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Pathogenic fungi associated with pre- and post-emergence

seedling blight of pine and cypress in Fars Province, Iran

By A. Zakeri1, H. Hamzeharghani2,3*, Z. Banihashemi2 and S. H. Saadati1

1Fars Agricultural and Natural Recourses Research Center, Zarghan, Shiraz, Iran; 2Department of

Plant Protection, Shiraz University, Badjgah 71444-65186, Iran; 3E-mail: [email protected]

(for correspondence)

Summary

Seed and root rot of pine and cypress seedlings cause heavy annual losses to forest nurseries in Fars

Province. Root and crown samples of various species of conifers, such as Tehran pine (Pinus eldarica),brutia pine (Pinus brutia), Arizona cypress (Cupressus arizonica), Shirazian cypress (Cupressussempervirens var. fastigiata), common cypress (C. sempervirens var. horizontalis) and oriental arbor-vitae (Thuja orientalis), showing chlorosis, necrosis, stunted growth, defoliation and root and crownrot symptoms were collected from pine and cypress nurseries across Fars province at various timeintervals. Infected tissues were washed and cultured on acidified potato dextrose agar (PDA) and cornmeal agar (CMA) amended with Delvocide and ampicillin, with and without surface sterilization,respectively, and incubated at 25C for 35 days. Decaying seeds of all plant species were also collectedfrom nursery seed stocks and cultured on PDA. Hyphal tip isolates were used for further studies.Species of Phytophthora, Pythium, Rhizoctonia and Fusarium were isolated from symptomaticseedlings of pine and cypress at different times during the growing season. Pathogenicity of isolates ofPhytophthora, Pythium and Rhizoctonia was confirmed on seedlings of all plant species, whereas

Fusarium sp. isolated from the seeds of Tehran pine was only pathogenic on seedlings of Tehran pineand Arizona cypress. Virulent isolates identified as Phytophthora nicotiana var. parasitica, Pythium

ultimum, Pythium paroecandrum, Rhizoctonia solani and binucleate Rhizoctonia-like fungus causedroot and crown rot of pine and cypress seedlings. Pathogenic isolates of Fusarium identified asFusarium proliferatum also caused pre-emergence seed decay of pine and cypress. Isolates ofPhytophthora and Pythium were the most virulent of these fungal isolates.

1 Introduction

Iran is considered a region of the world with low forest cover. Compared to othertemperate areas with better soil conditions, it is difficult to achieve afforestation on a largescale in Iran, and as a consequence, most of the afforestation is conducted with non-nativespecies (Mosadegh 1981). Establishment of conifer plantations is a top priority of theIranian National Forestry Department to optimize the use of marginal lands characterized

by low fertility and rocky, arid soil conditions. In addition to controlling soil erosion andrunoff and thus minimizing the loss of topsoil and delaying desertification, afforestationprovides an extra bonus of an increase of per capita green space and picnic areas for thepublic (Mosadegh 1981).

Management of forest nurseries to supply regional-scale outplantings with disease-freeand vigorous seedlings is essential for the success of aforementioned projects. Productionof conifer seedlings for outplanting has been limited by seed, root and crown rot of theseedlings by soilborne fungi in forest tree nurseries (Dixon et al. 1991) which has led to aneed to sow additional seeds to offset losses because of non-viable seeds and seedling

For. Path. doi: 10.1111/j.1439-0329.2010.00697.x 2010 Blackwell Verlag GmbH

*Also former faculty of Fars Agricultural and Natural Recourses Research Center,Zarghan, Shiraz 71555-617, Iran.

Received: 2.6.2010; accepted: 25.9.2010; editor: N. B. Klopfenstein

http://wileyonlinelibrary.com/

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blights. Different seedborne and soilborne fungi have been reported to cause seed decayand root and crown rot of conifer seedlings. Cram and Faeredrich (2009) provided acomprehensive list of relatively important seedborne pathogens of North American forestorchards and nurseries including the species of Fusarium, Lasiodiplodia theobromae,Caloscypha fulgens, Sirococcus conigenus and Diplodia pinea. Phytophthora parasitica,

Phytophthora cinnamomi, Phytophthora derchsleri (Heather and Pratt 1975; Kirby andGrand 1975; Kuhlman and Smith 1975; Heather et al. 1977; Webb and Dahm 1981;Barnard et al. 1985; Farr et al. 1989; Dumroese and James 2005), Pythium ultimum,Pythium irregular, Pythium aphanidermatum, Pythium mamillatum (Vaartaja andBumbieris 1961; Vaartaja et al. 1967; Edmonds and Heather 1973; Bumbieris 1974;Huang and Kulman 1990; Dumroese and James 2005), Fusarium solani, Fusariumoxysporum, Fusarium subglutinans, Fusarium moniliforme (Pawuk 1978; Brissette et al.1991; Dumroese and James 2005) Rhizoctonia solani, and binucleate Rhizoctonia-likefungus (English and Barnard 1981; English et al. 1986) and Cylindrocarpon(Dumroese and James 2005) have been associated with seedling blight of coniferousseedlings around the world. Macrophomina phaseolina, the cause of charcoal root disease,

occurs as one of the most important diseases in forest nurseries in the Western UnitedStates, and most conifer species (including pines) grown in warm lowland agricultural soilsare susceptible to the disease (Barnard 1994).

Fusarium in particular occurs as an important cause of pre- and post-emergencedamping-off, root rot and shoot blight on western larch and Douglas-fir in Canada(Peterson 2007). Fusarium oxysporum is considered the most prevalent of Fusaria withinthe F. oxysporum species complex occurring on conifer seedlings in the United States.

James and Dumroese (2007) evaluated the application of some commercially availablebiological control agents such as F. oxysporum Q12 as potential biological control againstpathogenic Fusarium and Cylindrocarpon species in nurseries.

The objective of this study was to identify pathogenic soilborne fungi associated withpine and cypress seed decay and root and crown rot of their seedlings.

2 Materials and methods

2.1 Isolation and identification

Seedlings of different species of conifers, including Tehran pine (Pinus eldarica), brutiapine (Pinus brutia), Arizona cypress (Cupressus arizonica), Shirazean cypress (Cupressussempervirens var. fastigiata), common cypress (C. sempervirens var. horizontalis) andoriental arborvitae (Thuja orientalis), with symptoms of damping-off, stunting, chlorosisand necrosis (Fig. 1e,f) were collected from pine and cypress nurseries across Farsprovince about once a month during various seasons in 2000 and 2001. Coniferousspecies from which fungal isolates were isolated are called source species to avoidconfusion with host species as not every specimen isolated from a coniferous speciesmay prove pathogenic. Root and crown samples from healthy seedling were alsocollected to serve as controls. Samples were stored in a cooler ice box to prevent heatingwhen transferring to the laboratory. Seed samples were also obtained from nursery seedstocks.

Seed, crown and root samples were surface-sterilized with 1% sodium hypochloride for1.5 min, rinsed with sterile distilled water and blotted dry in sterile paper towel. Sampleswere placed on acidified potato dextrose agar (PDA) (pH 4.2) and corn meal agar (CMA)amended with 10 lg ml)1 Delvocide (pimaricin) and 500 lg ml)1 ampicillin and incubatedat 25C for 35 days. Hyphal tips or single spores were taken from individual colonies on2% water agar (Singleton et al. 1990) and grown on potato carrot agar, hempseed

medium, V-8 medium (Van der Plaats-Niternk 1981; Ershad 1992), PDA, KCl agar

2 A. Zakeri, H. Hamzeharghani, Z. Banihashemi et al.

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and alfalfa leaf piece agar (Gerlach and Nirenberg 1982; Nelson et al. 1983; Sneh et al.1991, 1996; Burgess et al. 1994) for identification.

To make a conclusive identification of the genera Pythium and Phytophthora, aside fromtheir colony morphology and growth at various temperatures, their asexual and sexualreproductive structures were observed under a microscope at appropriate magnification(40 dry objective). Sporangial morphology includes shape, size, papilla formation andtheir size and number (only in Phytophthora), and 1015 randomly selected sporangia oroospores were examined under a microscope and oospore formation and morphology wererecorded. Identification ofFusarium isolates was attempted by recording colony characters(growth rate at 25C, aerial mycelium, the colour of the backside of the colony) on PDA,

morphology of macroconidia (mass, shape and septation of conidia and size and

(a) (b)

(c) (d)

(e) (f)

Fig. 1. (ad) Effect of inoculation of pine and cypress seedlings within 2030 days after inoculationwith (a), Phytophthora parasitica on Tehran pine (b), Pythium paroecandrum on Arizona cypress (c),Pythium ultimum on Arizona cypress (d), Binucleate Rhizoctonia-like fungus on Arizona cypress (Theright side pots in ad were filled with non-infested soil). (e) and (f) show a high rate of mortality ofvarious species of pine and cypress caused by pathogenic isolates of Pythium, Phytophthora and

Rhizoctonia under nursery conditions.

3Pre- and post-emergence seedling blight

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morphology of apical and basal cells), microconidia (their presence and abundance, shape,formation of false head) and chlamydospore (if formed, simple or in pairs or in chains).Rhizoctonia isolates were identified based on colony morphology on PDA, hyphalmorphology (width and the characteristic right-angle branching) and cellular number ofnuclei.

2.2 Pathogenicity tests

Ten to 15 isolates of each species of fungi isolated from necrotic conifer roots and crownsof approximately 205 isolates were selected randomly for pathogenicity testing (Table 2).Pathogenic isolates collected from the source species were used to inoculate the coniferspecies from which they were originally isolated, as well as from additional conifer species(Table 3). Six-inch (15.2-cm) pots were filled with steam-sterilized soil that was aged in thegreenhouse for 45 days before use. Seeds of pine andor cypress were surface-sterilized in1% sodium hypochloride for 15 s and kept in the dark at 5 C in moist sand (sterilized for1 h at 180C) for 7 weeks to germinate. Five to seven pots were sown with 510

germinated seeds of each coniferous species and grown for 20 days in a greenhouse(2025C, ambient light) prior to inoculation. The number of seedlings per pot varied, butthe total number of seedling per coniferous species used for inoculation was between 30and 50. The pots were watered as needed from the top. The percentage of dead seedlingswas calculated using the following formulae:

SM ndeadntotal

100

where seedling mortality (SM) is expressed as a percentage of the number of dead seedlings(ndead) to the total number of inoculated seedlings (ntotal). Inoculum was prepared onsubstrates of sterile barley and corn seeds, respectively, for the isolates of Pythium and

Phytophthora and sterile wheat kernels for Rhizoctonia and Fusarium isolates. Twohundred grams of each of inoculum substrate was first soaked in water for 24 h and thenautoclaved for three successive days (for 20 min each) in 0.5-l glass conical flasks (Carlingand Sumner 1992; Martin 1992; Windels 1992). Ten 5-mm blocks of fresh cultures of thefungal isolates were added to each flask and incubated for 23 weeks at 20 C, and flaskswere shaken every 3 days to homogenize the mixture and avoid caking.

For inoculation, 30 g of inoculum was added to 1 kg of sterile soil and mixed evenly tomake a uniform mixture. Six-inch (15.2-cm) plastic pots were filled with the inoculummixture and sown with 10 germinated seeds of each conifer species. Control pots were

Table 1. Taxonomic designations and sources of representative fungi isolated from seedlings of

different conifer species and their differential occurrence on various species of pine and cypress.

Coniferous species

Fungal isolates

Phytophthora Pythium Rhizoctonia Fusarium Macrophomina

Pinus eldarica + + + + +Pinus brutia + ) ) + )Cupressus arizonica ) + + + +Cupressus sempervirens

var. horizontalis) + ) + +

Cupressus sempervirensvar. fastigiata

+ + ) + )

Thuja orientalis ) + + + )


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filled with an even mixture of 30 g of sterile substrate and 1 kg of sterile soil. All pots werewatered thoroughly and transferred to greenhouse at 2025C where they were monitoredfor 35 weeks.

Symptoms of control and pathogen-challenged seedlings were recorded daily after7 days of inoculation and continued for 35 weeks. Samples of decayed seeds andorpieces of roots and crowns of symptomatic plants were cultured on general and selectivemedia as described in the isolation and identification section to recover the inoculatedfungi.

3 ResultsSeveral fungi were associated with root and crown rot of pine and cypress. Five genera ofknown root andor crown pathogenic fungi were isolated from roots and crowns ofdiseased seedlings. A summary of the occurrence of various fungi isolated from coniferousspecies used in this study is presented in Table 1.

3.1 Fungi associated with seedling blight

Isolates ofPhytophthora isolated from Tehran pine, brutia pine and common cypress haduni- and occasionally bi-papillate sporangia, 34.0743.10 lm long, with amphigynousantheridia. The isolates were all from A2 mating type and formed oogonia in dual cultures

with compatible isolates of the A1 mating type from Phytophthora capsici. Oospores

Table 2. Pathogenicity tests of fungi isolated from root and crown of various species of pine andcypress under greenhouse conditions.

Fungal isolates1 Source species2

No. ofisolatestested

Per centpathogenic

isolates

1 Pinus eldarica 15 1001 P. brutia 10 1001 Cupressus sempervirens var. fastigiata 10 1002-1 P. eldarica 10 02-1 C. arizonica 15 802-2 C. sempervirens var. horizontalis 15 702-3 C. sempervirens var. fastigiata 10 02-3 T. orientalis 10 03-1 C. arizonica 10 803-2 C. arizonica 10 503-2 P. eldarica 10 403-2 C. sempervirens var. horizontalis 10 0

3-2 T. orientalis 10 04 P. eldarica 10 04 P. brutia 10 04 C. arizonica 10 04 C. sempervirens var. horizontalis 10 04 C. sempervirens var. fastigiata 10 04 T. orientalis 10 0

11 = Phytophthora nicotianae var. parasitica, 2-1 = Pythium ultimum, 2-2 = Pythium paroecan-drum, 2-3 = Pythium sp., 3-1 = Rhizoctonia solani, 3-2 = Binucleate Rhizoctonia-like fungus,4 = Fusarium sp.2Source species is the conifer species from which the fungus was isolated, P = Pinus, C= Cupressus,T = Thuja.


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measured 20.827.4 lm diameter. Maximum, optimum and minimum temperatures for thehyphal growth of isolates were 39.5, 3032 and 10C, respectively. The isolates were

identified as Phytophthora nicotianae Breda de Haan var. parasitica Dasture.Isolates of Pythium from Arizona cypress were identified by morphology (globose

hyphal swellings and intercalary sporangia; antheridia monoclinous and occasionallydiclinous; oogonia terminal, smooth-walled; 2024 lm in diameters, oospores aplerotic,globose, wall 23 lm thick). Isolates of Pythium from common cypress possessed thefollowing morphology: globose, intercalary to terminal sporangia; globose hyphalswellings 30 lm diameter; oogonia intercalary, single or in chains of 34; antheridiamonoclinous; oospores aplerotic, 1618 lm diameter, smooth-walled, wall 0.51.5 lmthick. Arizona and common cypress isolates were identified as P. ultimum Trow andPythium paroecandrum Drechsler, respectively. Both identifications were verified byCABI, UK.

Rhizoctonia isolates from Tehran pine and Arizona cypress were identified as abinucleate Rhizoctonia-like fungus according to mean of hyphal diameter (4.4 and 4.15 lm,respectively). Another isolate of Rhizoctonia from Arizona cypress identified as R. solanihad more than two nuclei in each cell with a mean hyphal diameter of 6.5 lm and typicallyright-angled hyphal branching.

Identification of species of isolates ofFusarium and Macrophomina from the seedlings ofvarious conifer species was not attempted as all isolates were non-pathogenic.

3.2 Fungi associated with seeds

Rhizopus stolonifer, Aspergillus niger, Alternaria sp. and Penicillium sp. were associatedwith seed samples from all conifer species examined in this study. Only one isolate of

Fusarium was found on the seeds of Tehran pine and identified as Fusarium proliferatum

Table 3. Seedling mortality1 of various species of pine and cypress caused by the pathogenicisolates ofPythium, Phytophthora and Rhizoctonia under greenhouse conditions.

Fungal isolates2 Source species3 Conifer species tested

Per cent post-emergencedamping-off

After 15 days After 30 days

1 Pinus eldarica P. eldarica 60 901 P. eldarica C. s. var. fastigiata 40 801 P. brutia P. brutia 50 901 C. s. var. fastigiata C. s. var. fastigiata 50 702-1 C. s. arizonica C . arizonica 90 1002-1 C. arizonica C. s. var. fastigiata 80 1002-1 C. arizonica T . orientalis 30 502-2 C. s. var. horizontalis C. s. var. horizontalis 80 902-2 C. s. var. horizontalis C. arizonica 80 902-2 C. s. var. horizontalis T. orientalis 20 303-1 P. eldarica P. eldarica 50 70

3-1 C. arizonica C . arizonica 60 803-2 C. arizonica C . arizonica 50 80

1Measured as percentage of dead seedlings out of total number of seedlings inoculated. Seedlingswere 20 days old at the time of inoculation.21 = Phytophthora nicotianae var. parasitica, 2-1 = Pythium ultimum, 2-2 = Pythium paroecan-drum, 3-1 = Rhizoctonia solani, 3-2 = Binucleate Rhizoctonia-like fungus.3Abbreviations for coniferous species are P = Pinus, C. s. = Cupressus sempervirens,C = Cupressus, T = Thuja.


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(Matsushima) Nirenberg according to morphological characteristics (Gerlach andNirenberg 1982 and Nelson et al. 1983).

3.3 Pathogenicity tests

None of the plants grown in non-infested soils showed the symptoms of diseases. Duringthe first 2030 days of incubation, seedlings grown in infested soil showed blightsymptoms typical of those observed in nurseries (Table 2; Fig. 1ad). Isolates ofPhytophthora and Pythium caused the highest pine and cypress SM (Table 2). Among allfungi isolated from the seeds of pine and cypress, only F. proliferatum isolated fromTehran pine seeds was pathogenic. This isolate caused pre-emergence damping-off on 90and 86% of Tehran pine and Arizona cypress seedlings, respectively.

4 Discussion

Pre- and post-emergence soilborne diseases of pine and cypress in Fars Province are a

serious problem for forest nurseries. There are few studies on the causes of coniferseedlings mortality in Iranian conifer tree nurseries (Mirabolfathi 1991; Mirabolfathiand Ershad 1991), thereby very little is known about the causal agents of seedling root andcrown rot in Iran. In this study, different species of Phytophthora, Pythium, Rhizoctoniaand Fusarium were isolated from symptomatic seedlings of pine and cypress, and thepathogenicity of their isolates was confirmed under greenhouse conditions on seedlings ofall coniferous species used in the study.

Phytophthora nicotianae var. parasitica frequently isolated from the seedlings ofC. sempervirens var. fastigiata, P. brutia and P. eldarica was pathogenic on all threespecies (Tables 2 and 3). This pathogen caused highest losses to seedlings of P. eldarica andC. sempervirens var. fastigiata. Pythium ultimum and P. paroecandrum caused pre- andpost-emergence damping-off on seedlings ofC. arizonica, C. sempervirens var. fastigiata,

C. sempervirens var. horizontalis, P. eldarica and T. orientalis. Cupressus arizonica andC. sempervirens var. horizontalis were the most susceptible species to both species ofPythium, whereas P. ultimum and P. paroecandrum did not show much virulence againstT. orientalis (Table 3). Various species ofPythium and Phythophthora have been shown tooccur frequently in forest nurseries, and their potential significance to cause losses in toforest nurseries has been discussed previously (Bumbieris 1974; Webb and Dahm 1981;Barnard et al. 1985; Dixon et al. 1991). Our results are indicative of the universal spreadof these soilborne pathogenic fungi. The frequent isolation of these pathogens from theroots and crowns of blighted seedlings of pine and cypress indicates their capacity tosubsist in nursery soils for many years. Increased populations of pythiaceous fungi areassociated with continuous cropping of seedlings of the same coniferous species within

nurseries (Edmonds and Heather 1973; English and Barnard 1981). Anotherconsequence of continuous cropping of the same species in nurseries is the large amountsof plant debris left in the soil after site preparation which may contribute to the substantialbuildup of pythiaceous and other soilborne pathogens in nursery soils.

Isolates of Rhizoctonia spp. have been described as non-specialized and occasionallyaggressive pathogens (English et al. 1986). Isolates ofR. solani from Arizona cypress andbinucleate Rhizoctonia-like fungus from Tehran pine and Arizona cypress caused reducedgrowth, chlorosis, necrosis, defoliation and finally death of the seedlings. However, in ourstudy, these isolates were of minor importance and showed lower aggressiveness comparedto pythiaceous pathogens. Although Rhizoctonia isolates showed less virulence, most ofthe inoculated seedlings showed progressive symptoms of blight development.

Disease management programs are required to prevent or minimize losses in forest

nurseries, which impact nursery economics. Adequate knowledge of the causes of


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soilborne disease in forest nurseries is a prerequisite for any successful disease management.The identification of soilborne pathogens that cause seedling blight in particular is of primeimportance before any effective control method can be advised to nursery growers. Newtechniques such as DNA analysis are also now in widespread use for the identification ofthe fungal pathogens and may help expedite the identification. Cultural phenotypic, and

rDNA internal transcribed spacer (ITS) sequence-based identification was used to studythe fungal diversity in the rhizosphere of healthy and diseased clonal black spruce (Piceamariana) plants (Vujanovic et al. 2007). Identification of virulent isolates ofF. oxysporumbased on colony morphology appears to be inadequate as shown in recent molecular typingstudies. Molecular characterization ofF. oxysporum and Fusarium commune isolates from aconifer nursery revealed the insufficiency of colony morphology for the identification ofvirulent isolates of these fungi (Stewart et al. 2006).

One of the most useful management strategies for the soilborne diseases of forestnurseries is the implementation of cultural methods to avoid, exclude or eradicatepathogens. Our results and similar findings are particularly valuable to inform decisions onhow to establish and maintain conditions unfavourable to pathogens. For instance, while

Fusarium root rot can be minimized by preventing water stress through sufficientirrigation, root rot caused by Phytophthora and Pythium may be best managed byimproved seedbed drainage.

Acknowledgements

Financial support from former Research Center for Natural Resources and Animal Sciences of Farsprovince (Project No. 72-031098127-06) is appreciated. Authors appreciate the reviewers of the paperfor their valuable suggestions.

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