reduced due to contaminated red rice. Weedy rice infestation in rice fields is dangerous because seeds in seed bank increase over time with self-regeneration and there is no effective selective herbicide for controlling weedy rice. Recently, a new option for controlling weedy rice and also common weeds in rice fields has been initiated by exploring the integration of imidazolinone herbicides and tolerant trait containing variety (which is called CLEARFIELD TM rice). Imidazolinone herbicides controls weeds by inhibiting the plant specific enzyme acetohydroxyacid synthase (AHAS), which is involved in the biosynthesis pathway of the branched-chain amino acids as valine, leucine and isoleucine. This inhibition causes a disruption of protein synthesis, which interferes DNA synthesis and cell growth (Shanner and Connor 1991). CLEARFIELDTM rice has been developed by Louisiana State University Agricultural Center breeders through a combination of mutagenesis and conventional plant breeding, which is tolerant to imidazolinone herbicides. This is characterized as a non-GMO variety. In Vietnam, CLRRI plant breeders have successfully developed indica rice genotypes, which are tolerant to imidazolinone herbicides. The research aims at determining whether the integration of imidazolinone herbicides and tolerant trait in the genotypes can be used for controlling weedy rice and common weeds in rice fields


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Two experiments were conducted in clay soil lowland fields with good water supply in the Spring-Summer with dry seeded rice and Summer- Autumn with wet seeded rice of 2006 at the Experimental farm of CLRRI. Rice variety OM5749-5 is indica rice with IMI tolerance trait. The cultivar was bred by CLRRI plant breeders by crossing between a promising Vietnamese indica rice and IMI-tolerance japonica rice from Louisiana State University, USA. In dry seeded rice, 200 kg of cultivated rice seeds were mixed with 100 kg of weedy rice seeds and broadcasted randomly before incorporation into the soil surface. In wet seeded rice, 100 kgs of pre-germinated seeds were sown in line by drum seeder. Pre-germinated seeds of weedy rice at the rate of 100 kg /ha were broadcast randomly one day later. Field trials with plot size of 20m2 were established using randomized complete block design with four replications and seven treatments. Three active ingredients of imazapic, imazapyr and imazethapyr were used as solo and ready-mix formulations at various dose rate from 100 to 120 g a.i./ha . Herbicides were sprayed at 12 days after emergence (DAE) in dry seeded rice and 10 days after sowing (DAS) in wet seeded rice. Crop oil was added at 0.5% water volume as non-ionic surfactant.


Herbicidal activityThe population of weedy rice, Echinochloa crus-galli, Leptochloa chinensis, Cyperus iria

affected by treatments of imidazolinone herbicides have been presented in Table 1.All herbicide treatments brought down weedy rice densities significantly as compared to that

in untreated check (182.7 plants / m2). All treatments were very effective in controlling Echinochloa crus-galli except treatment T6 [Imazethapyr+imazapyr]@120. There was no difference between the treatments with weedy check. The density of Leptochloa chinensis remained at level of 22 plants / m2 in untreated check whereas in all herbicide treatments, this weed was killed completely. The same trend was observed in the case of Cyperus iria.

Table 1. Weed and weedy rice density (No. plants /m2) at 70 days after emergence (DAE) as affected by treatments (CLRRI, 2006 Spring-Summer)

Treatments Weedy rice (*) ECHCG LEPCH CYPIR

T1 Untreated check 182.7 12.0 22.0 17.3

T2 [Imazapic+imazapyr]@100 0.0 0.0 0.0 0.0



T5 Imazapyr@120 12.7 0.0 0.0 0.0

T6 [Imazethapyr+imazapyr]@120 0.0 14.0 0.0 3.3

T7 Imazapic@120 0.0 0.0 0.0 0.0

LSD(0.05) 81.4 6.1 12.8 14.1

(*) Weedy rice (Oryza sativa); ECHCG:Echinochloa crus-galli; LEPCH:Leptochloa chinensis; CYPIR:Cyperus iria.


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Table 2. Weed and weedy rice dry weight (g/m2) at 70 DAE as affected by treatments (CLRRI, 2006 Summer-Autumn)

Treatments Weedy rice (*) ECHCG LEPCH CYPIRT1 Untreated check 269.7 35.9 7.7 26.6T2 [Imazapic+imazapyr]@100 0.0 0.0 0.0 0.0T3 [Imazapic+imazapyr]@110 0.0 0.0 0.0 0.0T4 [Imazapic+imazapyr]@120 0.0 0.0 0.0 0.0T5 Imazapyr@120 21.7 0.0 0.0 0.0T6 [Imazethapyr+imazapyr]@120 0.0 8.0 0.0 12.0T7 Imazapic@120 0.0 0.0 0.0 0.0

LSD(0.05) 78.2 25.6 3.5 3.1

The density of Cyperus iria was lower statistically under all treatments except T6 [Imazethapyr+imazapyr]@120. In this treatment the weed remained at the level of 3.3 plants /m2.Dry matter accumulation of weedy rice and some common weeds observed in the experimental fields at 70 DAE have been presented in the Table 2.

All imidazolinone – herbicide treatments caused the reduction of weedy rice and weed dry weights statistically as compared to untreated check (269 g/m2). However, the treatment of T5 [Imazapyr]@120 could not control weedy rice completely resulting in the remaining weedy rice dry weight of 21.7 g. /m2. The Echinochloa crus-galli also was controlled successfully by all tested herbicides. However, some plants of this weed remained in the treatment of T6 [Imazethapyr+imazapyr]@120 resulting in the dry accumulation of 8.0 g/m2. Leptochloa chinensis was controlled completely by all treatments. Dry weights of Cyperus iria were brought down significantly under all treatments as compared to untreated check (26.6 g./m2) but the weed dry weight remained 12 g/m2 under treatment of T6 [Imazethapyr + imazapyr]@120.

Yield components and grain yieldThe imidazolinone herbicides successfully controlled weedy rice and common weeds in rice

fields and therefore the competition of weeds on rice plants was minimized and rice plants can grow better as compared with that under untreated plots. The data on yield components and grain yields as affected by treatments have been presented in Table 3.

Table 3. Yield components and yield of dry seeded rice as affected by treatments (CLRRI, 2006 Spring-Summer)

Treatment No. of panicle/m2

No.filled grains /panicle

1000-grain weight (g.) Yield (t/ha )

T1 Untreated check 286 51.1 26.6 0.9T2 [Imazapic+imazapyr]@100 311 63.5 26.4 1.7T3 [Imazapic+imazapyr]@110 323 62.1 26.7 1.9T4 [Imazapic+imazapyr]@120 325 63.2 26.7 1.8T5 Imazapyr@120 341 63.3 26.3 1.8T6 [Imazethapyr+imazapyr]@120 345 62.6 26.4 1.8T7 Imazapic@120 350 67.2 26.5 2.1

LSD(0.05) 48 4.2 0.9 0.5

Three treatments of [Imazapic+ imazapyr]@100; 110 and 120 g a.i./h tended to cause the increment of the number of panicles/m2 but had not reach the level of significance. The rest of the three treatments of T5 [Imazapyr]@120; T6 [Imazethapyr+imazapyr]@120 and T7 [Imazapic]@120 were superior to untreated check in terms of the increment of the number of panicles / m2 statistically. All herbicide treatments caused the increment of the number of filled grain per panicle significantly


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as compared to that of untreated check (51.1 grains / panicle). The average of the increment was 24.7%. There was no significant difference of 1,000-grain weights amongst treatments. Rice yields under treated plots were significantly higher as compared to that of untreated check (0.9t/ha). However, each herbicidal treatment was equal with the other in terms of yield. On the average of all herbicide treatments, rice yield increased 106% as compared to untreated check.

In wet seeded riceOne wet seeded experiment with the same design and treatments was conducted during Summer-

Autumn season of 2006.The population of weedy rice affected by herbicide treatments observed at 28, 56 and 84DAS have been presented in Table 4.

Table 4. Density (No.plants /m2) of weedy rice at 28; 56 and 84 days after sowing (DAS) as affected by treatments (CLRRI, 2006 Summer-Autumn)

TreatmentsWeedy rice density (No.plants /m2)

28 DAS 56 DAS 84 DAST1 Untreated checkT2 [Imazapic+imazapyr]@100 0.3 0 0T3 [Imazapic+imazapyr]@110 5.0 0 0T4 [Imazapic+imazapyr]@120 0.4 0 0T5 Imazapyr@120 10.3 0 0T6 [Imazethapyr+imazapyr]@120 1.8 0 0T7 Imazapic@120 0.0 0 0

LSD(0.05) 11.4 28 28

At 28 DAS, the density of weedy rice was 69.3 plants /m2 in untreated check. All herbicide treatments brought down the population of weedy rice at 28 DAS significantly as compared with that of check. However, some plants of weedy rice were countable at this stage because the remaining weedy rice showed the symptoms of injury but have not been killed completely yet. At 56 and 84 DAS, all weedy rice plants were susceptible and completely killed. In the case of no spraying imidazolinoe herbicides, weedy rice grew normally and reached the density of 245-246 plants /m2 at 56 DAS and 84 DAS, respectively. The yield components and grain yields of wet seeded rice as affected by treatments have been presented in Table 5.

Table 5. Yield components and grain yield as affected by treatments (CLRRI, 2006 Summer-Autumn)

Treatment No. of panicle/m2

No.filled grains /panicle

1000-grain weight (g.)

Yield(t/ha )

T1 Untreated check 142 57.4 26.5 0.88T2 [Imazapic+imazapyr]@100 301 62.6 26.5 2.22T3 [Imazapic+imazapyr]@110 339 76.7 27.2 2.25T4 [Imazapic+imazapyr]@120 317 66.6 25.7 2.15T5 Imazapyr@120 292 71.7 26.0 2.05T6 [Imazethapyr+imazapyr]@120 316 66.5 27.1 2.13T7 Imazapic@120 294 70.2 26.5 2.08

LSD(0.05) 46 16.7 2.1 0.48

All the imidazolinone herbicide treatments caused the increments of the number of panicles / m2 significantly as compared with untreated check (142 panicles /m2). The number of panicles / m2 in treatment T3 [Imazapic+imazapyr]@110 was the highest (339 panicles /m2) and higher than T5 [Imazapyr]@120 statistically (292 panicles/m2). However, treatment T3 [Imazapic+imazapyr]@110 was not higher than the rest of the other herbicide treatments. The average data of six treatments was


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310 panicles /m2 and increased 118% as compared to untreated check. The 1,000-grain weights were unchanged amongst all treatments including check. Rice grain yields under all herbicide treatments were superior to that of untreated check (0.88 t/ha) significantly. There was no significant difference amongst herbicide treatments. The average yield of 6 treated plots was 2.15 t/ha which increased 144 % as compared to untreated check (0.88 t/ha).

Acknowledement

Thanks are due to BASF Corporation, USA for granting the research funds and supporting other activities.

References

Chin, DV, Hien, T.V and Thiet. L.V. (2000). Weedy Rice in Vietnam. In Limited Proceedings No.2. “Wild and Weedy Rice in Ecosystem in Asia- A Review”,IRRI.

Shanner D.L and Connor, S.L.O. (1991). The Imidazolinone Herbicides. CRC Press. Pp 290 .


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Utilization of weeds in Thailand

Pensee Nantasomsaran Komson Nakornsri Patpitcha RujirapongchaiWeed Science Group, Plant Protection Research and Development Office, Department of Agriculture,

Chatuchak, Bangkok 10900, Thailand

Abstract

Thailand is in the tropical area with nourish humid climate. Weed is one of the biodiversity of plant community. The weed is a general pest to limit quantity and quality of yield in agricultural production. There are many methods to reduce weed problems by using chemical and non chemical control. However, utilization of weed is one strategy to minimize weed problems. Weed can be grown by naturally, weed utilization can be compiled in 16 purposes, such as traditional vegetables, medicinal plants, cosmetics, ornamental plants, artificial flowers, lawn, animal feeding, allelopathic substances, mulching materials, soil indicator, water pollution absorbent, green fuel and beautiful scenery.

Introduction

Weed is unwanted plant or need to be controlled, on the other hand, weed can be useful. Weed can be grown by naturally. Utilization of weeds were used for a long time in Thailand and many countries with various purposes (Vongsaroj et al, 1999). Weeds can be used for native vegetables, medicinal plants, cosmetics, ornamental plants, artificial flowers, lawn, animal feeding, allelopathic substances, mulching materials, soil indicator, water pollution absorbent, green fuel and beautiful scenery etc. There are so many vegetables which come from weeds (Aram and Pichet, 2005). Thailand is in the tropical, with high moisture content. There are so many plants including weeds to be biodiversity system. The farmers do not need to grow traditional vegetables such as Ipomoea aquatica Forsk.(morning glory), Marsilea crenata Presl., Aeschynomene javanica Miq., Momordica charantea L. (Watcharee Prachasuysoradej, 1999). Some weeds become medicinal plants such as Abutilon hirtum (Lam.) Sweet for the eurotic system, Centella asiatica (L.) Urb. For the protecting Alzimer, the tuber of Cyperus rotundus L. and the rhizome of Imperata cylindrica (L.) Reauschell to be in the ancient medicine recipes of Thailand and many countries in Asia and Pacific area (Nantasomsaran et al, 2009).

There are many purposes of weed usefulness. Therefore collection of the weed utilization are the database for research work and applied usage.

Materials and methods

The collection of data came from many references and from the survey work. After that classified in the group of weed utilization and compiled them by using references and some research work that the information will be useful in the future.

Results and Discussions

1.There were 27 species to be traditional vegetables. Watcharee et al, 1999 collected Weeds as a fresh vegetables and raw material for cooking as shown in Table 1. 2. There were 63 species to be medicinal plants which the Thai name, Scientific name, usage parts such as leaves, flowers, stem, root or the whole plants and the usage of indication of these plants.


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The user can find them in the field or gardener that weeds can be grown by planting or naturally ecology as shown in Table 2.

3. The weed can be used as homemade industry namely Eichhornia crassipes (Mart.)Solms for woven materials: bag, basket, mat, plate mat etc. The Aeschynomene aspera L. and Aeschynomene indica L.were done to be the wreath, artificial flower. The one species of fern used for fine quality lady bag, and Pennisetum spp. for paper material (Table 3).

4. The weed species showed the indicator of high soil fertility namely Monochoria vaginalis Presl for nourish nitrogen nutrient. The Xyris indica Linn. showed acidic soil and saline soil in the some eastern and some southern part of Thailand. The Fimbristylis miliacea Vahl. showed phosphorus deficiency (Table 4). 5. Weed can be built for the house. In the past, there are many weed species used for the roof such as Imperata cylindrica Raeuchel and for the wall namely Saccharum spontaneum Linn. (Table 5). 6. Weed can be used for assisting on purify water to absorb heavy metal in the swamp namely Eichhornia crassipes (Mart.)Solms. The pollution from household and some factory can eliminate by using Thypha angustifolia Linn. (Table 6). 7. Weed can be used for protection of soil erosion, in the northern area, the farmer used Vetveria spp. on the slope, mountain and roadsides for assisting land slopes (Table 7). 8.Weeds are used for house and garden decoration, There are so many species which use for suitable environments. Some have beautiful flowers, pretty shape, Some can be used dry flower to decorate for a long time namely Echinochloa crus-galli Beauv. and Eragrostis tenella P.Beauv.ex Roem.et Schult including aquatic weeds can be decorated many types of gardens such as Pistia stratiotes L. and Salvinia cuculata Roxb. (Table 8). 9. Weed can be used for mulching materials, to keep soil moisture content and control weed germination namely Imperata cylindrica Raeuchel in vegetable area of the central plain and Rottboellia cochinchinensis W.D. Clayton on seedling tobacco in the northern part of Thailand, including Azolla pinnata R.Br. and Lemna minor L. for controlling weed in paddy fields (Table 9).

10. Weed can be used for green fuel. Many weed species were done green fuel column and for charcoal namely Eichhornia crassipes (Mart.)Solms., Mimosa pigra L. and Aeschynomene spp. (Table 10). 11. Weed can be used for controlling other weeds by allelopathic mechanism such as Imperata cylindrica Raeuchel can grow only one species in the big area. Some species used for extract solution namely Ammania baccifera L. and Hyptis sauveolens Poit. to inhibit other weeds(Table 11). 12. Weed can be used for animal feed namely Brachiaria mutica Stapf and Echinochloa colona Link for cow buffalo sheep goat and hoarse (Table 12). 13. Weed can be used for fish protection from enemy and some species become food for herbivorous fish namely Eichhornia crassipes (Mart.)Solms., and Ceratophyllum demersum Linn. (Table 13). 14. Weed can be used for green manure and composed manure since weed can adsorb nutrient more than crop. Weed can incorporate in the soil for tillage system to increase soil fertility and good soil structure. 15. Weed can be used for genetic resources, there are many weeds used for father or mother lines to show the distinguish characteristics: disease resistance, drought tolerance such as Oryza rufipogon Griff. 16. Weed can be used for the name of location or place, province, temple and school such as the name of Ban nong pangpuay (Thai name of Ludwigia adscendens (L.)hara) in Nakon Pathom province (Table 14). The utilization of weed is one of the strategies to reduce weed problems and conserve the natural resources for human. Weed can be useful without buying from the market. There are various methods to use weeds especially medicinal weeds. The most importance, the user must understand well to use the correct plant by learning from the expert. The one weed control method is utilization from weed instead of eliminate alone. If some species are popular from the consumer. We can promote and plant them by using Good Agricultural Practice or GAP and recommend to the farmers to grow them for a good quality material to produce many products.


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Table 1 Weeds as the traditional vegetables (Watcharee,1999; Aram and Pichet,2005).

Thai name Scientific names Family Edible partsKrachab Xanthium indicum Koenig Compositae Seedling can be cooked for the soup

Krathin Leucaena leucacep0hala de Wit MimosaceaeYoung shoot, young leaves, young pod and seed for fresh vegetable, dip with chili paste or fry with flour, spicy with lemon and chili

Krapanghom Oxystelma esculentum Linn.R.Br. AsclepiadaceaeYoung shoot, young leaves, and flower for fresh vegetable, dip with chili paste, and with minced meat and chili

Kratokrok Passiflora foetida L. Passifloraceae Young leaves dip in warm water, fried in the hot oil

Khakhiat Monochoria vaginalis Presl Pontederiaceae young leaves as a fresh vegetable

Chaploo Piper sarmentosum Roxb. Piperaceae young leaves as a fresh vegetable and for coconut curry with shrimp fish and snail

Choomhetthai Cassia tora Linn. Caesalpiniaceae young leaves stream and take with some fish and chili paste

Tamlueng Coccinia grandis Voigt Cucurbitaceae Edible leaves and fruit for soup with minced pork

Talapatluesee Limoicharis flava Buch. Limnocharitaceae Flower and young fruit as fresh vegetable, and for traditional curry

Buabok Centella asiatica (L.) Urb. Umbelliferae Leaves, and upper ground level for fresh vegetable, and squeeze for juice drink

Phak kayang Limnophila aromatica Merr. Scrophulariaceae Leaves, and stem for frog and fish in the hot soup

Phak krad Spilanthes acmella (L.) Murr. Asteraceae leaves, and stem for fresh vegetable with chili fish paste, put in the bamboo shoot soup

Phak cheelom Oenanthe stolonifera Wall UmbelliferaeLeaves and young shootwith pork minced (lab) or dip in hot water and have it with chili paste

Phak top chava Eichhornia crassipes (Mart.) Solms Pontederiaceae Young shoot with chili pastePhak phet Spilanthes paniculata Wall. Ex DC. Asteraceae Leaves and flower for northern curry

Phak bung Ipomoea aquatica Forsk. Convolvulaceae Young shoot, leaves as fresh vegetable or put in the curry

Phak tubtao Mimulus orbicularis Benth. Scrophulariaceae Young shoot, leaves as fresh vegetable and have with minced meat in lemon and chili

Phak pang Basella rubra Linn. Basellaceae Young shoot, young flower for vegetable, dip with chili paste, and put in the chili curry

Phak wan Marsilea crenata Presl. Marsileaceae Young shoot as fresh vegetable with chili paste

Phak sean Cleome gynandra L. Capparidaceae Whole plant for fermentation to have with chili paste

Pang puay Ludwigia octovalis Hara Onagraceae Young shoot as fresh vegetable and cook with chili and lemon juice

Mara kheenok Momordica charantea L. CucurbitaceaeYoung shoot and fruit in a short time with hot water forchili paste, fry with oil, curry

Mawangton Solanum santiwongsei Craib Solanaceae young fruit as fresh vegetable with chili paste Yaanang Tiliacora triandra Diels Menispermaceae Squeezed leaves for juice put in the chili curry

Santawa baipay Ottelia alismoides (L.)Pers. Hydrocharitaceae Young shoot, leaves and young flower as fresh vegetable with meat chili paste

Sano kindog Sesbania javanica Miq. Papilionaceaeflower for fermentation, dip in hot water and have it with chili paste, fry with oil and flower for dessert

Hoo seua Coleus amboinicus Lour. Labiatae Leaves and young shoot as fresh vegetable with chili paste


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Table 2 Weeds as medicinal plant (Department of Medical Science,1990 ; Nantasomsaran et al , 2009).

Thai name Scientific names Part of plant Properties and action

Kratokrok Passiflora foetida Linn. Leaves stem flower root and fruit Diuretic

kameng Eclipta alba (L.) Hassk. Leaves stem flower root and whole plant Inflammation

Krathin Leucaena leucocephala (Lam.) de Wit Flower, root Flower for maintenance liver, root for

carminative

Krateepyod Biophytum sensitivum (L.) DC.

Whole plant, fresh and dry

Diuretic , maintenance liver,and antipyretic

Krapanghom Oxystelma esculentum Linn.R.Br. Stem leaves root Inflammation of throat and mouth

and laxative Koksay Cyperus iria L. Stolon Antipyretic, antidiarrhoea

Koktumhoo Cyperus kyllingia Endl. Tuber Antipyretic, antidiarrhoea Koksamleunhawkradan Scirpus grossus Linn.f. Root Antipyretic, antidiarrhoea

Khadmon Sida acuta Burm. Whole plantRelieve of cold, protect Inflammation of liver, bacterial diarrhea and gastro-intestinal infection

Crobjakawan Abutilon hirtum (Lam.) Sweet Leaves stem flower root

Antitussive, Diuretic, protect diabetes and digestant, blood tonic

Crobphansee Abutilon indicum (L.) Sweet Stem Antidiarrhoea, blood tonic

Cokkrasoon Tribulus terrestis L. Whole plant Diuretic

Chumhetthai Cassia tora Linn. Leaves, seed and whole plant

Laxative, skin cure, Diuretic, protect Inflammation of liver

Chumhetthes Cassia alata Linn. Leaves Laxative for dry leaves and vermifuge for fresh leaves

Tamleung Coccinia grandis Voigt Leaves root and fruitLeaves for maintenance eyes, antipyretic, expectorant, herpies, anti-toxin from insect; fruits for diabetes

Tongteng Physalis minima L. Whole plant Diuretic Namnom ratchasee Euphorbia hirta L. Whole plant

Fresh and dry Protect stomachache

Buabok Centella asiatica (L.) Urb. Leaves and over ground level

Diuretic, anti-bacteria, cough,antipyretic, anti diarrhoea and protect burn from the heat

Borapet Tinospora crispa (L.) Miers ex Hook.f. & Thomson Stem Antipyretic, drink fresh juice of stem to

assist edible tonic

Paka krong Lantana camara L. Leaves Stop blood from the wound, carminative, allergic and swollen

Phak crad hua wan Spilanthes acmella (L.) Murr. Whole plant Expectorant, asthma and cold

Phak chee lom Oenanthe stolonifera Wall Whole plant Expectorant, asthma and cold

Phak top chawa Eichhornia crassipes (Mart.) Solms Whole plant Inflammation

Phak phed Spilanthes paniculata Wall. Ex DC. Fresh flower Tooth ache


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Phak boong Ipomoea aquatica Forsk. Young shoot and leaves

Tonic, eye maintenance

Phak boong talae Ipomoea pes-caprae Sweet Root leaves and

whole plant

Root for diuretic, tooth ache; Leaves for diuretic, skin cure, haemorrhoid, Whole plant for jellyfish toxin

Phak pang Basella rubra Linn. Leaves stem flower root

Diuretic, dysentery, skin allergy,Laxative

Phak wan Marsilea crenata Presl. Whole plant Expectorant, antianagesic

Phak sean pee Cleome viscosa L. Whole plant Take care wound in lung liver, sprain, anthelmintic, dysentery and tonic

Pan ngu khao Achyranthes aspera L. Whole plant Diuretic, antipyretic

Pan ngu khiaw Stachytarpheta jamaicensis (L.) Vahl. Whole plant Antipyretic, diuretic, anthelmintic

Pang puay Ludwigia octovalis Hara stem flower and fruit

Diuretic, swollen, detoxifier, cough, skin allergy

Pha talay jone Andrographis paniculata (Burm.f.) Wall ex Nees

Root and Whole plant

Root for anthelmintic, anti malaria fever, Whole plant for Antipyretic, antiseptic of bactria diarrhea, Expectorant and cough

Mara kheenok Momordica charantea L. Stem flower fruit and seed

Anti bile duct infection, antipyretic, relief body heat, anti virus and cancer

Mawang kreao Solanum trilobum L. Root, fruitRoot for antipyretic, expectorant,diuretic, Fruit for cough, expectorant, and diabetes

Mawang ton Solanum santiwongsei Craib Fruit Diuretic, decrease sugar blood

Hing men Crotolaria spectabilis Roth. Root, extract fruit Root relief heat in the body,extract fruit for inhibition tumor

Maiyarab Mimosa pudica L. Leaves root Antipyretic, decrease debility, cure of wound

Yaanang Tiliacora triandra Diels Leaves root Antipyretic

Looktai bai Phylanthus niruri L. Fresh and dryWhole plant Antipyretic

Santawa bai paai Ottelia alismoides (L.) Leaves Relief heat in the body

Saray huamai-kheedphai Eriocaulon sexangular L. Stem Antipyretic

Sabraeng sabkaa Ageratum conyzoides L. Leaves shoot stem

root

Antipyretic, antianalgesic, sprainProtect cold and cough, stop blood, anti skin itchy

Sab suea Chromolaena odorata (L.) R.M. king Leaves stem Stop blood

Sano kindog Sesbania javanica Miq. Leaves stem flower root

Leaves ground with powder to external protection wound: detoxifier, inflammation, swollen Flower for intestine protection, Stem roast and put in the water to be alkaline solution for diuretic Root relief heat body, refrigerant

Yaa kled hoy Desmodium triflorum Dc. Whole plant Antipyretic, relief heat body, refrigerantYaa kled plaa Phyla nodiflora Greene Whole plant Diuretic

Saray huamai-kheedphai


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Yaa khaa Imperata cylindrica (L.) Reauschell Fresh and dry root Diuretic

Yaa nguang chang Heliotropium indicum R.Br. Whole plant Diuretic, swollen, carminative

Yaa channakad Panicum repens L. Fresh and dry tuber Diuretic Yaa sai Leersia hexandra Sw. Whole plant Diuretic Yaa teenka Eleusine indica Gaertn. Leaves DetoxifierYaa preak Cynodon dactylon (L.) Pers. Fresh whole plant Carminative, antipyretic, blood vomid

Yaa laong Vernonia cinerea Lees. Whole plant Antipyretic, antitussive, anti skin disease: elephant foot

Yaa hnuad maew

Orthosiphon aristatus (Blume) Miq. Leaves stem

Diuretic, take care of stone kidney, antianagesic, diabetes, hypertension, decrease uric acid

Mhak dib namkang Hedyotis biflora (L.) Lam. Whole plant Cure of skin

Haew moo Cyperus rotundus L. Tuber Maintenance digestive system

Hoo pla chon Emilia sonchifolia (L.) DC. Whole plant Antipyretic, tonsil gland, dysentery, diarrhea, allergy

Hoo seoa Coleus amboinicus Lour. Leaves Solution of leaves to protect inside ear, nasal congestion, carminative

Ngon kai pa Celosia argentea L. Root, leaves Root for blood toxic, antipyretic,expectorant, vomid and allergic

Uttapit Typhonium trilobatum (L.) Schott Root, tuber Root for haemorrhoid, tuber for oil to

cure wound

Table 3 Weeds as industrial plants (Department of Industry extension,1989).

Weed species location itemEichhornia crassipes (Mart.) Solms River canal lake swamp Toy, tray, basket, hand bag, Slippers,

cass soucer box, hat, earring, neglect Scirpus grossus Linn.f. canal swamp Mat, bag and toyCyperus corymbosus Linn. canal swamp Mat, bag and toyAeschynomene aspera L. paddy field Artificial flowerAeschynomene indica L paddy field Artificial flowerLygodium flexuosum(L.)Sw. Forest and rubber garden Bag ,bracelet ,neglectPennisetum spp. Non crop area paper

Table 4 Weeds as soil indicator (Vongsaroj et al, 1999).

Weed species location Soil indicator

Eleocharis dulcis (Burm.f.) Henschel Paddy field Acidic soil and saline soil

Xyris indica L. Paddy field Acidic soil and saline soilFuirena ciliaris (L.)Roxb. Paddy field Acidic soil and saline soilEriocaulon cinereum R.Br. Paddy field Acidic soil and saline soilScleria poaeformis Retz. Paddy field Acidic soil and saline soilScirpus juncoides Roxb. Paddy field Acidic soil and saline soilMonochoria vaginalis (Burm.f.) Presl Paddy field rich nitrogenAeschynomene spp. Paddy field Lack nitrogenFimbristylis miliacea (L.)Vahl Paddy field Lack nitrogen


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Table 5 Weeds as human house (Department of Industry extension,1989).

Weed species Weed in the crop usageImperata cylindrica (L.) Reauschell Fruit crop , non crop area RoofSaccharum spontaneum Linn. non crop area Wall

Table 6 Weeds as water purification (Department of Industry extension,1989).

Weed species Weed in the crop locationEichhornia crassipes (Mart.) Solms Canal Makkason swamp,Bangkok

Cyperus corymbosus Linn. Canal Lam Phakbia, Petchburi provinceScirpus grossus Linn.f. Swamp Lam Phakbia, Petchburi provinceTypha angustifolia L. Swamp Lam Phakbia, Petchburi province

Table 7 Weeds as soil erosion protection.

Weed species Weed in the crop location

Vetveria spp. non crop area, slope area Mountain slopes

Digitaria ciliaris (Retz.)Koel. Fruit crop, field crop, upland rice, vegetable, non crop area Roadside edge

Dactyloctenium aegyptium P.Beauv. Fruit crop, field crop, upland rice, vegetable Roadside edge

Chloris barbata (L.)Sw. Fruit crop, field crop, vegetable Roadside edge

Pennisetum polystachyon (L.)Schult. Fruit crop, field crop, non crop area Roadside edge

Pennisetum pedicellatum Trin. Fruit crop, field crop, non crop area Roadside edge

Pennisetum setosum (Swartz)L.C.Rich. Rubber crop, oil palm crop, non crop area Roadside edge

Chrysopogon aciculatus Trin. Fruit crop, non crop area Roadside edgeEleusine indica (L.)Gaertn. Fruit crop, non crop area Roadside edge

Table 8 Weeds as decoration of house and garden.

Weed species Weed in the crop Type of decoration

Pistia stratiotes L. Paddy field, swamp Put on the bowl or beautiful container

Salvinia cuculata Roxb. Fruit crop, swamp Put on the bowl or beautiful container

Echinochloa crus-galli Beauv. Paddy field Dry and decorate in the vastEragrostis tenella P.Beauv.ex Roem.et Schult Fruit crop, field crop Dry and decorate in the vast

Xyris indica L. Paddy field, non crop area Dry and decorate in the vast


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Table 9 Weeds as mulching materials.

Weed species Weed in the crop Part of plant

Imperata cylindrica (L.) Reauschell

Fruit crop, rubber crop, oil palm crop, non crop area

Cut at the ground level, cover on the soil to protect weed germination

Rottboellia cochinchinensis W.D.clayton Fruit crop , non crop area Mulching with whole plant in tobacco

seedbed ,red onion and garlic

Azolla pinnata R.Br. Paddy fieldGrow on the surface of water at 7 days after seeding or after transplanting, completely cover in 3 weeks

Lemna minor L. Paddy fieldGrow on the surface of water at 7 days after seeding or after transplanting, completely cover in 3 weeks

Table 10 Weeds as green fuel.

Weed species Weed in the crop method

Eichhornia crassipes (Mart.) Solms Swamp, canal, river Whole plant condense in a columnAeschynomene spp. Paddy field Whole plant condense in a column Mimosa pigra L. Non crop area Whole plant condense in a column

Table 11 Weeds as allelopathic substances.

Weed species Weed in the crop

Ammnania baccifera L. Paddy field Hyptis sauveolens Poit. Slope, non crop

Table 12 Weeds as animal feeds.

Weed species Weed in the crop Type of animals

Echinochloa colona (L.)Link Paddy field, field crop, fruit crop Cow buffalo hoarse

Eichhornia crassipes (Mart.) Solms Swamp, canal, river PigBrachiaria mutca (Forsk.)Stapf Fruit crop, non crop Cow buffalo hoarse

Brachiaria distachya (L.) Stapf Fruit crop, non crop Cow buffalo hoarse rabbitHerbivorous fish

Lemna minor L. Paddy field, swamp DuckHydrilla verticillata (L.f. )RoyLe Paddy field, canal PigDactyloctenium aegyptium P.Beauv. Fruit crop, field crop Cow buffalo hoarseIschaemum rugosum Salisb. Paddy field Cow buffalo hoarsePaspalum scrobiculatum L. Paddy field Cow buffalo hoarseHymenachne pseudointerrupta C.Muell Paddy field Cow buffalo hoarseIpomoea aquatica Forsk Paddy field PigAlternanthera philoxeroides (Mart.)Griseb.) Swamp Pig


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Table 13 Weeds as for fish and aquatic animals house.Weed species location

Eichhornia crassipes (Mart.) Solms Swamp, canal, river Alternanthera philoxeroides (Mart.)Griseb.) Swamp, canal, river Ottelia alismoides (L.)Pers. Fish aquariumHydrilla verticillata (L.f.) Royle Fish aquariumLimnophila heterophylla Benth Fish aquariumNajas graminea Del. Fish aquariumCeratophyllum demersum Linn. Fish aquariumIpomoea aquatica Forsk. Swamp, canal, river

Table 14 Weeds as the name of the places.

Thai name and weed species Crop Location name

kham(Saccharum arundinaceun Retz.) Non crop Nongkham, Bangkok

Jok(Pistia stratiotes L.)

Paddy field, swamp Nongjok, Bangkok

Phue(Scleria poaeformis Retz.) Paddy field Phue Amphur, Udonthani province

Yasai(Leersia hexandra Sw.) Paddy field Nongyasai Amphur,Suphanburi province

Yaplong(Hymenachne pseudointerrupta C.Muell) Paddy field

Yaplong Amphur,Petburi province: Yaplong school, NongYaplong temple Ratchaburi province:

Phakwaan(Marsilea crenata Presl.) Paddy field Nongphakwan home, Nakonratchasima

provincePhaktop(Monochoria hastate Solms Paddy field Phaktop home, Khon kaen province

Pangpuay(Ludwigia octovalis Hara) Paddy field Pangpuay canal, Ratchaburi province

Conclusions and Recommendations

1.There are many methods to utilize weed. The weed can be compiled for 16 purposed usage. There will be more than these ways for weed utilization. There are various methods to use the weeds, There are 27 weed species become traditional vegetables. The information can be done more than those references. 2. There were 63 species to be medicinal plants, They should have more than those references like the traditional vegetables. 3.The others purpose of weeds could be have a potential use, such as energy compensation, water purification, mulching material, decoration of house and garden. The study should be done from many references.


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References

Department of Medical Science. (1990). Traditional Medicinal Plants: Research and Development of Medicinal Plants. Ministry of Public Health. 199 p.

Department of Industry extension. (1989). The Products from Water Hyacinth. Homemade Industry. Ministry of Industry. 102 p.

Kumklang, A. and Wechwitan, P. (2005). Traditional Vegetables and Medicinal Plants vol.1. Siam Chareanpanich (Bangkok). 96 p.

Nantasomsaran, P., Nakornsri, K. and Ditchaiwong, C. (2009). Some Medicinal Weeds in Thailand. The ninth National Plant Protection of Thailand. Sunee Grand Hotel, Ubon Ratchathanee province. pp. 77-81.

Prachasuysoradej, W. 1999. Native Vegetables: North, Northeast, South area. Botany group, Botany and Weed Science Division. Department of Agriculture. 81 p.

Vongsaroj P., Nantasomsaran, P. and Nakornsri, K. (1999). Weed Utilization in Thailand. The 17 th Asian-Pacific Weed Science Society Conference, Bangkok, Thailand, p. 54.


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A General View of Weeds in Lowland Rice and Up-Land Crops in The South of Vietnam

Ho Van ChienSouthern Regional Plant Protection Center – PPD

Introduction

In recent decades, developmental physiology, genetics and molecular biology have helped biologists and researchers in particular have developed intensive contact with comparative morphology, taxonomy, systematics and phylogeny – have captured the human mind since earliest time. Since the beginning, farmers have been concerned with a special world of plants, the so-called “weeds”. When 10,000 years ago in the Middle East, man started to collect and grow wild plants, competition began between the cultivated “domestic” species and their wild companions. These weed populations escape absolute definition. Perhaps one can describe them as “plants growing in where and when they are not wanted”. This description implies that a plant species is only considered to be a weed under certain conditions, according to a subjective judgement. Although, according to our definition, any plant can be a weed, there are typical weeds characterized by special peculiarities promoting or maintaining their spread. They are very resistant and adaptable species which can grow in habitats created or modified by man. Their most important feature is their effective spread and survival under sometimes unfavorable conditions. Fortunately, weeds have kept their species diversity, despite intensive mechanical and chemical control. From the point of view of ecological interaction and genetic potential, it would be unfortunate to lose this treasure. Apart from their biological importance, their aesthetic richness is also fascinating.

Three books of publications of authors Toni J. Häfliger, Basel, editor Werner Püntener, Basel of CIBA-GEIGY – weeds are divided into three groups: Dicot weeds (copyright 1988) with 127 species from 13 families; Grass weeds (copyright 1981) with 137 species; Monocot weeds (copyright 1982) with two families: The Sedge family (Cyperaceae) and the Rush family (Juncaceae).

Recently, the book named “Common weeds in Vietnam” – second edition of authors: Suk Jin Koo, Yong Woong Kwon, Duong Van Chin and Hoang Anh Cung (2005) weeds are divided into three groups: Grasses (Poaceae), Sedges (cyperaceae) and Broadleaved weeds (66 families).

In the south of Vietnam, depending on the type of rice production system, farmers often contend with the same or similar weed species. These species are relatively small, but of great importance and includes many of the “world’s worst weeds”. Weeds undoubtedly cause great damage in rice cultivations and other crops. Weeds compete with crop plants for nutrients, water, and light, harbor other pests, reduce the quality of seed crops, increase production costs. Thus, needs to be developing and testing on-farm researches of weed control or management but there is no problem of developing resistance, no environmental pollution.

Rice growing areas and cultural methods of South VietnamThe largest areas for rice growing is in the Mekong River Delta. Most of rice varieties are

modern varieties with short growth durations. Direct seeding rice is popular in the south Vietnam, wherein around 85% with hand broadcast, 10% use of drum seeders and 5% transplanting. In the year 2011, total rice growing areas is around 4,521,685ha wherein Winter-Spring crop: 1,690,985ha; Summer-Autumn crop: 1,799,600ha; Autumn-Winter and Main crops: 1,031,100ha. There are more than 80 rice varieties with short growth durations are growing in the farmers practice of south Vietnam.

Current weed situations and common weedsVietnamese farmers well known about weeds compete with rice crops for sunlight, nutrients

and water, and when not controlled reduce yield. In addition to competitive yield loss, weed seeds can reduce rice quality and grade. Yield reductions as high as 46% caused by weeds in on-farm studies have been reported in weed-free plots (Chin and Sadohara, 1994). More than 400 weed species have been


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recorded in rice fields in Vietnam. The two most important families are the Poaceae and Cyperaceae, which constitute 42% of all weed species (total: 167). Other major families are Asteraceae (26 species), Scrophulariaceae (18 species), Fabaceae (14) species), Lythraceae (10 species), and Laminaceae (9 species) (Chin, 1995). In a survey of 197 farm sites in 11 provinces in the Mekong River Delta, the Echinochloa spp. complex was dominant species (higher than 70% of total plant density) at more than 50% of the sites surveyed, and was common (10-70%) at more than 35% of the rest sites. Leptochloa chinensis and Fimbristylis miliaceae were equally abundant at most sites and were ranked as the next most important weed species. Both were the most abundant at about 18% of the sites. Cyperus difformis was the dominant species at about 7% of the sites and was common at 41% of the sites. C. iria was much less. Weedy/red rice are an emerging pest in direct-seeded rice in Vietnam. In interviews of 4,397 respondents in 128 districts of 18 provinces, 64% of the farmers reported the presence of weedy/red rice in the rice fields. Compared with cultivated forms, these eco-types typically had a shorter duration, shuttered more easily. These eco-types have been postulated to have resulted from interspecific hybridization with O. rufipogon (Buu, 1998). Weed vary in their competitive effects with rice, rice grain yield reductions have ranged from 5-10 percent with barnyard grass (Echinochloa spp.) 20-30 percent with weedy/red rice. In direct-seeded rice, weeds are the major threat to crop protection. For major type of direct-seeding methods in rice practices in the south Vietnam are wet seeding, dry seeding, zero-tillage seeding, and submergence seeding. Infected weed area of rice growing area is around 5-7% meaning is about 75,000 – 100,000ha weed infestations per season. Chien et al., (1997) and Mai et al., (1998) reported that the quality of farm seed supplies is very poor, the average number of weed seeds per kilogram of rice seeds was 466, forty-seven-fold higher than the permitted national purity level. The corresponding number for weedy/red rice seed was 314 seeds per kg rice seed. Regulations on the level of impurity of weedy/red rice seeds in rice have not yet been established in Vietnam.

Of several weeds affecting rice production, the major weed complexes have been identified which require an integrated approach for their control. They are the complex formed by various Echinochloa species but two major species of them are Echinochloa crus-galli and Echinochloa colona. Leptochloa chinensis is too high populations on upland rice. Particular, the red/weedy rice complex occurred on both of low and upland rice. For the red/weedy rice: One of the major constraints to the production of rice in direct-seeded areas is the incidence of red rice, which is widespread all over the world. Crop losses due to red/weedy rice incidence may be as high as 60 percent where there is heavy field infestation (FAO, 2001). In the south of Vietnam, weedy rice infestation is highest under dry rice seeding followed by wet seeding and zero-tillage seeding. The most severe infestation is observed in Summer-Autumn. The typical characteristics of weedy/red rice, compared with popular modern rice varieties in south of Vietnam, are short duration, tall plants, weak culm, small seeds, easy shattering and red pericarp. The average yield loss due to weedy rice ranges from 15 to 17 percent. (Chin, D.V. and Mortimer, 1999). Barnyard grass: Echinochloa muricata, E. crus-galli is a variable annual species causing serious competition (Holm et al. 1977), (Abeysekera Anuruddhika, 2001). Asian sprangletop: Leptochloa chinensis: A strongly tufted annual or perennial grass, “associated with wetlands, swamps, or streams in open lowland regions. Can grow in heavy or light soils, along streams and watercourses, in marshy grounds and in lowland rice fields” (Holm et al., 1977).

Surveying of weed populations in five typical rice fields by weed infestations without herbicide control in Diem Hy village, Chau Thanh district, Tien Giang province we found out that Echinochloa crus-galli: 26 plants per sq.m. (4.9%), Leptochloa chinensis: 193.5 plants per sq.m. (36.6%), Cypreus difformis: 37.5 plants per sq.m. (7.1%), Fimbristylis miliacea: 249.3 plants per sq.m. (47.2%), and Ludwigia octovanlis: 22.3 plants per sq.m. (4.2%) at 45 DAS (SRPPC, 2011).

Besides, some common broad leaf species with their infestations are a concern for many farmers’ rice fields on lowland rice.

Trend of weed invasion by inter-trading, global warming by climate change

- Weed invasion by inter-tradingSince the invasive species frequently cause economic and ecological impact, invasion by exotic

plants is growing issue, contamination of weed seeds in agricultural products are the major causes of


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alien plant invasion, with the naturalization of deliberately introduced plants being the most common source of invasive plants. Grain trade is a major route of uninternational introduction of those weeds (Mack et al., 2000, Shimono & Konuma 2008). Some studies reported that various kinds of weed seeds were contained in imported grain seeds (Fay, 1990; Huelma et al., 1996). In Vietnam, very limited study has focused on understanding how many species are introduced the invasive plants. The economic losses associated with invasive plants such as water lettuce Pistia stratiotes, giant mimosa Mimosa pigra, water hyacinth Eichhomia crassipes, victoria grass Panicum repens and siam weed Chromolaena odorata in Vietnam were reported, not including damage to ecosystem function or loss of biodiversity, which also contribute to agricultural production (IUCN, 2006).

- Landscape ecologies and climate changeClimate change, diminished water resources, loss of rice or plant crop cultivations and other

changes in the future could have profound effects on the landscape ecology. Future research will need to address a number of issues regarding how to best rice in the background of these changes. How climate change will affect weed species in rice is not clear. Because flooding or drought we do not know which crop growing and weed species will be richness, whole community studies over multiple years will be necessary to understand the effects of changing climate in different environments. Shrinking irrigation water will certainly force farmers in many area to adopt irrigation practices that include periodic drying of the irrigated rice fields. Very strong increases in weed infestation were observed with both direct-seeding and poor water management. Therefore, research is needed to examine how changes in irrigation contribute to weed species richness and what measures might be available to mitigate the negative effects. When not enough water sources will also force farmers that now practice asynchronous cropping in many areas to shift to more water efficient synchronous cropping for brown plant hopper and weed management. Research is needed to determine how this change in planting scheme and how different intercropping manipulations or cropping rotations in weed management.

Herbicides for paddy field in the coming years

Weeds are at present the major biotic constraint to increased rice production of south Vietnam. The importance of their control has been emphasized in the past by various authors (De Data and Baltazar, 1996; Labrada, 1996). Chemical weed control has increased significantly over the past ten years. This is due to labor shortages, particularly in the Mekong River Delta, leading to an increased shift from transplanted rice to direct-seeding, with a subsequent increase in herbicide use. As has been stated in the past, Labrada (1996) shows that, although herbicide use has increased productivity, there are several weed problems that remain unsolved by the use of the herbicides commonly applied in rice cultivation.

Following Circular letter No. 10/2012/TT-BNNPTNT of Ministry of Agricultural and Rural Development, date: February 22, 2012 there were 195 active ingredient and 584 trade names of herbicides in Vietnam.

Weedy/red rice and other species, such as Echinochloa spp., Leptochloa chinensis and red/weedy rice, present the major weed problems for rice. The most popular methods of weed control are: i) field inundation at 4 DAS (giving 14.8-30.3% higher yields than with 12 DAS); ii) fields subjected to two rounds of rotovation (higher yields than with one round of rotovation, while zero tillage did not promise better yields for farmers); iii) use of herbicides (>82.2% of Vietnamese farmers), normally supplemented by hand weeding (85% of farmers); and iv) hand weeding, which takes about 150-200 work days/ha and is normally done at 10-15, 25-30 and 40-45 DAS (Chin, 2001).

Herbicide use in rice production has increased quickly in Vietnam over than last decade because of the increasing availability and rising cost of labor for hand weeding, lack labor in agriculture.

Herbicide resistanceIn the past two decades, evolution of newer herbicides provided wider user choice. Selection of

most promising prudent products intensified in use followed by genetically induced herbicide resistant crops. This is how the broad-spectrum herbicides created a great deal of impact on the stakeholders. The loss of herbicide effectiveness due to selection of herbicide-resistant weed populations has negative


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impact on farmers. Herbicide-resistance is the inability of a herbicide to effectively control a weed species that was previously controlled by the same herbicide. Herbicide-resistance is detected when a biotype within a weed species possessing a resistant trait increases in abundance while susceptible biotypes are controlled by use the same herbicide. The resistant trait is inheritable and therefore, is passed from one generation to the next. Once a herbicide-resistant population has been selected for, the likelihood of the weed population reverting back to a population dominated by the susceptible biotype is low. Resistant weed population become a serious constraint because it develops far faster (in 3 to 5 years) than the time and money investment on research, testing and registration for another newer chemical that meet modern environmental and health regulatory standards. As a result, herbicides with a new mode of action will not likely serve as a solution for herbicide-resistant weed populations.

For the red/weedy rice: Discussion and analysis regarding transgenic rice cultivars resistant to herbicides (HRC), particularly in the red/weedy rice in the south of Vietnam.

- There is no simple method for the control of weedy/red rice. Only through the integrated control approach can weedy/red rice infestation be effectively reduced.

- The main sources of weedy/red rice infestation are: rice seeds contaminated with weed seeds, and weedy/red rice seed bank in soil. Therefore, any control measure should be aimed at the reduction of infestation from these sources.

- In some countries the presence of weedy/red rice seeds is tolerated in rice seeds. However, experience in the control of this weed in countries that use advanced technologies shows that not even one single weedy/red rice seed should be tolerated in rice seeds.

- The reproduction of basic and foundation seed should be carried out in areas which are totally free of weedy/red rice infestation. Certified rice seeds should be free of weedy/red rice seeds.

- To date, the most efficient control measures have been those based on the combination of wet soil preparation (to bring about the emergence of weedy/red rice seeds), followed by the application of herbicide (e.g. glyphosate) over the weed stand and before rice seeding, and water management before and after seeding.

- Under upland and irrigation conditions, it is advisable to implement, whenever possible, minimum tillage or zero tillage combined with the use of non-selective herbicides. This practice is cheap and sustainable for rice farmers.

- The use of post-emergence herbicides in the process of land preparation needs improvement. It is necessary to find other chemical alternatives in order to avoid repeated use of the same chemical. This also applies to post-harvest application in rice areas.

- Within the context of integrated management, it is necessary to conduct regular studies of the behaviour of available rice cultivars in terms of: their ability to compete with weedy/red rice; life-cycle; and tolerance to submersion during flooding.

Agrochemical industry has founded an international body, Herbicide Resistance Action Committee (HRAC) primarily aiming to collect information and prepare a database on resistant biotypes (biotypes: Bubny, Jihlav, Karlín, Kochia - Kochia biotype belong to the C4, photo-synthesis class).

For types of resistance, herbicides target attack at one or more location in a weed plant. These locations can either be enzyme proteins, other non-enzyme proteins, cell division path etc. are called site of action. The generally focus of herbicide-resistance included interference to “ALS”=Acetolactate synthase (also called AHAS=Acetohydroxy acid synthase), ACCase inhibitors=Acetyl Co Enzyme-A Carboxylase, Synthetic auxins, “HRA”=Hill reaction activity, “PS (I)”=Photosystem I (activity), Photosystem II inhibitors, Ureas and Amides, Glycines, Chloroacetamide, Dinitroaniline, Thiocarbamates, Bipyridiliums, multiple resistance and weed-resistance to herbicide following these factors: weed biology, intensity of use, rate (incorrect), rain before herbicide is completely absorbed by the weed, drought or cold, no coverage of weeds below canopy, early morning applications, weed emerging after herbicide application.

In order to understand which species of weed resistance to what organo-chemical of herbicides and where the country is, look for in a handbook of “A Practical Field Guide to: Weeds of Rice in Asia”, IRRI publication, 2010.


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Application of GMO in weed managementThe risks of genetically modified crops: Some concerns that have been raised by scientists,

community groups and members of the public include:- Cross-breeding - other risks include the potential for cross-breeding between GM crops and

surrounding vegetation, including weeds. This could result in weeds that are resistant to herbicides and would thus require a greater use of herbicides, which could lead to soil and water contamination. The environmental safety aspects of GM crops vary considerably according to local conditions.

- Herbicide tolerant (HR) crops - the introduction of the Glyphosate resistant soybean in 1996 was the start of crops that gave farmers an opportunity to reduce the cost of their herbicide use. However, the increasing acreage of HR crops (such as soybean and canola) has resulted in an increase in the types of weeds that are now Glyphosate resistant (GR). These GR weeds may have a major environmental influence on crop production in years to come.

The extensive use of herbicides and insect resistant crops could result in the emergence of resistant weeds and insects. This has often occurred as a consequence of conventional herbicide and insecticide spraying. Several weed species have developed resistance to specific herbicides which are extensively used in combination with herbicide-resistant genetically modified crops. Insect-resistant Bt-crops similarly could lead to the emergence of Bt-resistant insects. The extent and possible severity of impacts of resistant weeds and insects are subject to continuing scientific investigation.

- Pest and weed resistance - Scientists agree that extensive long-term use of Bt crops and Glyphosate and Gluphosinate, the herbicides associated with herbicide tolerant (HT) crops, can promote the development of resistant insect pests and weeds. Similar breakdowns have routinely occurred with conventional crops and pesticides and, although the protection conferred by Bt genes appears to be particularly robust, there is no reason to assume that resistant pests will not develop. Worldwide, over 120 species of weeds have developed resistance to the dominant herbicides used with HT crops, although the resistance is not necessarily associated with transgenic varieties. Because the development of resistant pests and weeds can be expected if Bt and Glyphosate and Gluphosinate are overused, scientists advise that a resistance management strategy be used when transgenic crops are planted. Scientists disagree about how effectively resistance management strategies can be employed, particularly in developing countries.

Trend of weed management on these crops in the coming yearsWeeds undoubtedly cause great damage in agriculture. However, for definition of “weed” in

some situations such plants can be used by farmers to their advantage. They have learned to manage populations of weeds and to strike a balance between competition, soil protection and weeding costs.

Integrated weed management (IWM) is the control of weeds through a long-term management approach, using several weed management techniques such as: Physical control, Cultural control Chemical control, Biological control.

Physical control is the removal of weeds by physical or mechanical means, such as mowing, grazing, mulching, tilling, burning or by hand. The method used often depends on the area of weeds to be managed, what the land is used for, physical characteristics and the value of the land. It is important that, when using physical control, any item that can move from a weed-infested site to an un-infested site, such as machinery, vehicles, tools and even footwear, is cleaned free of weed seed before moving, to stop the spread of weeds to new areas. As with most control methods long-term suppression of weeds requires follow up weed prevention.

Cultural control is usually associated with farming systems, although some elements are relevant to landscape and bushcare practices. It largely involves manipulating farming practices to suppress weed growth and production, while promoting the development of the desired plant. The principles and techniques used to prevent weed spread are relevant to cultural control methods to limit the spread of weeds between different land areas.


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- Cultural control methods:Encourage the competitiveness of desired species that are more competitive and fast growing.

This suppresses weed growth by reducing access to available sunlight, nutrients and moisture and can include:

. Choose plant and crop species or cultivars that are naturally more competitive. This can include using plant species that suppress other plant species by the release of toxins.

. Use high quality seeds, as they are more likely to produce vigorous and competitive plants.

. Use shallow seeding techniques, where possible, to allow the desired species to grow above the soil surface more quickly.

. Ensure the desired plant is placed in the optimum growing environment.

. Use fertilizers in the optimal growth period to encourage rapid growth of the desired species.

. If possible grow nectar flowers on the bunds using “Ecological Engineering” for pest management and on the other hand, to avoid some weed species can grow up on the bunds following spread to the rice fields.

Make it hard for weeds to adapt to weed management techniques. Using the same land management routines year after year may result in weeds adapting to these practices. Some practices that make it hard for weeds to adapt and therefore reduce their spread and vigor include:

. Rotate crops: if a weed has adapted to grain crops continuously being sown, then alternating with a broadleaf crop will remove the environmental condition to which the weed has adapted.

. Rotate species with different seasonal and growing cycles.

. Rotate herbicides with different modes of action to help delay the development of herbicide resistance.

- Weed preventionPrevention is the most effective method of dealing with weeds. Once a weed has entered an area

and become established, eradication is far more expensive and it is likely that greater resources will be required to control its further spread and reduce its impact.

The first step in weed prevention, and the most cost effective means of managing weeds, is preventing the entry of new weeds into the country.

Early detection and eradication requires an awareness and understanding of the factors that favor the establishment and spread of weeds, and applying appropriate management practices that can prevent or reduce the risks.

- Chemical controlAlthough the use of chemicals is not always essential, herbicides can be an important and

effective component of any weed control program.In some situations herbicides offer the only practical, cost-effective and selective method of

managing certain weeds. Because herbicides reduce the need for cultivation, they can prevent soil erosion and water loss, and are widely used in conservation farming.

In some cases, a weed is only susceptible to one specific herbicide and it is important to use the correct product and application rate for control of that particular weed. Common mistakes include incorrect identification of the weed or using inappropriate products.

In most cases, weeds must be actively growing to be vulnerable to herbicide treatments.Herbicide resistance can also be an issue with some species.

- Biological controlPlants that have become weeds in Australia are rarely invasive and troublesome in their natural

range. This is often because natural populations are regulated by a variety of natural enemies such as insects and pathogens (disease-causing organisms like fungi and bacteria) that attack the seeds, leaves, stems and roots of a plant. If plants are introduced to a new region that does not have these natural enemies, their populations may grow unchecked to the point where they become so prevalent that they are regarded as weeds. It is critical that the biological control agents introduced into Vietnam do not become pests themselves. Considerable testing is done prior to the release of biological control agents to ensure they will not pose a threat to non-target species such as native and agricultural plants.


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Although in the long term, biological control can be cost effective and can reduce the need for less desirable management practices, not all weeds are suitable for biological control.

There have been several other successful biological control programs in Vietnam such as leaf beetle coconut – importation of parasitoid have been mass-rearing and released. There is also major research being undertaken on biological control for a number of other weed species.

- Procedure to import and release a biological control agentTo avoid such problems in future, the process for approving biological control agents is much

more rigorous now than it has been in the past.Before commencing the search for biological control agents, agreement needs to be sought

from the MARD–Vietnam, with initial application through the PPD to target the weed species for bio-control. The next step is to undertake host specificity testing. This is a requirement under the MARD and PPD. Once host specificity testing is completed, biological control agents must be assessed and meet the requirements of the Quarantine and the Environment Protection before approval can be given for their introduction and release in practice.

To meet the requirements of the Quarantine - Biosecurity assesses the risk regarding proposed importation and release of biological control agents. PPD - Biosecurity assesses the risk of release of exotic biological control agents via a risk analysis, based on the results of host specificity testing. Host specificity testing is required under the process to ensure that the agent will not damage native flora or agricultural stock or crops.

Advantages and Disadvantages of Weed ManagementBy using several techniques to control weeds you reduce the chance that weed species will

adapt to the control techniques, which is likely if only one technique is used. For example, if a herbicide is used over a long period of time, a weed species can build up a resistance to the chemical.

A long-term integrated weed management plan, that considers all available management control techniques or tools to control weeds, can be developed for a particular area. Any integrated weed management plan or strategy should focus on the most economical and effective control of the weeds and include ecological considerations.

The long term approach to integrated weed management should reduce the extent of weeds and reduce the weed seed stock in the soil. It should consider how to achieve this goal without degrading the desirable qualities of the land, such as its native ecology or agricultural crops.

The soil seed bank refers to the natural storage of seeds, often dormant, within the soil of most ecosystems. Many cases have been classified according to the longevity of their seeds in the soil seed bank. Seeds of transient species remain viable in the soil seed bank only to the next opportunity to germinate, while seeds of persistent species can survive longer than the next opportunity - often much longer than one year.

A study of weed seed banks in rice field cultivation of Go Cong district, Tien Giang province, (SRPPC. 2011). The results show that weed seed banks in surface soil layer (0-10 cm depth) with 65,681 plants per sq.m. and in lower surface soil layer (10-20 cm depth) with 22,272 plants per sq.m., wherein seven major weed species are: Leptochloa chinensis,Echinochloa crus-galli, Oryza sativa, Fimbristylis miliacea, Cyperus difformis, Ageratum conyzoides and Hedyotis corymbosa.

Longevity of seeds is very variable and depends on many factors; In typical soils the longevity of seeds can range from nearly zero (germinating immediately when reaching the soil or even before) to several years. The mortality of seeds in the soil is one of the key factors for the persistence and density fluctuations of plant populations, especially for annual plants. There are indications that mutations are more important for species forming a persistent seed bank compared to those with only transient seeds. The increase of species richness in a plant community due to a species-rich and abundant soil seed bank is known as the storage effect.


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