rajasthan agricultural university, bikaner...sri karan narendra agriculture university, jobner...

Natural and Chemical Induced Resistance against Sclerotinia Rot of Indian Mustard

[Brassica juncea (L.) Czern & Coss]

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Thesis

Submitted to the

Sri Karan Narandra Agriculture

University, Jobner

In partial fulfillment of the requirement

for the degree of

Master of Science

In the Faculty of Agriculture

(Plant Pathology)

By

Arjun Lal Yadav

2014

Sri Karan Narendra Agriculture University, Jobner

S.K.N. College of Agriculture, Jobner

CERTIFICATE-I

Dated :_______2014

This is to certify that Mr. Arjun Lal Yadav has successfully

completed the comprehensive examination held on ----------2014 as required

under the regulation for Master’s degree.

(K.S. SHEKHAWAT) Professor & HEAD

Department of Plant Pathology S.K.N. College of Agriculture,

Jobner



CERTIFICATE-II

Dated :________2014

This is to certify that the thesis entitled “Natural and Chemical

Induced Resistance against Sclerotinia Rot of Indian Mustard

[Brassica juncea (L.) Czern & Coss]” submitted for the degree of Master

of Science in the subject of Plant Pathology embodies bonafide research

work carried out by Mr. Arjun Lal Yadav under my guidance and

supervision and that no part of this thesis has been submitted for any other

degree. The assistance and help received during the course of investigation

have been fully acknowledged. The draft of the thesis was also approved by

the advisory committee on 09.12.2014.

(K.S. Shekhawat)

Professor & HEAD (R.P. Ghasolia) Department of Plant Pathology Major Advisor S.K.N. College of Agriculture, Jobner

(G.L. KESHWA) DEAN




CERTIFICATE-III

Dated :______2014

This is to certify that the thesis entitled “Natural and Chemical

Induced Resistance against Sclerotinia Rot of Indian Mustard

[Brassica juncea (L.) Czern & Coss]” submitted by Mr Arjun Lal Yadav to

Sri Karan Narendra Agriculture University, Jobner, in partial fulfillment of the

requirements for the degree of Master of Science in the subject of Plant

Pathology after recommendation by the external examiner, was defended

by the candidate before the following members of the examination

committee. The performance of the candidate in the oral examination on his

thesis has been found satisfactory. We therefore, recommend that the thesis

be approved.

(R.P. Ghasolia) (A.C. Mathur) Major Advisor Advisor

(K.C. Kumawat) (K. Ram Krishna) Advisor Director Education Nominee (K.S. Shekhawat) Professor & HEAD (G.L. KESHWA) Department of Plant Pathology Dean S.K.N. College of Agriculture, S.K.N. College of Agriculture, Jobner Jobner Approved

DIRECTOR EDUCATION S.K.N. Agriculture University, Jobner



CERTIFICATE-IV

Dated : _____ 2014

This is to certify that Mr Arjun Lal Yadav of the Department of Plant

Pathology, S.K.N., College of Agriculture, Jobner has made all corrections/

modifications in the thesis entitled “Natural and Chemical Induced

Resistance against Sclerotinia Rot of Indian Mustard [Brassica juncea

(L.) Czern & Coss]” which were suggested by the external examiner and

the advisory committee in the oral examination held on ______2014. The

final copies of the thesis duly bound and corrected were submitted on

______2014 and forwarded herewith for approval.

(R.P. Ghasolia) Major Advisor

(K.S. Shekhawat) PROFESSOR & HEAD

Department of Plant Pathology S.K.N. College of Agriculture, Jobner

(G.L. KESHWA)

DEAN S.K.N. College of Agriculture, Jobner

APPROVED

DIRECTOR EDUCATION SKNAU, Jobner

ACKNOWLEDGEMENT

In the ecstasy. I express my esteem and profound sense of gratitude to Dr. R.P.

Ghasolia, major advisor, Asstt. Professor, Department of Plant Pathology, S.K.N. College

of Agriculture, Jobner, for his valuable and inspiring guidance, constant encouragement

and keen interest during the course of present investigation and preparation of this

research report.

I convey the feeling of gratitude to the members of my advisory committee Dr. A.C.

Mathur, Professor, Deptt. of Plant Pathology, Dr. K.C. Kumawat, Professor, Deptt. of

Entomology and Dr. K. Ram Krishna, Professor, Deptt. of Plant Breeding & Genetics,

Dean P.G. Nominee for the help rendered during the course of investigation.

I also deep and heartful greatfulness to Dean, S. K. N. College of Agriculture, Jobner

and Dr. K.S. Shekhawat, Head , Deptt. of Plant Pathology for providing necessary

facilities during the course of investigation.

I am again ecstatic to express my inherent sense of gratitude to Dr. R.R. Ahir, Dr. S.

Godika, Dr. Mahaveer Choudhary and all the staff members, Deptt. of Plant Pathology,

S.K.N. College of Agriculture, Jobner whose cooperation made this investigation smooth

and easy.

Down the memory lane. I am very much thankful to my dear friends Sajjan, Dinesh,

Ganpat, Ramchandra, Pawan my seniors Rekha ji, Sanju ji, Mukesh ji, Suresh ji, Gopal ji

and my lovely juniors Jitendra, Vishambar, Sukh Lal, Roshan, Rajesh, Raj kumar,

Pramod and Suresh with whom I went hand in hand during my study and research work.

My vocabulary falls short to express heartiest regards to my grandfather Sh.

Bansidhar Yadav, grandmother Smt. Mangli Devi, father Sh. Laxmi Narayan, lovely

mother Smt. Mohani Devi, Jiju Bhadhaurji, elder brother Mr. Dinesh, Rajesh and

Ranjeet Yadav, elder sisters Hansa di, younger bro. Mahesh, Rakesh, Ajay, Vinod, Ashok,

Mahendra and Shivam younger sis. Kanchan, Komal, Bhabhi Shakhshi and Lalita and

lovely nephew Aditya and Tanish, whose consistent encouragement and blessing are

beyond my expression that brought me here up to dream without which it could not have

been sketched.

I am also grateful to Sh. Shanker Yadav of Shivam Computer Centre, Jobner, who

typed this manuscript.

Last but not the least, a million of thanks to God. The Almighty who made me to this

task and made every job a success for me.

Place: Jobner

Dated: (ARJUN LAL YADAV)

CONTENTS

CHAPTER NO.

PARTICULARS PAGE NO.

1.

INTRODUCTION

………..

2. REVIEW OF LITERATURE ………..

3. MATERIALS AND METHODS ………..

4. RESULTS ………..

5. DISCUSSION ………..

6. SUMMARY ………..

LITERATURE CITED ………..

ABSTRACT (English) ………..

ABSTRACT (Hindi) ………..

LIST OF TABLES

Table No.

Particulars Page No.

3.1 Different SAR activators and their concentrations ……

3.2 Plant extracts and their concentrations

4.1 Biochemical changes in leaves of healthy and infected

(S. sclerotiorum) plants of Indian mustard varieties

……

4.2 Efficacy of SAR activators against Sclerotinia

Sclerotiorum by poisoned food technique after 7 days

of incubation at 25 + 1 0C

……

4.3 Efficacy of SAR activators against Sclerotinia rot of

Indian mustard

……

4.4 Fungitoxicity of different oil cakes against Sclerotinia

sclerotiorum by poisoned food technique after 7 days

of incubation at 25 + 1 0C

……

4.5 Fungitoxicity of different plant extracts against

Sclerotinia sclerotiorum by poisoned food technique

after 7 days of incubation at 25 + 1 0C

……

LIST OF FIGURES

Figure No.

Particulars Between Page No.

4.1 Biochemical changes (total soluble sugar) in leaves of

healthy and infected (S. sclerotiorum) plants of Indian

mustard varieties

……

4.2 Biochemical changes (total protein content) in leaves

of healthy and infected (S. sclerotiorum) plants of

Indian mustard varieties

……

4.3 Biochemical changes (phenol content) in leaves of

healthy and infected (S. sclerotiorum) plants of Indian

mustard varieties

…….

4.4 Efficacy of SAR activators against Sclerotinia

Sclerotiorum by poisoned food technique after 7 days

of incubation at 25 + 1 0C (in vitro)

…….

4.5 Efficacy of SAR activators against Sclerotinia rot of

Indian mustard (in vivo) …….

4.6 Fungitoxicity of different oil cakes against Sclerotinia

sclerotiorum by poisoned food technique after 7 days

of incubation at 25 + 1 0C (in vitro)

…….

4.7 Fungitoxicity of different plant extracts against

Sclerotinia sclerotiorum by poisoned food technique

after 7 days of incubation at 25 + 1 0C (in vitro)

…….

LIST OF PLATES

Plate

No.

Plate Between

Page No.

1 Pathogenicity test ……

2 Efficacy of different SAR activators against S.

sclerotiorum (in vitro)

…….

3 Fungitoxicity of different oil cakes against S.

sclerotiorum(in vitro)

…….

4 Fungitoxicity of different plant extracts against S.


…….

CHAPTER-1 INTRODUCTION

Indian mustard [Brassica juncea (L.) Czern & Coss] is an important oilseed crop. It

belongs to family (Brassicaceae) Crucifereae. Indian mustard is natural amphidiploids

having 36 chromosomes (2n). It is self-pollinated but certain amount of pollination (2-15%)

occurs due to insects and other factors.

The largest cultivation of Brassica crops is done for edible vegetable oil production.

They also play a pivotal role in world’s agricultural economy and are recognized for their

long history of cultivation and varied uses. They are widely cultivated throughout the world

as condiments and spices for improved flavour of human diet and as fodder crop for

livestock feeding. The oilseed Brassica crops are generally grouped as rape and mustard.

Commonly cultivated species are B. compestris var. Sarson Prain. Yellow and brown

sarson; B. campestris var. toria; B. juncea Czern and Coss; rai and Eruca sativa Lam.:

taramira. They are second most important oilseed crops of India after groundnut in terms of

area and production. Indian mustard [Brassica juncea (L.) Czern and Coss] is the most

important in Indian subcontinent because of its relative tolerance to biotic and abiotic

stresses and inherent high yield potential. The black and brown seeds possess about 40

per cent oil content. The oil is mainly used for direct human consumption. However, its use

in different industries can’t be rules out. After the extraction oil, the cake containing over 40

per cent high quality protein is utilized as a feed for livestock and poultry and also as

organic manure for different field and plantation crops.

India is the third largest country in edible oil economy after USA and China. In

respect of rapeseed and mustard production, the country commands a premier position with

an annual production of 7.8 million tonnes from 6.5 million ha area (Anonymous, 2013-14a).

Rapeseed and mustard crops are extensively grown in northern and western parts of India

viz., Rajasthan, Uttar Pradesh, Madhya Pradesh, Haryana, Gujarat, West Bengal, Assam,

Bihar, Punjab and Jammu & Kashmir. Among these, Rajasthan state ranks first both in area

and production i.e., 2.78 million ha and 3.62 million tonnes, respectively with an average

productivity of 1301 kg ha-1

(Anonymous, 2013-14b). In Rajasthan, Tonk district ranks first

both in area and production (2,66,482 lakh ha and 3,85,847 lakh tonnes, respectively)

followed by Sriganganagar, Alwar, Bharatpur and Hanumangarh (Anonymous, 2013-14b).

The problem of diseases and pests is the most important factor causing yield

instability in rape and mustard. Many diseases have so far been reported from different

parts of the world. Among these, diseases caused by fungi take heavy toll of the crop.

Some economic important diseases of rape and mustard in India are leaf spot and blight

(Alternaria brassicae and Alternaria brassicicola), white rust (Albugo candida), downy

mildew (Peronospora brassicae), powdery mildew (Erysiphe cruciferarum), root gall smut

(Urocystis brassicae), stem rot (Sclerotinia sclerotiorum), Rhizoctonia leaf blight

(Rhizoctonia solani), black leaf canker (Leptosphaeria maculans), grey mold (Botrytis

cinerea), Fusarium wilt (F. oxysporum f. sp. conglutinans), Macrophomina wilt (M.

phaseolina), damping off (Pythium butleri), black rot (Xanthomonas campestris pv.

campestris) and mosaic (Saha et al., 1989).

Sclerotinia sclerotiorum (Lib.) de Bary, is a ubiquitous, omnivorous, soil borne and

destructive plant pathogen, inciting disease on more than 400 plant species (Boland and

Hall, 1994). The fungus is an Ascomycete, in the order Helotiales and family

Sclerotiniaceae, characterized by the formation of hard blackish sclerotia, which on

germination produce cup-shaped brown coloured apothecia.

Stem rot or stem blight or white blight or white rot of Indian mustard incited by

Sclerotinia sclerotiorum (Lib.) de Bary is an economically important yield reducing disease

that has been widely reported in the last few years in India and elsewhere. It was earlier

considered to be a disease of minor importance but it has now become a serious disease of

crucifers in some parts of India like Rajasthan, Haryana, Madhya Pradesh and Bihar

(Aggarwal et al., 1997). The disease is monocyclic and symptoms usually appear four to six

weeks after initial infection or at post-flowering stage, when significant damage has already

been done to the crop. Sudden drooping of leaves followed by drying of plants area

characteristics features of the disease. Hyphae from myceliogenically germinated sclerotia

infect basal part of stems causing stem rot. Air-borne ascospores produced by carpogenic

germination of sclerotia during periods of high moisture and favourable temperature

conditions are the major source of inoculums for primary infection in the standing crop. The

petals act as a substrate for ascospore-bearing petals are deposited on leaves. White

cottony, fungal growth is visible near collar portion of the stem with black sclerotial bodies

measuring from 2-20 x 3-7 mm on and inside the stem. When the infected stem of the host

is splitted, large cavities lined by fluffy mycelial growth and numerous black sclerotia of the

fungus are seen which are the main structure responsible for fungus to overwinter and

disseminate.

This disease is gaining importance in the raya-growing areas, which may lead to

disastrous crop failure as the disease incidence was recorded up to 73.8 per cent in some

districts of Punjab and Harayana and at a few locations it went up to 80 per cent (Kang and

Chahal, 2000 and Sharma et al., 2001). Maximum incidence was recorded at Dausa

(29.2%) district in Rajasthan followed by Rohtak (24.8%) districts in Haryana and minimum

7.0 and 7.4 per cent at Jaipur and Bharatpur districts, respectively (Yadav et al., 2013).

Control of this disease mainly depends on fungicides (Tripathi and Tripathi, 2010).

However, fungicidal applications cause hazards to human health and increase

environmental pollution. Induction of host resistance through chemical inducers and use of

resistant cultivars are the effective solutions to overcome the problem. Therefore, keeping

in view all these facts, the investigations were carried out with the following objectives.

(i) To know the chemical basis of susceptibility and resistance in Indian mustard

cultivars against Sclerotinia sclerotiorum.

(ii) To evaluate resistance inducer chemicals against Sclerotinia sclerotiorum in vitro.

(iii) To elicit systemic acquired resistance by chemical inducers against Sclerotinia

sclerotiorum.

(iv) To test the fungitoxicity of organic amendments and phytoextracts against

Sclerotinia sclerotiorum

CHAPTER-2

REVIEW OF LITERATURE

In the beginning, the pathogen was described for the first time from Belgium by

Libert (1837) as Peziza sclerotiorum Libert. Later, it was transferred to a new genus

Sclerotinia by Fuckel (1870) who changed the specific name to Sclerotinia libertiana Fuckel.

Later, de Bary (1884) changed the binomial of Peziza sclerotiorum to Sclerotinia

sclerotiorum. Saccardo (1884) retained the genus Sclerotinia Fuckel. But, Wakefield (1924)

showed it to be inconsistent with the International Rules of Botanical Nomenclature and

cited G.E. Massee as the proper authority for S. sclerotiorum (Lib.) Massee, because he

had used that binomial in 1885, but de Bary used it in his contribution in 1884. Therefore,

the proper name and authority for the fungus seems to be Sclerotinia sclerotiorum (Lib.) de

Bary. Whetzel (1945) described S. sclerotiorum as type spices of Sclerotinia and named 14

other spices, which included S. trifoliorum and S. minor as members of the genus

Sclerotinia. Korf and Dumont (1972), however, suggested the name Whetzelinia in honour

of Professor Whetzel but the special committee of International Association of Plant

Taxonomist recommended that the generic name Sclerotinia be used (Kohn, 1979).

In India, Sclerotinia stem rot reported for the first time by Shaw and Ajerakar (1915)

on several hosts including rapeseed and mustard.

Sclerotinia sclerotiorum reported to be one of the most omnivorous plant pathogen.

It has wide host range that includes 64 families, 225 genera, 361 species and 22 other

cultivars for a total of 383 speceis (Purdy, 1979).

From Rajasthan, the pathogen S. sclerotiorum was first reported on gaillardia (Rai

and Agnihotri, 1970) from Jobner. Later on, it was reported on fennel, potato, sunflower,

pea, brinjal, mustard etc. from other parts of the state (Sehgal and Agrawal, 1971 and Singh

and Agrawal, 1989).

Morrall et al. (1976) reported infection level of Sclerotinia rot up to 62% on Brassica

in Saskatchewan, Canada. Roy and Saikia (1976) reported an outbreak of S. sclerotiorum

from Assam in mustard under cool and wet environmental conditions. Infection at early

growth stages resulted in failure of the crop. Gladders et al. (1991) reported Sclerotinia

epidemics in winter rape oilseed in England and Wales, where the infection level was up to

46%. From trace to 50% losses in seed yield vary with poor quality and quantity of oil has

been reported by Singh (1998) due to Sclerotinia stem rot on mustard.

Management of soil borne pathogen is very difficult but various methods of disease

control viz. chemical, biocides, cultural practices and host plant resistance etc. may reduce

disease incidence.

2.1 To know the chemical basis of susceptibility and resistance in Indian mustard

varieties against Sclerotinia sclerotiorum.

Yarwood (1957) found that bean plants (Phaseolus vulgaris) infected

with (Uromyces phaseoli) showed high accumulation of carbohydrates

around the rust lesions.

Imman (1962) reported that infection of great northern strain 59 bean

(Phaseolus vulgaris) with Uromyces phaseoli typical race 3 resulted in an

increase about two to three folds in soluble sugars and starch, accompanied

by qualitative changes.

Mohamed et al. (1976) found that stems of wheat and barley cultivars

showed the maximum amounts of total carbohydrates in the milk stage then

decreased with the exception of tosson variety in which the maximum

amount was in the tillering stage. Inoculation with stem rust pathogen

accounted to decrease in the amounts of total carbohydrates compared

with uninoculated healthy plants. This decrease was more pronounced in

moderate susceptible or susceptible cultivars than in resistant or moderate

resistant cultivars especially in the milk stage when infection with stem rust

is usually severe.

Basyouni et al . (1976) found that plants susceptible to wheat leaf rust contained

higher level of total phenols, however, the free forms of phenols were higher in amount and

or proportion in the resistant plants. They added that the phenolic level required for

resistance varied from one entry to another and against one race to another. Therefore,

plant phenols and particularly the free forms, were a critical factor in determining adult plant

resistance in wheat to leaf rust.

Helal et al. (1978) found that resistant cucumber variety poinsettia

contained low level of total soluble sugars which prevents further

establishment of Erysiphe cichoracearum within host tissues of the infected

leaves. They also found higher amount of preformed phenols which hinder

fungal infection in the same resistant variety.

Rai et al. (1980) screened cultivars against S. sclerotiorum and the most resistant

and susceptible were selected for study. Healthy stems of the former contained relatively

more phenolics than those of the latter. In infected stems phenolics accumulated at the

infection site in the resistant and there was a relatively low degree of enzyme activity

compared with that in susceptible. The pH was also lowered but to a lesser extent in

resistant than in susceptible infected tissues.

Dhawan (1980) isolated four isolates of S. sclerotiorum and tested on two cultivars

of Chinese mustard which produced significant amount of protease. Protease activity was

greater in the susceptible than in the resistant cultivar.

Kiessling and Hoffmann (1985) reported that inhibition of

photosynthesis, accumulation of starch, glucose and sucrose around the site

of infection until the end of the latent period by infection of rust pathogen on

bean and other crops.

Manners (1989) revealed that sucrose is one of the most important

carbohydrates in plants especially during host parasite interaction as a

transport for carbohydrates satisfying the requirements of leaf and leaf-

borne pathogens.

Tetlow and Farrar (1992) demonstrated a decrease in sucrose

phosphate synthase in Hordeum distichum after infection with Puccinia

hordei. Infection promoted hydrolysis of sucrose to glucose and fructose to

starch.

Scholes et al. (1994) stated and verified the alterations in

carbohydrate status during infection with obligate fungi. Changes in

carbohydrates in the early stages of infection might influence the regulation

of the Calvin cycle.

Wagner and Boyle (1995) investigated the changes in carbohydrate metabolism

and senescence during the development of the bean-rust fungus, Uromyces appendiculatus

(Pers.) Link at different stages of urediospore and teliospore production. Samples were

taken from infected and non-infected bean leaves beginning with the flecking stage. The

unsoluble carbohydrate, sucrose, glucose and fructose content, as well as fluctuations in

soluble acid invertase activity and parameters of leaf senescence were determined. While

fructose remained constant without significant differences between infected leaf sections

and the control. Differences in the other carbohydrates and in soluble invertase activity

could be detected. Parameters indicating leaf senescence were retarded and decreased

with the age, except for peroxidase activity which was enhanced and increased with the age

in infected tissue to a higher degree than in the control .

Lee et al. (2000) compared two Glycine max near-isolines, GL2415 (glyphosate

sensitive) and GL2600RR (glyphosate resistant) for susceptibility to Sclerotinia

sclerotiorum. Results from these studies indicated no differences in fungal growth, disease

development or G. max yield.

Starzycka et al. (2000) reported resistance of 78 top yielding strains of bean against

S. sclerotiorum by inoculating 1551 plants using the “pin-up” method in a field experiment

conducted in Borowo, Poland. They recognized the most resistant (PNG 2170, MA 1615-1,

MZL 236, BK 2466/93, MA 1649-1) and the most susceptible strains (MA 0742-2, PN

610z/98, MZL 189, MZL 201, PN 9011AB/97).

Abd-EL-Magid et al. (2004) revealed that citric acid, tartaric acid, sodium citrate,

sodium salicylate, sodium benzoate and ammonium tartarate decreased the radial growth

of Sclerotium cepivorum Berk, and decreased number of sclerotia. The chemical analysis of

healthy or infected onion bulbs or garlic cloves indicated a higher phenolic content in

healthy onion bulbs than in the infected ones and higher amounts of the amino acids in

healthy samples than infected ones both in onion and garlic.

Ghasolia and Shivpuri (2005) screened 32 rapeseed and mustard genotypes for

resistance to Sclerotinia stem rot (Sclerotinia sclerotiorum) under artificial inoculation

conditions in the field for three consecutive cropping seasons. Among them, eight (Hyola-

401, PBN-9501, PWR-9541, Kiran, RH-9401, RH-492, RW-8410 and PAB-9511) showed

resistant to moderately resistant reaction to S. sclerotiorum.

Guox and Stotz (2007) reported that genotypic differences in susceptibility of

Arabidopsis thaliana to Sclerotinia sclerotiorum have not been reported due to the extreme

susceptibility of this cruciferous plant. To overcome this limitation, two coi1 mutant alleles

conferred hypersusceptibility to S. sclerotiorum. The plant defensin gene PDF1.2 was no

longer induced after challenging the coi1-2 mutant with S. sclerotiorum. Hypersusceptibility

of the coi1-2 mutant to S. sclerotiorum was not correlated with oxalate sensitivity. The

mutants npr1 and ein2 were also hypersusceptible to S. sclerotiorum. Induction of PDF1.2

and the pathogenesis-related gene PR1 was reduced in ein2 and npr1 mutants,

respectively. Actigard, a commercial formulation of the systemic acquired resistance

inducer benzothiadiazole, reduced susceptibility to S. sclerotiorum. Based on histochemical

analysis of oxalate-deficient and wild-type strains of S. sclerotiorum, oxalate caused a

decrease in hydrogen peroxide production but no detectable changes in plant superoxide

production or gene expression.

Jing-Fan et al. (2013) reported that Chinese cabbage (Brassica campestris sp.

chinensis Makino) varieties (003-R-6, Y3-R-4, 109-S-9 and 003-S-7) with different

resistance to Sclerotinia sclerotiorum were selected and used to analyze the physiological

and biochemical changes such as the content of malondialdehyde (MDA), free amino acid

(FAA), the activity of chitinase and beta -1, 3-glucanase. The result showesd that increase

of MDA, FAA in the susceptible variety 003-S-7 was higher than that of resistant variety

003-R-6. On the contrary, the increase of chitinase and beta -1, 3-glucanase activity in the

resistant varieties was higher than that of the susceptible ones.

2.2 To evaluate resistance inducer chemicals against Sclerotinia sclerotiorum in

vitro

Shahda (2000) reported that benzoic acid, salicylic acid and ascorbic acid

significantly reduced the linear growth of the three fungi and spore germination of Fusarium

spp. at 20 mM. The antioxidants used exhibited growth promoting effect where they

increased shoot and root length and dry weight. The three antioxidants significantly reduced

damping off when used as soil drench at 20 mM. The antioxidants were superior to Rizolex-

T either in controlling the disease or in promoting the plant growth.

EL-Ganaieny et al. (2002) evaluated antioxidants like aminobutyric acid (ABA),

potassium salicylate (PS), oxalic acid (OA), salicylic acid (SA) and ascorbic acid (AA) for

their effect on the Fusarium oxysporum, F. solani and F. moniliforme. Mycelial growth was

reduced by the antioxidants. Spore germination was greatly reduced by many tested

chemicals at 8 mM concentration or less. The inhibitory effect of the antioxidants increased

with increasing concentrations. ABA was the only antioxidant which induced protection in all

onion cultivars against all tested Fusarium species.

Hilal et al. (2006) performed a laboratory experiment to evaluate the

antifungal activity of Bion, chitosan, oxalic acid and salicylic acid at 5

concentrations against Sclerotinia sclerotiorum on PDA. All of the

concentrations of the tested compounds significantly reduced linear growth

of S. sclerotiorum compared with the control.

Abdel-Monaim et al. (2012) reported the effectiveness of some chemical inducers

viz. ethephon, hydrogen peroxide (H2O2), mannitol, salicylic acid at three different

concentrations (50, 100 and 200 ppm) on root rot and wilt. All tested chemical inducers

were significantly reduced mycelia linear growth of all tomato root rot and wilt tested fungi

compared with control. The highest decrease in linear growth was noticed with ethephon at

concentration 200 followed by SA at 200 ppm.

Rahamah et al. (2012) evaluated the effect of single and combined application of

calcium ion (Ca2+

), copper ion (Cu2+

) and salicylic acid (SA) on growth and sporulation of

Ganoderma boninense. In poison medium test, T7 (Ca+Cu+SA) showed effective control of

G. boninense in vitro.

2.3 To elicit systemic acquired resistance by chemical inducers against


Shalaby et al. (2001) revealed that the efficacy of helichrysum flower extract, castor

bean oil, aminobutryic acid, potassium salicylate, oxalic acid and salicylic acid in controlling

Macrophomina phaseolina on sesame and sun flower was investigated in laboratory,

greenhouse and field. The evaluated compounds, except helichrysum flower extracts, did

not inhibit the growth of M. phaseolina in vitro. In the greenhouse, all treatments reduced

M.phaseolina incidence and increased plant height, peroxidase activity and IAA content of

sesame and sunflower plants.

Reglinski et al. (2001) stated that salicylic acid (SA) and pre-inoculation with

Sclerotinia sclerotiorum, and the endogenous SA and phenylalanine ammonialayse (PAL)

activity during the period between pathogen inoculation and the onset of systemic

resistance, were investigated under field conditions in Newzealand. Pretreatment of leaves

with SA or pre-inoculation with S. sclerotiorum induced resistance to a challenge inoculation

with S. sclerotiorum 4 days later. Total SA level in the inoculated and Inoculation-1 leaves

did not increase significantly 96 h after inoculation. PAL activity started to increase in the

inoculated leaf after 48 h post-inoculation, and by 96 h was 7 fold greater than the basal

level, while no change was observed in Inoculation- 1 and Inoculation +1 leaves.

Pankaj and Choudhary (2001) tested cupric chloride, ferric chloride, lithium sulfate,

manganese sulfate and sodium molybdate against Sclerotium rot of chickpea. Ferric

chloride and manganese sulfate were the most effective and sodium molybdate was the

least effective. In such effective treatments, the infected host tissue showed marked

reductions in polygalacturanase activity and oxalic acid contents and also moderate

increase in calcium and magnesium levels.

Application of acetyl salicylic acid, amino isobutyric acid, IAA, DL-beta amino-

butyric acid and salicylic acid resulted in lesion-free leaves 72 h after inoculation with

Rhizoctonia solani. Seed and root tip treatment and foliar spray with gamma- amino n-

butyric acid, DL-beta-amino-butyric acid and isonicotinic acid, respectively, gave the highest

control of the disease (Dantre et al., 2003).

Molloy et al. (2004) measured the susceptibility of carrots to Sclerotinia

sclerotiorum after treating with chitosan hydrolysate (0.2%). Sclerotinia infection of carrot

was declined after treating with chitosan hydrolysate due to induced host resistance.

EL-Mougy et al. (2004) revealed that some salts as potassium chloride

,monopotassium sulphate and potassium sulphate are reported to have antifungal activities

against several fungi also used salicylic acid (SA) or acetyl salicylic acid (ASA) as seed

dressing or soil drench which reduced root rot infection of lupine plants under green house

conditions . Furthermore resistance against plant pathogens can be also increased by

salicylic acid or acetyl salicylic acid.

EL-Mougy (2004) evaluated salicylic acid (SA) and acetylsalicylic acid

(ASA) in addition to Rizolex-T as seed dressing or soil drench against lupin

root rot pathogens under greenhouse conditions. No significant reduction in

seed germination was observed when lupin seeds were treated with SA and

ASA up to 2 and 3 g/ kg, respectively. Raising the applied dosages has

reflected negatively on seed germination. SA, ASA and Rizolex-T as seed

dressing or soil drench at the rate of 2, 3 and 3 g/ kg, respectively, have

significantly reduced the percentage of root rot incidence at both pre- and

post –emergence stages of lupin plants growth comparing with untreated

control in artificially infested soil with Fusarium solani, Rhizoctonia solani

and Sclerotium rolfsii. He suggested the use of SA and ASA as seed

dressing or soil drench for controlling such soil borne diseases.

Basha and Chatterjee (2007) reported that foliar application of plant growth-

promoting rhizobacterial strains, i.e. Pseudomonas fluorescens and P. aeruginosa, and two

non-conventional chemicals, i. e. zinc sulfate (10-3

mM) and oxalic acid (4 mM), induced

synthesis of phenylalanine ammonia-lyase (PAL) in wheat. Increased PAL activity followed

by accumulation of different phenolic acids demonstrated that activation of the

phenylpropanoid pathway was one of the reasons for resistance in wheat against

Sclerotinia sclerotiorum and thereby serves as a non-host. Greater accumulation of

phenolics due to increased PAL activity offered protection against diseases (Grey et al.,

1997; Singh et al., 2002, 2003).

EL-Bana (2007) tested antioxidants as seed treatment to induce resistance in chick

pea plants against Sclerotinia sclerotiorum infection. Ability of antioxidants to induce

resistance varied with chickpea cultivars. Malic acid seed treatment was the most

resistance inducer followed by salicylic acid and cumaric acid.

Hathout et al. (2010) applied jasmonic acid as seed soaking that

significantly reduced root rot incidence in bean plants. Production of reactive

oxygen species especially H2O2 was highly increased in both roots and

shoots of the plants infected with Rhizoctonia solani, as compared with non-

infected control. Jasmonic acid significantly increased the total phenolic

compounds in bean roots.

Abdel-Monaim et al. (2012) reported the effectiveness of some chemical inducers

viz. ethephon, hydrogen peroxide (H2O2), mannitol, salicylic acid (SA) at three different

concentrations (50, 100 and 200 ppm) on root rot and wilt diseases incidence as well as

their influence on growth, quantity and quality parameters of tomato plants. On the other

hand, F. solani more affected with chemical inducers than F. oxysporum or R. solani. Under

field conditions, the treatments were accompanied with significant increase in tomato

growth, yield quantity and quality parameters.

2.4 To test the fungitoxicity of organic amendments and phytoextracts against


Meena et al. (2006) proved that application of aqueous garlic clove extract as seed

treatment (1% w/v) followed by a foliar spray at 50 days after sowing could be a user-

friendly, effective and economic non-chemical means of managing Sclerotinia rot of

mustard.

Chattopadhyay et al. (2007) reported that treatment with GR isolate of Trichoderma

harzianum and Allium sativum clove extract caused significant increase in seed germination

and radicle length of Indian mustard by reducing Sclerotinia rot.

Yadav et al. (2009) evaluated ten plant extracts for their antimicrobial

capacity against 5 isolates of S. sclerotiorum. All the plant extracts tested

were found effective in controlling the radial growth of S. sclerotiorum.

However, the Calotropis gigantea and Azadirachta indica extracts were very

effective in reducing the radial growth of S. sclerotiorum.

Tripathi and Tripathi (2009) used different plant extracts viz. Allium

sativum, Eucalyptus globosus, Azadirachta indica, Ocimum sanctum [O.

tenuiflorum], Parthenium hysterophorus, Bougainvillea spectabilis, Lantana

camara, Datura stramonium, Calotropis procera and Capsicum annum

against Sclerotinia sclerotiorum causing stem rot of Indian mustard

(Brassica juncea). Maximum inhibition of pathogen colony growth was

observed in Allium sativum (71.11%) followed by Azadirachta

indica (50.37%), O. sanctum (38.15%) and C. procera (22.96%). The

remaining plant extracts were ineffective in controlling the mycelial growth of

the pathogens.

Yadav (2009) reported that five botanical plant extracts (1%) viz., Allium sativum, A.

cepa, Azardirachta indica, Calotropis procera and Eucalyptus globosus were tested for their

biopesticidal activity on mustard diseases. A. sativum (garlic) and Eucalyptus were found

more effective than Calotropis (aak) and Azardirachta indica (neem).

Tripathi et al. (2011) tested six organic amendments viz., sunflower cake, safflower

cake, groundnut cake, mustard cake, neem cake and farmyard manure against Sclerotinia

sclerotiorum and found maximum inhibition of mycelial growth (30.37%) in sunflower cake

while minimum (2.22%) in groundnut cake.

CHAPTER-3 MATERIALS AND METHODS

3.1 To know the chemical basis of susceptibility and resistance in Indian mustard varieties against Sclerotinia sclerotiorum.

Sclerotinia rot infected samples of Indian mustard were collected from

Bassi Tehsil of Jaipur. Stem rot pathogen was isolated from infected tissues.

The pathogen was identified as Sclerotinia sclerotiorum on the basis of

morphological, cultural characters and pathogenicity was proved (Plate 1).

3.1.1 Bio-chemical studies

The following experiments were conducted in order to study the effect

of Sclerotinia rot on some biochemical constituents (proteins, carbohydrates,

phenols) of mustard varieties, namely, Bio-902, Kranti, Varuna, Manihari,

Aravali, RRN-505, Navgold, NRCDR-2, Laxmi, RGN-48 and NRCDR-601.

These varieties were grown in pots (9x12 inches) with three replication in

cage house. Healthy leaves from above varieties were collected 30 days

after sowing (DAS) than washed and dried in shade for biochemical

analysis. After it, inoculum (multiplied on sorghum grains) were added @ 20

g/pot. Leaves from infected plants were collected 45 DAS and disease

incidence were also noted.

For the quantitative estimation of primary metabolites following different

protocols were used.

3.1.1.1 Determination of total soluble sugar

Leaf material (0.1-0.5 g) without midrib were extracted in 10 ml of

80% ethanol in a mortar pestle, the extract obtained was centrifuged at 8000

rpm for 10 min. Extraction was repeated four times with 5 ml of 80% ethanol

each time and supernatants were collected into same beaker. Volume of the

extract was made to 50 ml with 80% ethanol. A 0.1 to 0.5 ml of aliquot from

supernatant was added to 4.0 ml of Anthrone reagent (0.2% : 200 mg

anthrone dissolved in 100 ml H2SO4, prepared fresh before use) and the test

tubes were placed in ice cold water. The intensity of colour was read at 620

nm on spectrophotometer. A standard curve was prepared using glucose

(100 µg/ml) (Dubois et al., 1951).

3.1.1.2 Determination of phenol content

One ml of supernatant was taken from ethanol extract prepared for

total soluble sugar analysis (3.1.1.1) and evaporated to dryness in water

bath. One ml of milipore water in each test tube and 0.5 ml of Folin &

Ciocalteu reagent (1:1 with water) was added and kept for three min. After

this, 2 ml of 20% Na2CO3 was added and mixed thoroughly. The tubes were

placed in boiling water for exactly one minute and cooled in ice water. The

absorbance was read at 650 nm against a reagent blank (Malik and Singh,

1980).

A standard graph was prepared using pyrocatachol ranging between 0-25 µg concentration.

The amount of phenols present in the sample was calculated as: Phenol (mg/g) = Sample O.D. × Standard O.D. × Dilution factor Where O.D. = Optical density

3.1.1.3 Determination of total protein

Protein concentration of extract was estimated by method of Lowry et al. (1951).

(a.) Reagents for Lowry’s method

(i) Solution A: 2% Na2CO3 in 0.1 N NaOH

(ii) Solution B: (a) 1% CuSO4.5H2O solution

(b) 2% sodium potassium tartrate solution

Working solution of B: Prepared fresh before use by mixing equal

volume of solution B (a) and B (b).

(iii) Solution C: Prepared fresh before use by mixing 50 ml of solution A

and 1ml of working solution of B.

(iv) Solution D: Folin & Ciocalteu reagent (1N).

(b.) Procedure

Extracts of different samples (25µl) were taken in separate test tubes

and volume was made up to 1 ml in each tube with milipore water. A tube

with 1 ml of water served as blank. Five ml of solution C was mixed in each

tube by vortexing and kept for 10 min. Then 0.5 ml of solution D (Folin &

Ciocalteu reagent) was added in each tube and vortexed. The tubes were

allowed to stand at room temperature for 30 min. Absorbance was read at

660 nm. A standard curve was prepared using bovine serum albumin (BSA)

in the concentration range of 10-80 µg.

3.2 To evaluate resistance inducer chemicals against Sclerotinia


The following systemic acquired resistance (SAR) activators in addition

to carbendazim (standard check) were used.

Table :3.1 Different systemic acquired resistance activators

S.No. SAR activators

1. β-amino butyric acid

2. Salicylic acid

3. Hydrogen peroxide

4. 2,6-Di Chloroisonicotinc acid

5. Azoxystrobin

The SAR activators (Table 3.1) in addition to carbendazim (standard

check) were evaluated with three (100, 200 and 500 ppm) concentrations

against the pathogen under laboratory conditions to find out their relative

efficacy in inhibiting the growth of the pathogen in culture by poisoned food

technique (Schmitz, 1930). Requisite quantity of each chemical was

incorporated in sterilized two per cent potato dextrose agar medium,

thoroughly mixed by shaking prior to pouring in sterilized Petriplates and

were allowed to solidify. These Petriplates were inoculated with 5 mm disc

of four day old culture in the centre of the plate and incubated at 25 + 10C.

Each treatment was replicated three with a suitable control. The efficacy of

chemicals in each treatment and average of three replications was

calculated. Per cent inhibition over control was calculated by the following

formula (Bliss, 1934).

C - T

Per cent inhibition over control = ———— x 100

C

C = growth of fungus in control

T = growth of fungus in treatment

3.3 To elicit systemic acquired resistance by chemical inducers against

Sclerotinia sclerotiorum (in vivo)

The experiment was carried out in earthen pots (9 x 12 inches) with

cultivar T-59 (Varuna). The pathogen multiplied on sorghum grains at 25 + 1

0C for one week was used as the soil inoculum. Prior to sowing, pots were

sterilized with copper sulphate solution and filled with sterilized soil (soil :

vermicompost 3 :1). The soil was sterilized at 1.045 kg/cm2 for one hour for

three consecultive days. Varuna, the susceptible variety of Indian mustard

was sown in these pots with four replications. The SAR activators viz., β-

amino butyric acid (100 ppm), Salicylic acid (100 ppm), Hydrogen peroxide (100 ppm), 2,6-

Di Chloroisonicotinc acid (100 ppm), Azoxystrobin (2000 ppm) and carbendazim (1000

ppm) were tested by applying as seed soaking (for 30 minutes), foliar spray (30 DAS) and

seed soaking-cum-foliar spray.

These pots were inoculated with inoculum multiplied on sorghum

grains 45 days after sowing. For inoculation the upper 5 cm layer of soil of

each pot was thoroughly mixed with inoculum @ 20 g/pot. The pots were

covered with polythene bags and kept for 24 hours in cage house.

To assess the stem rot intensity the following slightly modified

disease rating (0-4) scale (Lesovoi et al., 1987 and Sansford, 1995) was

followed.

Grade/numerical

scale

Description/lesion lenghth on stem

0 Healthy (no visible lesion)

1 0.1-2.0 cm lesion length on stem



4 > 6 cm lesion length on stem or complete dried plant

The length of lesion on infected stem was considered for recording the disease intensity (Sharma, 1987). The infected area was calculated from 10 randomly selected plants at 80 DAS. In each pot and the average for each treatment was worked out. The intensity was calculated using the formula of Wheeler (1969).

Sum of individual ratings Per cent disease intensity = ----------------------------------------------------------- x 100 No. of plants observed x maximum disease rating

3.4 To test the fungitoxicity of organic amendments and phytoextracts against Sclerotinia sclerotiorum

3.4.1 Organic amendments

Five organic amendments viz., neem cake, mustard cake, castor

cake, groundnut cake and sesame cake were screened in vitro to evaluate

their inhibitory effect on radial growth of the pathogen. Hundred g

oil cake was taken in 1000 ml water and preserved in earthen pot for

extraction. Pots were wrapped by polythene bags to preserve moisture. Oil

cake extract was filtered with cheese cloth, mixed @ 3% in PDA in conical

flask and autoclaved. Twenty ml PDA was poured in each sterilized

Petridish and allowed for solidify. The each plate was inoculated with 5 mm

diameter bit of 7 days old culture of fungus. Inoculated plates were

incubated at 25 + 10C for 7 days. The linear growth of test fungus was

recorded and per cent growth inhibition was calculated as per Bliss (1934)

formula referred under 3.2.

3.4.2 Phytoextract

To find out the fungitoxicity of six plant extracts (Table 3.2) against

the pathogen were evaluated. Hundred gram from each was collected and

washed 2-3 times with water and allowed to dry at room temperature

(25±10C) for six hours. Before extraction leaves of each plant (100g) were

crushed separately with 100 ml sterilized distilled water. The extract was

filtered through muslin cloth and centrifuged at 5000 rpm for 30 min. The

extract were then sterilized by passing them through a Millipore filter using a

swimming filter adapter.

The extract of each plant species was diluted in order to achieve

three concentrations viz., 5, 10 and 15 per cent. Petri plates containing PDA

supplemented with different phyto-extracts, each with three concentrations

and replicated three times were inoculated with 5-day-old culture (5 mm dia

disc). A suitable check (without plant extract) was also maintained. Fungal

colony was measured after 7 days of incubation at 25 + 1 0C. The linear

growth of test fungus was recorded and per cent growth inhibition was

calculated by Bliss (1934) formula referred under 3.2.

Table : 3.2 Plant extracts and their concentrations

S.No. Common Name

Botanical Name Part used

Concentration (%)

1 Garlic Allium sativum Clove 5, 10, 15

2 Neem Azadirachta indica Leaves 5, 10, 15

3 Tulsi Ocimum sanctum Leaves 5, 10, 15

4 Alstonia (devil’s tree)

Alstonia scholaris Leaves 5, 10, 15

5 Ginger Zingiber officinalis Rhizome 5, 10, 15

6 Turmeric Curcuma longa Rhizome 5, 10, 15

CHAPTER-4

RESULTS

4.1 To know the chemical basis of susceptibility and resistance in

Indian mustard cultivars against Sclerotinia sclerotiorum

Biochemical studies: Changes in total soluble sugars, total protein content

and total phenols were estimated in leaves of healthy and infected (S.

sclerotiorum) plants of different Indian mustard varieties (Table 4.1).

The resistance of a plant as a result of host pathogen interaction

involves morphological and biochemical changes which depends upon the

plant response to infection. The fungal infection induces the oxidative and

hydrolytic reactions along with hormonal imbalance in the host tissue

affecting the normal metabolism.

4.1.1 Total soluble sugar

Total soluble sugar (Table 4.1 and Fig. 4.1) in leaves of healthy and

infected (Sclerotinia sclerotiorum) plants of Indian mustard varieteis was

estiamted at 30 & 45 DAS, respectively. The total soluble sugar was

decreased (20.17 – 37.79%) in all the infected plants as compared to

healthy ones. The total soluble sugar was decreased maximum (37.79%) in

infected plants of Manihari variety followed by Varuna (33.33%), NRCDR-

601 (31.00%), RGN-48 (29.34%) and minimum in Bio-902 (20.17%) as

compared to healthy ones. Minimum disease incidence (Table 4.1) was

recorded in Kranti (10%), as it also resulted in minimum reduction in total

soluble sugars.

4.1.2 Total protein content

Total protein content (Table 4.1 and Fig. 4.2) in leaves of healthy and


estiamted at 30 and 45 DAS, respectively. The total protein content was

decreased in all infected plants as compared to healthy ones. The total

protein content was decreased maximum (5.56%) in infected plants of

Manihari variety followed by Varuna (5.21%), NRCDR-601 (5.02%) and it

was found minimum in Kranti (2.21%).

4.1.3 Total phenol

Phenol content (Table 4.1 and Fig. 4.3) in leaves of healthy and


estimated at 30 and 45 DAS, respectivley. The total phenol content was

increased in infected plants as compared to healthy ones. Phenol content

was increased maximum (4.71%) in infected plants of Kranti variety followed

by Laxmi (4.17%) and it was increased minimum in Novgold (1.12%),

Manihari (1.27%), Aravali (1.35%), Bio-902 (1.44%) and Varuna (1.53%).

It is cleared (Table 4.1) that variety Kranti showed lowest disease

incidence (10.00%) as it had highest amount of total phenol (5.10 mg/g dry

leaf) in healthy and infected leaves (5.34%). Varieties like Novgold,

Manihari, Aravali, Bio-902, Varuna had low level of increased phenol content

and showed maximum disease incidence (20.00 to 36.66%).

4.2 To evaluate resistance inducer chemicals against Sclerotinia

sclerotiorum in vitro

The efficacy of SAR activators in addition to carbendazim was

evaluated against Sclerotinia sclerotiorum on PDA by poisoned food

technique. The data suggested (Table 4.2, Fig. 4.4 and Plate 4.2) that

increase in concentration of the SAR activators caused increased inhibition

of mycelial growth of the fungus. Among these, salicylic acid was found cent

per cent inhibitory at 200 ppm. This was followed by β-amino butyric acid

(77.77, 86.66 and 92.00 %) at 100, 200 and 500 ppm, respectively.

Azoxystrobin (18.48, 50.22 and 73.33%) and 2, 6-Dichloroisonicotinic acid

(24.44, 38.52 and 80.00%) were found to be least effective at 100, 200 and

500 ppm, respectively against Sclerotinia sclerotiorum.

4.3 To elicit systemic acquired resistance by chemical inducers

against Sclerotinia sclerotiorum (in vivo)

4.3.1 Seed soaking

A perusal of data (Table 4.3 and Fig.4.5) revealed minimum disease

intensity with azoxystrobin (26.00%) followed by salicylic acid (35.32%), as

compared to control (52.20%). Maximum reduction in disease intensity over

control was osberved with azoxystrobin (50.19%) followed by salicylic acid

(32.34%) over control. β-amino butyric acid (28.93%) was found at par with

salicylic acid. Minimum reduction in disease intensity was observed in 2, 6-

Dichloroisonicotnic acid (22.22%).

4.3.2 Foliar spray

Perusal of data (Table 4.3 and Fig. 4.5) revealed a similar trend of

results as in 4.3.1. The highest reduction in disease intensity over control

was observed in azoxystrobin (60.73%) followed by salicylic acid (49.82%),

β-amino butyric acid (45.45%), hydrogen peroxide (38.73%) and minimum in

2, 6 Dichloroiso-nicotinic acid (34.55%).

4.3.3 Seed-cum-foliar spray

A perusal of data (Table 4.3 and Fig. 4.5) revealed minimum disease

intensity in azoxystrobin (14.65%) followed by salicylic acid (21.90%) and β-

amino butyric acid (23.90%) over control (48.12%).

Maximum reduction in disease intensity over control was observed in

azoxystrobin (69.56%) followed by salicylic acid (54.49%), β-amino butyric

acid (50.33%) and minimum in 2, 6 Dichloroiso nicotinic acid (42.02%).

4.4 To test the fungitoxicity of organic amendments and

phytoextracts against Sclerotinia sclerotiorum

4.4.1 Organic amendements

Efficacy of five organic oil cakes was tested in vitro against

Sclerotinia sclerotirum. The castor cake (Table 4.4) was found significantly

superior over all the tested oil cakes with maximum (44.44%) inhibition of

mycelial growth of Sclerotinia sclerotiorum over control followed by neem

cake (40.00%). Groundnut cake was found least effective (5.55%) in

inhibiting mycelial growth of S. sclerotiorum (Table 4.4, Fig. 4.6 and Plate

4.3).

4.4.2 Phytoextracts

The efficacy of six plant leaf extracts (Table 4.5) was tested in vitro at

three concentrations viz., 5, 10 and 15 per cent against S. sclerotiorum on

PDA by poisoned food technique. Among six plant extracts, extract of garlic

cloves was found most effective in inhibiting mycelial growth (52.22, 65.66

and 88.00 %) of S. sclerotiorum at 5, 10 and 15 per cent, respectivley

followed by neem (50.74, 67.20 and 80.0%) over control. Extract of Alstonia,

tulsi and turmeric were found least effective in inhibiting mycelial growth of

S. sclerotiorum over control.

All the cocentrations (5, 10 and 15%) of garlic extract were found

significantly superior over each other while 10 and 15 per cent of Alstonia,

tulsi and turmeric were found at par to each other.

Table:4.1 Biochemical changes in leaves of healthy and infected (S. sclerotiorum) plants of Indian mustard varieties

Varieties Total soluble sugar (mg/100mg of dry leaf)

Per cent decrease

Total protein content (mg/g of dry leaf)

Per cent decrease

Phenol content (mg/g of dry leaf)

Per cent increase

Disease incidence

(%)# Healthy* Infected** Healthy* Infected** Healthy* Infected**

Bio-902 11.90 9.50 20.17 20.5 20.00 2.44 4.85 4.92 1.44 30.00 (33.21)

Kranti 12.90 10.13 21.47 22.6 22.10 2.21 5.10 5.34 4.71 10.00 (18.43)

Varuna 10.80 7.20 33.33 21.1 20.00 5.21 3.92 3.98 1.53 36.66 (37.26)

Aravali 11.25 8.75 22.22 22.4 21.70 3.13 4.46 4.52 1.35 21.11 (27.35)

RRN-505 10.90 8.05 26.15 19.9 19.30 3.02 4.62 4.74 2.60 22.22 (28.12)

Navgold 12.40 9.45 23.79 21.9 21.15 3.42 4.45 4.50 1.12 20.00 (26.57)

Manihari 13.02 8.10 37.79 21.6 20.40 5.56 3.95 4.00 1.27 33.33 (35.26)

NRCDR-2 11.25 8.15 27.56 21.7 21.00 3.23 4.58 4.65 1.53 27.77 (31.80)

Laxmi 11.62 9.15 21.26 22.4 21.90 2.23 5.03 5.24 4.17 13.33 (21.41)

RGN-48 13.02 9.20 29.34 19.7 19.10 3.05 4.20 4.32 2.86 24.44 (29.63)

NRCDR-601 11.45 7.90 31.00 21.9 20.80 5.02 4.02 4.10 1.99 30.00 (33.21)

SEm+ 0.350 0.221 - 0.518 0.523 - 0.156 0.105 - 0.426 CD (p=0.05) 1.033 0.653 - 1.529 1.543 - 0.460 0.311 - 1.257

* Leaves collected 30 DAS and inoculum applied after it ** Leaves collected 45 DAS # Average of three replications

Table:4.2 Efficacy of SAR activators against Sclerotinia sclerotiorum by poisoned food technique after 7 days of incubation at 25 + 1 0C

SAR activators Per cent growth inhibition at various concentration (ppm)

100 200 500 Mean

β-amino butyric acid (BABA)

77.77

(61.87)

86.66

(68.58)

92.00

(73.57)

88.14

(68.01)

Salicylic acid 90.37

(71.92)

100.00

(90.00)

100.00

(90.00)

96.79

(83.93)

Hydrogen peroxide 16.66

(24.09)

38.88

(38.57)

100.00

(90.00)

51.85

(50.89)

2,6-Di Chloroisonicotinic

acid

24.44

(29.63)

38.52

(38.36)

80.00

(63.43)

35.43

(43.81)

Azoxystrobin 18.48

(25.46)

50.22

(45.13)

73.33

(58.91)

47.34

(43.17)

Carbendazim (Standard

check)

100.00

(90.00)

100.00

(90.00)

100.00

(90.00)

100.00

(90.00)

Control 0.00

0.00

0.00

0.00

SEm+ CD (p=0.05)

A 1.00 2.87

C 0.66 1.88

AxC 1.74 4.97

* Average of three replications

Figures given in parenthesis are angular transformed value

Table:4.3 In vivo efficacy of SAR activators against Sclerotinia rot of Indian mustard SAR activators Concentration used

(ppm) Seed soaking Foliar spray Seed soaking-cum- foliar spray

Disease intensity

(%)*

Per cent disease control

Disease intensity

(%)*


Disease intensity (%)*


β-amino butyric acid 100 37.10 (37.52)

28.93 30.00 (33.21)

45.45 23.90 (29.27)

50.33

Salicylic acid 100 35.32 (36.46)

32.34 27.60 (31.69)

49.82 21.90 (27.90)

54.49

Hydrogen peroxide 100 39.60 (39.00)

24.14 33.70 (35.49)

38.73 27.00 (31.31)

43.89

2,6-Di Chloroisonicotinic acid

100 40.60 (39.58)

22.22 36.00 (36.87)

34.55 27.90 (31.88)

42.02

Azoxystrobin 2000 26.00 (30.66)

50.19 21.60 (27.69)

60.73 14.65 (22.50)

69.56

Carbendazim (Standard check)

1000 10.82 (19.20)

79.27 12.65 (20.83)

77.00 6.32 (14.56)

86.87

Control - 52.20 (46.26)

0.00 55.00 (47.87)

0.00 48.12 (43.92)

0.00

SEm+ 0.69 - 0.87 - 0.41 - CD (p=0.05) 2.19 - 2.77 - 1.31 -

* Average of four replications


Table:4.5 Fungitoxicity of different plant extracts against Sclerotinia

sclerotiorum by poisoned food technique after 7 days of incubation at

25 + 1 0C

Plant extract Part used Per cent growth inhibition at different

concentration

(%)*

5 10 15 Mean

Garlic Clove 52.22

(46.27)

65.66

(54.13)

88.00

(69.73)

68.63

Turmeric Rhizome 25.96

(30.36)

61.11

(51.42)

67.82

(55.42)

51.63

Neem Leaves 50.74

(55.39)

67.20

(45.17)

80.00

(63.43)

66.01

Ginger Rhizome 41.00

(39.82)

56.20

(48.56)

71.04

(57.44)

56.08

Tulsi Leaves 45.07

(42.17)

49.30

(44.60)

60.26

(50.92)

51.54

Alstonia Leaves 35.82

(36.76)

50.93

(45.53)

62.96

(52.51)

49.90

Control - 0.00 0.00 0.00

0.00

SEm+ CD (p=0.05)

E 1.44 4.11

Con. 0.94 2.69

E x Con. 2.49 7.12



Table:4.4 Fungitoxicity of different oil cakes against Sclerotinia sclerotiorum by poisoned food technique after 7 days of incubation at 25 + 1

0C

Organic amendments Concentration (%)

Radial mycelial

growth (mm)*

Per cent growth

inhibition

Mustard cake 3.00 83.00 7.77

(16.17)

Neem cake 3.00 54.00 40.00

(39.23)

Castor cake 3.00 50.00 44.44

(41.81)

Groundnut cake 3.00 85.00 5.55

(13.63)

Sesame cake 3.00 70.00 22.22

(28.12)

Control - 90.00 0.00

(0.00)

SEm+ - - 0.28

CD (p=0.05) - - 0.88



CHAPTER-5

DISCUSSION

Indian mustard [Brassica juncea (L.) Czern & Coss] has gained importance in

different parts of Rajasthan as a potential oilseed crop and is grown in almost every district

of the state. This crop suffers from vagaries of fungal, bacterial and viral diseases. Among

the fungal diseases, Sclerotinia rot caused by Sclerotinia sclerotiorum (Lib.) de Bary, earlier

considered to be a minor disease, is now becoming increasingly destructive and widely

damaging in recent years. Particularly in areas of heavy soils receiving four or more

irrigation (Ghasolia et al., 2004).

The total soluble sugars level decreased in all the cultivars of Indian mustard after

infection by S. sclerotiorum. After infection by S. sclerotiorum, varieties showing higher

reduction in total soluble sugars, expect few, were highly susceptible & depicted higher

disease incidence while varieties with less reduction were moderately resistant. These

results are in accordance with the findings of Teltow and Farrar (1992) and Jaypal and

Mahadevan (1968). Teltow and Farrar (1992) reported post infectional decrease in sugar

levels in Puccinia infected barley may be due to rapid hydrolysis of sugars during

pathogenesis through enzymes secreted by the pathogen. The invading pathogens may

utilize the sugar leading to decrease in its content. Mohamed et al. (1976) also recorded

reduction in total amount of carbohydrates in infected (Puccinia sp.) wheat and barley plants

compared with healthy plants. This reduction was more pronounced in moderate susceptible

cultivars than in resistant or moderate resistant cultivars. After infection by S. sclerotiorum,

Jing-fan et al. (2013) estimated higher biochemical changes in the susceptible varieties of

Chinese cabbage than that of resistant ones.

Total protein content in infected leaves was lower as compared to healthy ones.

After infection, varieties showing higher reduction in total protein content, except few were

highly susceptible and showed higher disease incidence while varieties with less reduction

were moderately resistant. These results are confirmatory with the findings of Sempio and

Marte (1968), who reported that protein content was decreased in susceptible variety of

beans against rust pathogen. During host pathogen interaction, between barley and

Septoria sp. amino acid act as a substrate for the pathogen (Titarenko et al., 1993).

Plants have developed an arsenal of defense mechanisms to protect themselves

against pathogen attacks. In the present investigation, phenol content in leaves of infected

plants was higher as compared to healthy ones. It is cleared that varieties showing high

increase in phenol content after infection were highly resistant and showed lowest disease

incidence while varieties with low increase were highly susceptible. Singh et al. (2011)

concluded that presence of phenolics in high concentration in the plant cell gives them

resistance to pathogens. After infection by pathogen plant cells synthesize phenol oxidizing

enzymes that oxidize phenols to toxic quinines. Hence the activity of these enzymes

increases in infected cell. Basyouni et al. (1976) also reported increase in the phenolic

content of wheat cultivars after infection. Helal et al. (1978) reported increase in the amount

of total phenols in both resistant and susceptible cultivars of cucumber infected with

Erysiphe cichoracearum. Various workers (Patil and Dimond, 1967; Ravise and Trique,

1972 and Rai et al., 1980) recorded increase in the amount of phenolic substances in

different host pathogen system after infection. Our results are also in accordance with the

findings of Basha and Chatterjee (2007), who reported that increased level of phenolics

activate the phenylpropanoid pathway which is one of the reasons for resistance in wheat

against S. sclerotiorum. They also explained that phenylpropanoid pathway may be utilized

to induce resistance in the hosts of S. sclerotiorum by biotic and abiotic agents (Singh et al.

2011).

All the SAR activators (β-amino butyric acid, salicylic acid, hydrogen peroxide, 2, 6

Dichloroiso nicotinic acid and azoxystrobin) in addition to carbendazim were tested at 100,

200 and 500 ppm concentration and inhibited mycelial growth of S. sclerotiorum. Our

observations are in agreement with El. Ganaieny et al. (2002), Shahda (2002), Hilal et al.

(2006), Abdel-Monaim et al. (2012) and Rahaman et al. (2012).They reported that all the

tested abiotic agents were found significantly superior in reducing linear growth of many

fungal pathogens including S. sclerotiorum.

Protection of plants against pathogens depends on constitutive and induced

defense mechanisms (Nurnberger and Lipka, 2005). Extensive research has shown that

biotic and abiotic agents including salicylic acid play a key role in local and systemic

acquired resistance (SAR) to biotrophic and necrotrophic pathogens (Durrant and Dong,

2004; Gaffney et al., 1993; Thomma et al., 1998 and Conarth et al., 2002). Amongst

different SAR activators used in the present investigation were applied through seed

soaking, foliar and seed-cum-foliar spray. In these methods, seed soaking-cum-foliar spray

of SAR activators was found most effective to control disease by reducing disease intensity,

followed by foliar spray and seed soaking alone. All SAR activators tested were able to

reduce the disease intensity significantly over control. Azoxystrobin, a fungicide-cum-SAR

inducer was the most effective in reducing the disease intensity followed by salicylic acid, β-

amino butyric acid, hydrogen peroxide and 2, 6 Dichloroisonicotinc acid. These results are

in agreement with the results of Dantre et al. (2003), Abdel-Monaim et al. (2012), El Bana

(2007), El-Mougy et al. (2004) and Shalaby et al. (2001). They reported effectiveness of

various SAR inducer in disease control against many fungal pathogens including S.

sclerotiorum.

Extracts of five oil cakes were screened in vitro for fungitoxicity against S.

sclerotiorum and observed that castor and neem cake were found most effective in inhibiting

mycelial growth. Earlier workers have also been reported oil cakes as a source for inhibition

of the fungal growth. Chand and Rai (2008), reported saw dust, castor cake and neem cake

significantly superior in inhibiting mycelial growth of S. sclerotiorum. Inhibitory effect of six

organic amendments on mycelial growth of S. sclerotiorum have also been reported by

Tripathi et al. (2011).

Six plant extracts namely garlic, neem, ginger, turmeric, tulsi and Alstonia were

tested at 5, 10 and 15 per cent concentration and inhibited mycelial growth of S.

sclerotiorum in vitro. Garlic gave maximum inhibition of mycelial growth at higher

concentration. Similar results have been observed by Tripathi and Tripathi (2009) while

working with S. sclerotiorum in vitro. Extracts of ten plants have also been evaluated in vitro

against S. sclerotiorum by Yadav et al. (2009) and reported effective in inhibiting mycelial

growth

CHAPTER-6

SUMMARY

Indian mustard (Brassica juncea L.) has gained importance in different parts of

Rajasthan as potential oilseed crop and is grown in almost every district of Rajasthan. This

crop suffers from many disease among which stem rot caused by Sclerotinia sclerotiorum

(Lib.) de Bary, has become a serious problem in recent years in Rajasthan and in other

mustard growing parts of India.

Total soluble sugars, total protein content and phenol content have received

considerable attention in relation to resistance in plants against diseases. The biochemical

estimation is carried out to study the host parasite relationship in Indian mustard cultivars

infected with S. sclerotiorum. Leaves of infected and healthy plants were taken in to

consideration. Low disease incidence was recorded in varieties showing decreased level of

sugars and protein content and increased level of phenols after infection. This may impart

resistant power to plants to fight against pathogen.

Studies on the relative efficacy of SAR activators were tested in vitro and found that

salicylic acid was observed to be most effective in inhibiting mycelial growth followed by β-

amino butyric acid.

To activate defense mechanism of plants, five SAR activators were evaluated as

seed soaking, foliar and seed soaking-cum-foliar sprays. Azoxystrobin proved to be most

effective against Sclerotinia sclerotiorum followed by salicylic acid, β-amino butyric acid,

hydrogen peroxide and 2, 6 Dichloroiso nicotinic acid in reducing per cent disease intensity.

Castor cake was found most effective followed by neem cake in inhibiting mycelial

growth in vitro.

Garlic extract was found most effective followed by neem extract in inhibiting

mycelial growth in vitro.

Natural and Chemical Induced Resistance against Sclerotinia Rot

of Indian Mustard [Brassica juncea (L.) Czern & Coss]

Arjun Lal Yadav * Dr. R. P. Ghasolia ** (Research Scholar) (Major Advisor)

ABSTRACT

Sclerotinia rot infected samples of Indian mustard were collected from Bassi tehsil of Jaipur district. Pathogen isolated, purified and identified as Sclerotinia sclerotiorum & its pathogenicity was proved.

Biochemical changes in total soluble sugars, total proteins content and phenol content played very important role in relation to resistance in plants against disease. An increase in total phenols and decline in total soluble sugars and total protein content were observed in the leaves of infected plants with S. sclerotiorum compared to the healthy ones which impart resistance to plants.

Five SAR activators were tested by poisoned food technique and inhibited the growth of fungus. Salycilic acid checked mycelia growth completely at 200 ppm followed by β-amino butyric acid

SAR activators were used as seed soaking, foliar spray and seed soaking-cum-foliar spray. Azoxystrobin and salicylic acid were observed to be most effective in reducing the disease intensity.

Among oil cakes, castor cake was found most effective followed by neem cake in inhibiting mycelial growth.

Garlic extract was found most effective in inhibiting mycelial growth followed by neem.

* Post graduate student, M.Sc. (Ag.), Department of Plant Pathology, S.K.N. College of Agriculture, Jobner.

** Thesis submitted in partial fulfillment of the requirement for M.Sc. (Ag.), degree in Plant Pathology under supervision of Dr. R.P. Ghasolia, Assistant Professor, Department of Plant Pathology, (SKN Agriculture University, Jobner), S.K.N. College of Agriculture, Jobner, Jaipur.

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