For. Path.

2010 Blackwell Verlag GmbH

doi: 10.1111/j.1439-0329.2010.00697.x

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
1

Fars Agricultural and Natural Recourses Research Center, Zarghan, Shiraz, Iran; 2Department of Plant Protection, Shiraz University, Badjgah 71444-65186, Iran; 3E-mail: zarghani@shirazu.ac.ir (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 (Cupressus sempervirens var. fastigiata), common cypress (C. sempervirens var. horizontalis) and oriental arborvitae (Thuja orientalis), showing chlorosis, necrosis, stunted growth, defoliation and root and crown rot symptoms were collected from pine and cypress nurseries across Fars province at various time intervals. Infected tissues were washed and cultured on acidied potato dextrose agar (PDA) and corn meal 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 collected from 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 symptomatic seedlings of pine and cypress at different times during the growing season. Pathogenicity of isolates of Phytophthora, Pythium and Rhizoctonia was conrmed on seedlings of all plant species, whereas Fusarium sp. isolated from the seeds of Tehran pine was only pathogenic on seedlings of Tehran pine and Arizona cypress. Virulent isolates identied as Phytophthora nicotiana var. parasitica, Pythium ultimum, Pythium paroecandrum, Rhizoctonia solani and binucleate Rhizoctonia-like fungus caused root and crown rot of pine and cypress seedlings. Pathogenic isolates of Fusarium identied as Fusarium proliferatum also caused pre-emergence seed decay of pine and cypress. Isolates of Phytophthora 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 other temperate areas with better soil conditions, it is difcult to achieve afforestation on a large scale in Iran, and as a consequence, most of the afforestation is conducted with non-native species (Mosadegh 1981). Establishment of conifer plantations is a top priority of the Iranian 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 and runoff and thus minimizing the loss of topsoil and delaying desertication, afforestation provides an extra bonus of an increase of per capita green space and picnic areas for the public (Mosadegh 1981). Management of forest nurseries to supply regional-scale outplantings with disease-free and vigorous seedlings is essential for the success of aforementioned projects. Production of conifer seedlings for outplanting has been limited by seed, root and crown rot of the seedlings by soilborne fungi in forest tree nurseries (Dixon et al. 1991) which has led to a need to sow additional seeds to offset losses because of non-viable seeds and seedling *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

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

blights. Different seedborne and soilborne fungi have been reported to cause seed decay and root and crown rot of conifer seedlings. Cram and Faeredrich (2009) provided a comprehensive list of relatively important seedborne pathogens of North American forest orchards 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 and Grand 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 and Bumbieris 1961; Vaartaja et al. 1967; Edmonds and Heather 1973; Bumbieris 1974; Huang and Kulman 1990; Dumroese and James 2005), Fusarium solani, Fusarium oxysporum, Fusarium subglutinans, Fusarium moniliforme (Pawuk 1978; Brissette et al. 1991; Dumroese and James 2005) Rhizoctonia solani, and binucleate Rhizoctonia-like fungus (English and Barnard 1981; English et al. 1986) and Cylindrocarpon (Dumroese and James 2005) have been associated with seedling blight of coniferous seedlings 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 United States, and most conifer species (including pines) grown in warm lowland agricultural soils are susceptible to the disease (Barnard 1994). Fusarium in particular occurs as an important cause of pre- and post-emergence damping-off, root rot and shoot blight on western larch and Douglas-r in Canada (Peterson 2007). Fusarium oxysporum is considered the most prevalent of Fusaria within the F. oxysporum species complex occurring on conifer seedlings in the United States. James and Dumroese (2007) evaluated the application of some commercially available biological control agents such as F. oxysporum Q12 as potential biological control against pathogenic Fusarium and Cylindrocarpon species in nurseries. The objective of this study was to identify pathogenic soilborne fungi associated with pine and cypress seed decay and root and crown rot of their seedlings.

2 Materials and methods

2.1 Isolation and identication Seedlings of different species of conifers, including Tehran pine (Pinus eldarica), brutia pine (Pinus brutia), Arizona cypress (Cupressus arizonica), Shirazean cypress (Cupressus sempervirens var. fastigiata), common cypress (C. sempervirens var. horizontalis) and oriental arborvitae (Thuja orientalis), with symptoms of damping-off, stunting, chlorosis and necrosis (Fig. 1e,f) were collected from pine and cypress nurseries across Fars province about once a month during various seasons in 2000 and 2001. Coniferous species from which fungal isolates were isolated are called source species to avoid confusion with host species as not every specimen isolated from a coniferous species may prove pathogenic. Root and crown samples from healthy seedling were also collected to serve as controls. Samples were stored in a cooler ice box to prevent heating when transferring to the laboratory. Seed samples were also obtained from nursery seed stocks. Seed, crown and root samples were surface-sterilized with 1% sodium hypochloride for 1.5 min, rinsed with sterile distilled water and blotted dry in sterile paper towel. Samples were placed on acidied 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 incubated at 25C for 35 days. Hyphal tips or single spores were taken from individual colonies on 2% 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

Pre- and post-emergence seedling blight

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 1. (ad) Effect of inoculation of pine and cypress seedlings within 2030 days after inoculation with (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 (The right side pots in ad were lled with non-infested soil). (e) and (f) show a high rate of mortality of various species of pine and cypress caused by pathogenic isolates of Pythium, Phytophthora and Rhizoctonia under nursery conditions.

and alfalfa leaf piece agar (Gerlach and Nirenberg 1982; Nelson et al. 1983; Sneh et al. 1991, 1996; Burgess et al. 1994) for identication. To make a conclusive identication of the genera Pythium and Phytophthora, aside from their colony morphology and growth at various temperatures, their asexual and sexual reproductive structures were observed under a microscope at appropriate magnication (40 dry objective). Sporangial morphology includes shape, size, papilla formation and their size and number (only in Phytophthora), and 1015 randomly selected sporangia or oospores were examined under a microscope and oospore formation and morphology were recorded. Identication of Fusarium 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. Zakeri, H. Hamzeharghani, Z. Banihashemi et al.

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 identied based on colony morphology on PDA, hyphal morphology (width and the characteristic right-angle branching) and cellular number of nuclei. 2.2 Pathogenicity tests Ten to 15 isolates of each species of fungi isolated from necrotic conifer roots and crowns of approximately 205 isolates were selected randomly for pathogenicity testing (Table 2). Pathogenic isolates collected from the source species were used to inoculate the conifer species from which they were originally isolated, as well as from additional conifer species (Table 3). Six-inch (15.2-cm) pots were lled with steam-sterilized soil that was aged in the greenhouse for 45 days before use. Seeds of pine and or cypress were surface-sterilized in 1% sodium hypochloride for 15 s and kept in the dark at 5C in moist sand (sterilized for 1 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, but the total number of seedling per coniferous species used for inoculation was between 30 and 50. The pots were watered as needed from the top. The percentage of dead seedlings was calculated using the following formulae: SM ndead 100 ntotal

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 on substrates of sterile barley and corn seeds, respectively, for the isolates of Pythium and Phytophthora and sterile wheat kernels for Rhizoctonia and Fusarium isolates. Two hundred grams of each of inoculum substrate was rst soaked in water for 24 h and then autoclaved for three successive days (for 20 min each) in 0.5-l glass conical asks (Carling and Sumner 1992; Martin 1992; Windels 1992). Ten 5-mm blocks of fresh cultures of the fungal isolates were added to each ask and incubated for 23 weeks at 20C, and asks were 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 to make a uniform mixture. Six-inch (15.2-cm) plastic pots were lled with the inoculum mixture 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. Fungal isolates Coniferous species Pinus eldarica Pinus brutia Cupressus arizonica Cupressus sempervirens var. horizontalis Cupressus sempervirens var. fastigiata Thuja orientalis Phytophthora + + Pythium + ) ) + + + ) + + ) Rhizoctonia + + ) ) ) Fusarium + + + + + + Macrophomina + + + )

) )

Pre- and post-emergence seedling blight

Table 2. Pathogenicity tests of fungi isolated from root and crown of various species of pine and cypress under greenhouse conditions. No. of isolates tested 15 10 10 10 15 15 10 10 10 10 10 10 10 10 10 10 10 10 10 Per cent pathogenic isolates 100 100 100 0 80 70 0 0 80 50 40 0 0 0 0 0 0 0 0

Fungal isolates1 1 1 1 2-1 2-1 2-2 2-3 2-3 3-1 3-2 3-2 3-2 3-2 4 4 4 4 4 4

Source species2 Pinus eldarica P. brutia Cupressus sempervirens var. fastigiata P. eldarica C. arizonica C. sempervirens var. horizontalis C. sempervirens var. fastigiata T. orientalis C. arizonica C. arizonica P. eldarica C. sempervirens var. horizontalis T. orientalis P. eldarica P. brutia C. arizonica C. sempervirens var. horizontalis C. sempervirens var. fastigiata T. orientalis

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

lled with an even mixture of 30 g of sterile substrate and 1 kg of sterile soil. All pots were watered thoroughly and transferred to greenhouse at 2025C where they were monitored for 35 weeks. Symptoms of control and pathogen-challenged seedlings were recorded daily after 7 days of inoculation and continued for 35 weeks. Samples of decayed seeds and or pieces of roots and crowns of symptomatic plants were cultured on general and selective media as described in the isolation and identication section to recover the inoculated fungi.

3 Results
Several fungi were associated with root and crown rot of pine and cypress. Five genera of known root and or crown pathogenic fungi were isolated from roots and crowns of diseased seedlings. A summary of the occurrence of various fungi isolated from coniferous species used in this study is presented in Table 1. 3.1 Fungi associated with seedling blight Isolates of Phytophthora isolated from Tehran pine, brutia pine and common cypress had uni- and occasionally bi-papillate sporangia, 34.0743.10 lm long, with amphigynous antheridia. 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

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

Table 3. Seedling mortality1 of various species of pine and cypress caused by the pathogenic isolates of Pythium, Phytophthora and Rhizoctonia under greenhouse conditions. Per cent post-emergence damping-off Fungal isolates2 1 1 1 1 2-1 2-1 2-1 2-2 2-2 2-2 3-1 3-1 3-2
1

Source species3 Pinus eldarica P. eldarica P. brutia C. s. var. fastigiata C. s. arizonica C. arizonica C. arizonica C. s. var. horizontalis C. s. var. horizontalis C. s. var. horizontalis P. eldarica C. arizonica C. arizonica

Conifer species tested P. eldarica C. s. var. fastigiata P. brutia C. s. var. fastigiata C. arizonica C. s. var. fastigiata T. orientalis C. s. var. horizontalis C. arizonica T. orientalis P. eldarica C. arizonica C. arizonica

After 15 days 60 40 50 50 90 80 30 80 80 20 50 60 50

After 30 days 90 80 90 70 100 100 50 90 90 30 70 80 80

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

measured 20.827.4 lm diameter. Maximum, optimum and minimum temperatures for the hyphal growth of isolates were 39.5, 3032 and 10C, respectively. The isolates were identied as Phytophthora nicotianae Breda de Haan var. parasitica Dasture. Isolates of Pythium from Arizona cypress were identied by morphology (globose hyphal swellings and intercalary sporangia; antheridia monoclinous and occasionally diclinous; oogonia terminal, smooth-walled; 2024 lm in diameters, oospores aplerotic, globose, wall 23 lm thick). Isolates of Pythium from common cypress possessed the following morphology: globose, intercalary to terminal sporangia; globose hyphal swellings 30 lm diameter; oogonia intercalary, single or in chains of 34; antheridia monoclinous; oospores aplerotic, 1618 lm diameter, smooth-walled, wall 0.51.5 lm thick. Arizona and common cypress isolates were identied as P. ultimum Trow and Pythium paroecandrum Drechsler, respectively. Both identications were veried by CABI, UK. Rhizoctonia isolates from Tehran pine and Arizona cypress were identied as a binucleate Rhizoctonia-like fungus according to mean of hyphal diameter (4.4 and 4.15 lm, respectively). Another isolate of Rhizoctonia from Arizona cypress identied as R. solani had more than two nuclei in each cell with a mean hyphal diameter of 6.5 lm and typically right-angled hyphal branching. Identication of species of isolates of Fusarium and Macrophomina from the seedlings of various 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 associated with seed samples from all conifer species examined in this study. Only one isolate of Fusarium was found on the seeds of Tehran pine and identied as Fusarium proliferatum

Pre- and post-emergence seedling blight

(Matsushima) Nirenberg according to morphological characteristics (Gerlach and Nirenberg 1982 and Nelson et al. 1983). 3.3 Pathogenicity tests None of the plants grown in non-infested soils showed the symptoms of diseases. During the rst 2030 days of incubation, seedlings grown in infested soil showed blight symptoms typical of those observed in nurseries (Table 2; Fig. 1ad). Isolates of Phytophthora and Pythium caused the highest pine and cypress SM (Table 2). Among all fungi isolated from the seeds of pine and cypress, only F. proliferatum isolated from Tehran pine seeds was pathogenic. This isolate caused pre-emergence damping-off on 90 and 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 conifer seedlings mortality in Iranian conifer tree nurseries (Mirabolfathi 1991; Mirabolfathi and Ershad 1991), thereby very little is known about the causal agents of seedling root and crown rot in Iran. In this study, different species of Phytophthora, Pythium, Rhizoctonia and Fusarium were isolated from symptomatic seedlings of pine and cypress, and the pathogenicity of their isolates was conrmed under greenhouse conditions on seedlings of all coniferous species used in the study. Phytophthora nicotianae var. parasitica frequently isolated from the seedlings of C. sempervirens var. fastigiata, P. brutia and P. eldarica was pathogenic on all three species (Tables 2 and 3). This pathogen caused highest losses to seedlings of P. eldarica and C. sempervirens var. fastigiata. Pythium ultimum and P. paroecandrum caused pre- and post-emergence damping-off on seedlings of C. arizonica, C. sempervirens var. fastigiata, C. sempervirens var. horizontalis, P. eldarica and T. orientalis. Cupressus arizonica and C. sempervirens var. horizontalis were the most susceptible species to both species of Pythium, whereas P. ultimum and P. paroecandrum did not show much virulence against T. orientalis (Table 3). Various species of Pythium and Phythophthora have been shown to occur frequently in forest nurseries, and their potential signicance to cause losses in to forest 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 spread of these soilborne pathogenic fungi. The frequent isolation of these pathogens from the roots and crowns of blighted seedlings of pine and cypress indicates their capacity to subsist in nursery soils for many years. Increased populations of pythiaceous fungi are associated with continuous cropping of seedlings of the same coniferous species within nurseries (Edmonds and Heather 1973; English and Barnard 1981). Another consequence of continuous cropping of the same species in nurseries is the large amounts of plant debris left in the soil after site preparation which may contribute to the substantial buildup of pythiaceous and other soilborne pathogens in nursery soils. Isolates of Rhizoctonia spp. have been described as non-specialized and occasionally aggressive pathogens (English et al. 1986). Isolates of R. solani from Arizona cypress and binucleate Rhizoctonia-like fungus from Tehran pine and Arizona cypress caused reduced growth, chlorosis, necrosis, defoliation and nally death of the seedlings. However, in our study, these isolates were of minor importance and showed lower aggressiveness compared to pythiaceous pathogens. Although Rhizoctonia isolates showed less virulence, most of the 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

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

soilborne disease in forest nurseries is a prerequisite for any successful disease management. The identication of soilborne pathogens that cause seedling blight in particular is of prime importance before any effective control method can be advised to nursery growers. New techniques such as DNA analysis are also now in widespread use for the identication of the fungal pathogens and may help expedite the identication. Cultural phenotypic, and rDNA internal transcribed spacer (ITS) sequence-based identication was used to study the fungal diversity in the rhizosphere of healthy and diseased clonal black spruce (Picea mariana) plants (Vujanovic et al. 2007). Identication of virulent isolates of F. oxysporum based on colony morphology appears to be inadequate as shown in recent molecular typing studies. Molecular characterization of F. oxysporum and Fusarium commune isolates from a conifer nursery revealed the insufciency of colony morphology for the identication of virulent isolates of these fungi (Stewart et al. 2006). One of the most useful management strategies for the soilborne diseases of forest nurseries is the implementation of cultural methods to avoid, exclude or eradicate pathogens. Our results and similar ndings are particularly valuable to inform decisions on how to establish and maintain conditions unfavourable to pathogens. For instance, while Fusarium root rot can be minimized by preventing water stress through sufcient irrigation, root rot caused by Phytophthora and Pythium may be best managed by improved seedbed drainage.

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

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