THE SENSITIVITY OF ASPERGILLUS NIGER AND FUSARIUM OXYSPORUM f. sp. CEPAE TO FUNGISTASIS IN ONION-GROWING SOILS

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1 015_JPP459Ozer_ :09 Pagina 401 Journal of Plant Pathology (2009), 91 (2), Edizioni ETS Pisa, THE SENSITIVITY OF ASPERGILLUS NIGER AND FUSARIUM OXYSPORUM f. sp. CEPAE TO FUNGISTASIS IN ONION-GROWING SOILS N. Özer 1, M. Koç 2 and B. Der 2 1 Department of Plant Protection, Faculty of Agriculture, Namık Kemal University, Tekirdag, 59030, Turkey 2 Graduate School of Natural and Applied Sciences, Namık Kemal University, Tekirdag, 59030, Turkey SUMMARY Twenty-seven soil samples were collected from the onion (Allium cepa L.) fields of Tekirdag province, Turkey. These samples were investigated for the sensitivity of Aspergillus niger V. Tieghem (AN) and Fusarium oxysporum Schlechtend.: Fr. f. sp. cepae (H. N. Hans.) W. C. Snyder H. N. Hans (FOC), known as the causal agents of black mould and basal rot of onion, respectively, to soil fungistasis. Fungistasis was evaluated using two methods: inhibition of pathogen spore germination by volatile compounds from the soil and determination of the antagonistic fungal population of soil samples. Volatile compounds in twelve of the soil samples strongly (>70%) inhibited spore germination of only AN. Inhibition rates of volatile compounds were not correlated with physical and chemical characters of the soils. Fungi isolated from soil samples were evaluated for their antagonism to both pathogens using dual cultures. The population of species causing over 70% inhibition of radial growth on pathogens was calculated in soil samples. The presence of both volatile compounds inhibiting spore germination of AN and populations of fungi antagonistic for AN and FOC were observed in six of the soil samples. The possible effects of two fungistatic mechanisms in soils on disease development by these pathogens are discussed. Key words: antagonist fungus population, Aspergillus niger, Fusarium oxysporum f. sp. cepae, onion (Allium cepa L.), soil volatile compounds. INTRODUCTION Aspergillus niger V. Tieghem (AN) and Fusarium oxysporum Schlechtend.: Fr. f. sp. cepae (Hans.) Snyder and Hansen (FOC) cause black mould and basal rot, respectively, in onion (Allium cepa L.). Both fungi are Corresponding author: N. Özer Fax: nurayozer@hotmail.com seed- and soil-borne and are generally present in soils where onion is grown extensively (Havey, 1995; Sumner, 1995; Özer and Köycü, 2004). They can infect during seedling and bulb development, and in storage depending upon the time of year, environmental conditions, and cultivars (Özer and Köycü, 2004). To control these diseases seed or seedling application of fungicides is routine but this causes pollution of the environment (Özer and Köycü, 1998; Cramer, 2000). An alternative is the use of biological control and organic soil amendments. Trichoderma spp. can reduce diseases caused by AN and FOC (Rajendran and Ranganathan, 1996; El Neshawy et al., 1999; Srivastava and Tiwari, 2003; Coskuntuna and Özer, 2008). Organic amendments with especially sunflower stalks, but also alfalfa and Hungarian vetch, reduce set rot by AN and FOC (Özer et al., 2002). A limitation to the use of biocontrol and organic amendments is insufficient knowledge of the ecological interactions taking place in the soil and root environment (Whipps, 2001). In this regard, soil fungistasis is an attractive addition to biological or organic control and to finding new biocontrol agents. Fungistasis, first described by Dobbs and Hinson (1953), is the suppressiveness of natural soils to the germination and growth of fungi. Plant pathogenic fungi appear to be more sensitive to fungistasis than saprophytic fungi. Therefore, there is a positive correlation between fungistasis and disease suppression (Kao and Ko, 1983; Lockwood, 1986; Larkin et al., 1996; Knudsen et al., 1999). Certain aspects of fungistasis toward soil-borne fungi causing wilt disease on plants other than onion have been described. Some studies have suggested that soil fungistasis can be attributed to the presence of high populations of antagonistic microorganisms (fluorescent pseudomonads and saprophytic fungi) (Bora and Nemli, 1973; Johri et al., 1975; Tamietti and Pramotton, 1990; Larkin et al., 1993; De Boer et al., 2003). Among these microorganisms, biocontrol fungi have a much greater potential than bacteria to grow and spread through soil and in the rhizosphere by means of hyphal growth (Whipps, 2001). Other studies have explained the role of both microbiological and physicochemical properties of soil on fungistasis (Mishra and Kanaujia, 1973; Bora et al., 1982; Toy-

2 015_JPP459Ozer_ :09 Pagina Sensitivity of A. niger and F. oxysporum f.sp. cepae to fungistasis Journal of Plant Pathology (2009), 91 (2), ota et al., 1996; Mondal and Hykumachi, 1998; Wahid et al., 1998). An additional aspect of fungistasis is the presence in soil of volatile or nonvolatile compounds that inhibit fungal spore germination. This mechanism and its relationships with soil properties such as texture and ph have been recorded for different soil-borne fungi (Romine and Baker, 1973; Hora and Baker, 1974; Ko and Hora, 1974; Roth and Griffin, 1980; Liebman and Epstein, 1992; Chuankun et al., 2004). No data have been presented on the mechanism of fungistasis against the onion pathogens, AN and FOC. Our objective was to test the sensitivity of FOC and AN to two types of fungistasis, the presence of volatile compounds and saprophytic fungi in onion-growing soils, and their possible interactions with physical and chemical characters of the soil. The interactions between candidate fungi and pathogens were determined to gain a preliminary understanding of the biological control mechanism and to discover new fungi antagonistic against FOC and AN. MATERIALS AND METHODS Fungal pathogens. Aspergillus niger (AN6) and Fusarium oxysporum f. sp. cepae (FOC16) isolates obtained from naturally infected onion seeds were used (Köycü and Özer, 1997). These isolates were selected for their high level of aggressiveness in a previous study (Özer and Köycü, 1997). Soil samples. Twenty-seven soils were collected from onion fields located in Tekirdag province, Turkey. Samples were taken from a depth of 2-20 cm when the onions were at the seedling stage. Sites were selected on the basis of the occurrence of either symptoms of damping-off or symptomless plants (Table 1). Each sample consisted of a mixture of 3 soil sub-samples taken in a diagonal section of the field and combined in a paper bag. The soils differed in texture, ph, salt content, Ca- CO 3, organic matter and N (Table 2). The soils were passed through a sieve, air-dried and stored at 4 o C. Prior to use, soil moisture was adjusted to 20% (wt/wt). Soil volatiles fungistasis. Volatiles fungistatic activity of the 27 soils was assessed using the technique of Chuankun et al. (2004). Ten grams of soil were added to an empty 6.0 cm diameter Petri dish. The top of the plate was replaced with the bottom portion of another Petri dish with a 5 mm thickness of 1% water agar (WA). The plate bottoms were sealed together with Parafilm so that volatile compounds could diffuse into the WA. After incubation at 25 C for 4 days, the WA was inoculated with 10 µl of fungal spore suspension (approximately 300 spores/ml) and re-sealed with Parafilm. As a control, all soils were treated by autoclaving (twice, each time for 30 min at 121 C) to kill soil microorganisms and were then assayed for fungistasis by the same method. All plates were incubated at 25 C for 24 h and the percentage of spore germination inhibition (SGI %) was calculated as SGI% = [(sc-sd)/(scx100)], where sc is spore germination of control and sd is spore germination in the presence of volatiles. Inhibition >70% was considered as strong fungistasis, 50-70% as moderate, 25-50% as slight, and 25% as no fungistasis. Isolation and selection of candidate antagonist fungi. Fungal populations of soil samples were determined on two different media. The first was potato dextrose agar (PDA, Oxoid) acidified with 0.5 ml l -1 of 1 N lactic acid and modified with 0.1 ml l -1 of Igepal, streptomycin (100 mg l -1 ) and chlortetracycline (50 mg l -1 ) (MPDA) (Latorre et al., 1997), final ph 5.0. The second was modified TSM, a medium selective for Trichoderma and Gliocladium spp., containing 1 g Ca(NO 3 ) 2, 0.26 g KNO 3, 0.26 g MgSO 4 7H 2 O, 0.12 g KH 2 PO 4, 1 g CaCl 2. 2H 2 O, 0.05 g citric acid, 2 g sucrose, 20 g agar, 1ml Igepal, 0.05 g chlortetracycline, 0.04 g captan/l (Smith et al., 1990). In the laboratory, a 10 g portion from each soil sample was added to 90 ml of sterile distilled water and shaken. This soil suspension was diluted 1: with sterile distilled water and 10 ml of the dilution was added to 190 ml of medium. This mixture was poured into ten Petri dishes (20 ml in each dish). The plates were incubated for 4 days at 25 C. The total number of colonies of each fungus in 1 g soil sample was determined and recorded. Isolates were grouped to species based on morphological characters (spore forming units, shape, color, size of conidia and conidiophores, and appearance) and odour of mycelia on plates. One isolate of each species was selected to test its antagonistic activity toward AN6 and FOC16. Antagonistic effects of candidate fungal species isolated from soil samples. Antagonistic effects of candidate fungi on AN6 and FOC16 were studied by dual culture in Petri dishes on two different culture media: malt extract agar (MEA) and potato dextrose agar (PDA). Two assays were conducted: the candidate fungus was inoculated 48 h before (deferred inoculation) and at the same time (simultaneous inoculation) as the pathogen. The first assay showed the presence of antifungal metabolites, whereas the second assessed its antagonistic capacity (Rodriguez et al., 2006). In both tests, two 5-mm diameter plugs of candidate fungus and pathogen were inoculated at equal distance from the periphery. Control dishes were inoculated with the pathogen isolate on each medium being assessed. Four replicates were included for each test. All Petri dishes were incubated at 25 C in the dark. The percentage of

3 015_JPP459Ozer_ :09 Pagina 403 Journal of Plant Pathology (2009), 91 (2), Özer et al. 403 radial growth inhibition (RGI%) was calculated as RGI% = [(rc-rd)/(rcx100)] where rc is the control pathogen colony radius and rd is the pathogen colony radius in dual culture. Inhibition zones were observed and alterations in hyphae or conidia were examined. The mycelial mats of FOC16, in contact with an antagonist or in the inhibition zone on dual culture plates, were gently lifted by a needle, placed in a drop of 0.05% cotton blue on a slide, and observed under a microscope for hyphal alterations. Conidia of AN6 were examined using the same process because of the absence of mycelial growth. Strong antagonistic fungus populations of soil samples. The population densities of fungal species that strongly antagonized (>70%) the pathogenic fungi, for at least one of the dual culture conditions (media or inoculation time) were evaluated by calculating total colony number of these species in soil samples tested on TSM or MPDA. The others (<70%) were accepted to be insignificant for soil fungistasis. Statistical analysis. Quantitative data on inhibition of spore germination (%) by volatile substances from soil samples, radial growth inhibition of pathogen (%) and population densities of strong antagonist fungi were statistically evaluated with analysis of variance (one way ANOVA) procedures of SPSS (Statistical Package for Social Sciences, Inc., 2001, Model Chicago). The Duncan Multiple Range test was used to compare means of treatments at P= 0.05 level. Correlation analysis (Pearson) was used to determine percent inhibition Table 1. Distribution of soil samples based on the presence of seedling damping-off in Tekirdag province. District Soils with symptomless seedlings Soils with diseased seedlings Total Malkara (M) M Central (C) C2-3, C6-7, C9-10 C1, C4, C5, C8 10 Saray (S) S1, S3-4 S2 4 Total Table 2. Some physical and chemical characters of soil samples. Soil sample Texture ph Salt (%) CaCO 3 (%) Organic matter (%) Total N (%) M1 Loamy clay M2 Sandy clay loam M3 Loamy clay M 4 Loamy clay M5 Clay loam M 6 Loamy M7 Loamy clay M8 Loamy M9 Loamy clay M10 Loamy clay M11 Loamy clay M12 Clay M13 Clay loam C1 Clay loam C2 Clay loam C3 Loamy clay C4 Clay loam sandy C5 Loamy clay C6 Loamy clay C7 Loamy clay C8 Loamy clay C9 Loamy clay C10 Loamy clay S1 Loamy clay S2 Loamy clay S3 Loamy clay S4 Loamy clay

4 015_JPP459Ozer_ :09 Pagina Sensitivity of A. niger and F. oxysporum f.sp. cepae to fungistasis Journal of Plant Pathology (2009), 91 (2), of spore germination by volatile substances, antagonistic fungus populations and physicochemical properties of the soil. RESULTS Soil volatiles fungistasis. A total of 27 soils from onion cultivated fields were evaluated for volatile compounds causing fungistasis against AN and FOC (Table 3). Results showed that twelve soil samples (M3-7, M9, M11, C1-3, C8 and C10) were strongly inhibitory, four soil samples (M1, M2, C9 and S4) were moderately inhibitory, and two soil samples (C7 and S3) were slightly inhibitory for spore germination of AN6; among the strongly inhibitory samples, M9 caused maximum spore germination inhibition followed by M3. Inhibition of spore germination in these two soils differed significantly compared to ten soil samples (M8, M10, M12-13, C4-6 and S1-3). Nine soil samples were not inhibitory to spore germination of this pathogen. FOC16 was not sensitive to soil volatiles from any of the samples. Inhibition of spore germination of FOC16 was generally <25%. Maximum inhibition (25.86%) was observed in one soil sample, M5. Inhibition of AN6 and FOC16 spore germination was not correlated with physicochemical characters of the soil, e.g. ph, salt, CaCO 3 or organic matter. Antagonistic effect of the candidate fungal species. Thirty-seven fungal species (3 Aspergillus spp., 24 Penicillium spp., 1 Thysanophora sp. and 9 Trichoderma spp.) were isolated. Occurrence of these species varied with different soil samples and media (data not shown). In dual culture experiments, isolates AS3, PEN13, PEN15, PEN17-18, TRIC2-4 and TRIC6-9 inhibited radial growth of AN6 more than 70% in one or more combinations of medium and inoculation time (Table 4). Among these isolates, TRIC4, TRIC7 and TRIC9 also had a hyperparasitic effect on AN6 with deferred inoculation on MEA or PDA. AN6 was highly susceptible to Trichoderma isolate TRIC3 and was inhibited over 70% by this isolate in all inoculation times and media. When the selected fungal species were assayed for antagonistic effect against FOC16, isolates AS3, PEN6, PEN18, TRIC5-6, and TRIC8-9 inhibited radial growth more than 70% with both media and inoculation modes (Table 5). Isolates AS2, PEN4-5, PEN9-11, PEN13-17, Table 3. Spore germination inhibition (SGI%) for A. niger (AN6) and F. oxysporum f. sp. cepae (FOC16) isolates by volatile compounds from soil samples. Soil sample SGI (%) AN6 FOC16 M a-e ab M a-d 6.03 bc M ab 0.00 c M a-c 2.58 bc M a-c a M a-c 8.98 a-c M a-c 5.75 bc M fg 0.07 c M a 0.13 c M g 0.00 c M a-c 5.09 bc M fg 5.64 bc M fg 0.00 c C a-c a-c C a-c 4.25 bc C a-c a-c C fg 0.00 c C g 1.19 c C e-g 2.75 bc C b-f 1.75 c C a-c a-c C a-d 5.75 bc C a-c 9.50 a-c S fg 0.00 c S d-g 0.00 c S c-g 1.25 c S a-d 2.75 bc Means in each column followed by the same letter do not differ significantly at P<0.05 according to the Duncan Multiple Range Test.

5 015_JPP459Ozer_ :09 Pagina 405 Journal of Plant Pathology (2009), 91 (2), Özer et al. 405 Fig. 1. Alterations in the spores of A. niger isolate AN6 by antagonistic fungal species. A. Control; B. Increased size and coagulation of the cytoplasm ; C. Abnormal morphology. Bar = 4 µm. Table 4. Radial growth inhibition (RGI %) for A. niger isolate AN6 in dual cultures with different fungal species isolated from soil samples, using simultaneous inoculation (A) and deferred inoculation (B) on MEA and PDA. MEA PDA Fungus RGI (%) RGI (%) species A B A B AS f-j m-o g-k m-o AS ij i-o g-k m-o AS bc a-d b a-d PEN c-g f-h g-k g-l PEN d-j g-m jk no PEN f-j h-n d-h j-n PEN d-j i-o h-k l-o PEN g-j l-o k l-o PEN c-e f-k b-e ef PEN h-j g-m g-k m-o PEN c-j f-k f-k j-n PEN c-h f-j f-k f-h PEN c-f ef e-ı f-j PEN c-j f-k f-k g-k PEN c-j g-l f-k m-o PEN c-e de bc fg PEN f-j de c-g h-n PEN d-j fg g-k b-d PEN d-j f-i f-k i-n PEN ab c-e f-k j-n PEN c-j c-e e-j cd PEN c-i k-o i-k g-l PEN c-i 4.56 no g-k l-o PEN c-j fg g-k h-n PEN g-j j-o g-k k-n PEN c g-m g-k j-n PEN c-j g-m h-k m-o Thysanophora sp c-j fg f-k g-m TRIC j 3.06 o 9.71 k o TRIC bc b-e b a-d TRIC a ab a a-c TRIC a a b-d a-c TRIC e-j 5.94 no b-e f-i TRIC ab a-c c-g a-d TRIC a a b a TRIC c-g a c-f de TRIC a a b ab+ AS: Aspergillus, PEN: Penicillium, TRIC: Trichoderma, +: Hyperparasitism Means in each column followed by the same letter do not differ significantly at P<0.05 according to the Duncan Multiple Range Test.

6 015_JPP459Ozer_ :09 Pagina Sensitivity of A. niger and F. oxysporum f.sp. cepae to fungistasis Journal of Plant Pathology (2009), 91 (2), Table 5. Radial growth inhibition (RGI %) for F. oxysporum f. sp. cepae isolate FOC16 in dual cultures with different fungal species isolated from soil samples, using simultaneous inoculation (A) and deferred inoculation (B) on MEA and PDA. MEA PDA Fungus RGI (%) RGI (%) species A B A B AS i-k d-i b-e e-j AS c-h a-g b-e e-k AS ab a-f a-c ab PEN d-i jk ıj k-m PEN l k j m PEN kl i-k f-i h-m PEN kl k ab a-e PEN bc a h-j e-j PEN a-c a-g a-c a-f PEN i-k jk f-i j-m PEN j-l jk h-j g-m PEN e-i ab f-i e-k PEN i-k g-j f-i b-h PEN b-g a-g ab b-h PEN i-k b-h f-i h-m PEN a-c a-g c-f a-g PEN a a e-i ab PEN b-f f-j f-i c-j PEN a-c a-g h-j g-m PEN b-d a-g c-g k-m PEN ab a-e ab a-c PEN b-g e-i ij h-m PEN ab a-d * b-e d-j PEN f-i g-j g-j a-f PEN i-k h-k ij lm PEN ab a-d h-j a-g PEN g-j c-i d-h c-i Thysanophora sp h-k i-k g-j i-m TRIC b-g a-g b-e f-l TRIC a-c a-e a-d * a-c * TRIC b-g a-f a-c a-e TRIC b-g a-g * a-d ab TRIC a-c a a a-c + TRIC bc ab a-c a-d TRIC b-e * a-e a-d a-f * TRIC a-c a-c a-c a-c TRIC a-c a-c ab a AS: Aspergillus, PEN: Penicillium, TRIC: Trichoderma, + Hyperparasitism * Inhibition zones: 8 mm for PEN20, 2 mm for TRIC 2, TRIC4 and TRIC7. Means in each column followed by the same letter do not differ significantly at P<0.05 according to the Duncan Multiple Range Test. PEN20-21, PEN23, TRIC1-4 and TRIC7 also strongly inhibited radial growth of the pathogen, although their effectiveness differed with inoculation time and medium. Among them, PEN20, TRIC2, TRIC4 and TRIC7 caused inhibition zones probably due to antifungal metabolites. Additionally, TRIC5 showed a hyperparasitic effect on this pathogen. Microscopic examination of dual cultures showed alterations of spores and mycelia of AN6 and FOC16 respectively, where they were in contact with antagonists or within the inhibition zone. AN6 spore alterations included increased size and coagulation of the cytoplasm (Fig.1b) and deformation of the shape (Fig. 1c). All types of AN6 spore alteration were observed in the presence of isolates AS3, PEN13, PEN15, PEN18, TRIC3, TRIC8; however, PEN17 and TRIC6 caused only morphological abnormalities. Microscopic observation of the mycelium of FOC16 generally revealed coagulation of the fungal cytoplasm, and collapse of the hyphae (Fig. 2). Collapse and coagulation of cytoplasm were observed with antagonists AS2-3, PEN5-6, PEN11, PEN13-16, PEN18, PEN20, PEN23 and TRIC1-9. No differences were found in either of the media or inoculation modes. Distribution of strongly antagonistic fungal species

7 015_JPP459Ozer_ :09 Pagina 407 Journal of Plant Pathology (2009), 91 (2), Özer et al. 407 Table 6. The total colony forming units (CFU) of fungal species strongly antagonistic for A. niger (AN6) and F. oxysporum f. sp. cepae (FOC16) isolates in soil samples tested on MPDA and TSM media. Soil sample Total CFU of fungal species strongly antagonistic for AN6 (X 10 4 /g soil)* Total CFU of fungal species strongly antagonistic for FOC16 (X 10 4 /g soil)** MPDA TSM MPDA TSM M1 0.6 d 0.8 c 9.3 c 5.6 de M2 0.0 d 0.3 c 0.6 kl 3.8 e M3 0.0 d 8.1 a 0.0 l 14.5 cd M4 0.0 d 8.2 a 0.0 l 9.3 de M5 0.0 d 3.2 b 0.6 kl 5.1 de M6 0.0 d 6.4 a 0.1 l 20.1 c M7 0.0 d 0.8 c 0.6 kl 1.7 e M8 0.2 d 6.8 a 0.3 l 9.0 de M9 0.1 d 4.5 b 4.1 e-g 19.5 c M d 0.2 c 13.9 b 19.7 c M d 0.0 c 0.5 kl 73.2 a M d 0.0 c 0.1 l 62.3 a M d 0.0 c 0.2 l 0.0 e C1 0.3 d 0.0 c 1.1 kl 1.1 e C2 1.0 d 0.0 c 2.0 g-l 0.5 e C3 7.0 a 0.0 c 34.1 a 7.2 de C4 0.2 d 0.1 c 0.8 kl 0.4 e C5 0.2 d 0.0 c 3.6 f-h 0.0 e C6 2.7 c 0.6 c 6.3 de 2.3 e C7 4.3 b 0.6 c 5.8 d-f 2.1 e C8 0.2 d 0.7 c 1.5 kl 2.8 e C9 0.0 d 0.0 c 7.3 cd 1.4 e C d 0.0 c 5.4 d-f 7.6 de S1 0.2 d 0.0 c 0.3 l 0.1 e S2 0.9 d 0.0 c 2.9 g-k 0.0 e S3 0.0 d 0.0 c 0.0 l 0.0 e S4 0.0 d 0.0 c 0.0 l 0.0 e * The sum of CFUs of AS3, PEN13, PEN15, PEN17, PEN18, TRIC2-4, TRIC6-9. ** The sum of CFUs of AS2-3, PEN4-6, PEN9-11, PEN13-18, PEN20-21, PEN23, TRIC1-9. Means in each column followed by the same letter do not differ significantly at P<0.05 according to the Duncan Multiple Range Test. among soil samples. Table 6 shows the population counts (cfu/g soil) of fungal species strongly antagonistic (inhibition >70%) for AN6 and FOC16 in soils tested on MPDA and TSM media. Soils M3-6, M8-9, C3, C6 and C7 accommodated significantly higher total counts of fungi strongly antagonistic to AN6 than other soils, varying with the testing medium. Soil samples M11-12, C9, S3-4 did not contain any antagonist species for this pathogen. The total counts of species strongly antagonistic against FOC16 revealed that soil sample C3 contained the largest population of strongly antagonistic fungal species when it was tested on MPDA, followed by M10, M1, C9, C6-7, C10. Samples M11 and M12 tested on TSM resulted in the largest population, followed by M6, M9 and M10 with statistically different effects compared to other soil samples. DISCUSSION Several researchers have used direct fungistasis tests for different fungi, measuring spore germination rate on membrane filters or agar discs placed directly on soil (Bora and Nemli, 1973; Roth and Griffin, 1980; Bora et al., 1982; Kao and Ko, 1983; Tamietti and Pramotton, 1990; Knudsen et al., 1999) and have suggested that spore germination was reduced by some soils. In our soil volatiles fungistasis test, there was no direct contact with soil, but strong fungistasis toward AN6 still occurred with 12 soil samples. In many fungistasis cases, soil volatile compounds served an important role in spore germination of soil-borne pathogens such as Helminthosporium sativum and Fusarium solani f. sp. phaseoli (Romine and Baker, 1973), Penicillium frequentans (Ko and Hora, 1974), Helminthosporium victoriae, Cochliobolus sativus and Verticillium sp. (Liebman and Epstein, 1992), Paecilomyces lilacinus, Pochonia chlamydospora, Clonostachys rosea (Chuankun et al., 2004). In these studies, it was also suggested that direct soil fungistasis and soil volatiles fungistasis were positively correlated. It is known that volatile fungistatic compounds are

8 015_JPP459Ozer_ :09 Pagina Sensitivity of A. niger and F. oxysporum f.sp. cepae to fungistasis Journal of Plant Pathology (2009), 91 (2), Fig. 2. Alteration of the hyphae of F. oxysporum f. sp. cepae isolate FOC16 induced by antagonistic fungal species. A. Control; B. Coagulation of the cytoplasm; C and D.Collapse of the hyphae. Bar = 35 µm. generally present in sandy, silty, clay soils of alkaline character (Hora and Baker, 1974; Ko and Hora, 1974). Soil samples M3, M9, C1, C3 and C10, which contained strongly inhibitory volatile compounds toward AN, were loamy-clay. Among them, C3 and C10 were acidic (ph 5.53 and 5.71, respectively), but the others had a neutral ph. These results are in agreement with observations showing that volatile fungistatic compounds are present in soils of different ph ( ) and textures (Romine and Baker, 1973; Liebman and Epstein, 1992; Chuankun et al., 2004). In the current study, volatile compounds from soil samples did not affect FOC spore germination. Differences in inhibitory effect of soil containing volatile compounds on spore germination of FOC and AN may be due to the different genetic properties of the pathogenic fungi. This is consistent with suggestions of Papavizas and Lumsden (1980), Kao and Ko (1983) and Chuankun et al. (2004) who reported that volatile compounds from soil did not play an important role in soil fungistasis toward all fungal species. Specific antagonistic strains of soil fungus rather than the general population may be involved in suppression of pathogenic fungi (Larkin et al., 1993). Our assays showed that 12 of the fungal species isolated from soil samples were markedly antagonistic against AN in dualculture tests, producing strong growth inhibition (>70%), at least for one experimental condition. Most of the fungal species isolated reduced FOC mycelial growth with two different types of inoculation or media. Similar results with Trichoderma spp. for FOC and AN were obtained in previous studies (Rajendran and Ranganathan, 1996; El Neshawy et al., 1999; Srivastava and Tiwari, 2003; Coskuntuna and Özer, 2008). Different species of Penicillium have also been reported to cause growth inhibition of F. oxysporum f. sp. lycopersici and F. oxysporum f. sp. melonis (Bora et al., 1982; De Cal et al., 1995). However, our study is the first report of Penicillium and Aspergillus spp. limiting radial growth of AN and FOC. Microscopic alteration in conidia and hyphae of AN and FOC, respectively, by some of the strongly antagonistic isolates, were observed in dual culture. These isolates caused coagulation and collapse of FOC hyphae and coagulation and morphological abnormalities of AN spores. The same alterations were obtained for Fusarium oxysporum f. sp. lycopersici, the causal agent of tomato wilt,

9 015_JPP459Ozer_ :09 Pagina 409 Journal of Plant Pathology (2009), 91 (2), Özer et al. 409 challenged by Penicillium oxalicum, P. purpurogenum and Aspergillus nidulans (De Cal et al., 1995) and F. oxysporum f. sp. albenidis, the causal agent of bayoud on date palm, challenged by Bacillus pumilus, B. terreus and Rhizobium aquatilis (El Hassni et al., 2007), although there was no report of the alterations in the spores of A. niger. The most popular explanation for soil fungistasis toward Fusarium nivale, Curvularia lunata (Mishra and Kanaujia, 1973), Alternaria tenuis, C. geniculata, Helminthosporium rostratum and Pestalotia sp. (Johri et al., 1975) is the presence of strongly antagonistic fungi with high inoculum potential and faster colonizing capacity. Suppression may not be due to an individual antagonist, but to a group of antagonists integrally acting together and belonging to diverse taxonomic groups (Wahid et al., 1998). In the current study, fungal species that strongly antagonized the pathogenic fungi, at least for one of the dual culture conditions, were evaluated for their population densities in the soil samples. Some soil samples (C1, C4, C5, C8 and S2), in which damping-off was observed, contained fungi strongly antagonistic for AN and FOC. Among these soils, C1 and C8 contained volatile compounds strongly inhibiting AN spore germination. These data show that fungus populations in these soil samples antagonistic towards the two pathogenic fungi were present at densities insufficient to account for soil fungistasis. In addition, the present study demonstrates that a combination of volatile compounds inhibiting spore germination and strongly antagonistic fungus populations are responsible for inducing fungistasis toward AN. Soil samples, M3-6, M9 and C3 had a greater number of fungi strongly antagonistic against AN and FOC than the values mentioned above, and also had volatile compounds inhibitory toward AN. These soils, where damping off was not observed at the seedling stage, would be expected to have a high level of fungistasis toward both pathogens during the onion vegetative phase. Fungus populations strongly antagonistic for both pathogens were high in three other soil samples, M8, C6 and C7. However, AN was not highly sensitive to volatile compounds from these samples. Furthermore, M1, M2, M10, M11-12, C9 and C10 contained larger numbers of fungi strongly antagonistic toward FOC only. These soil samples, which included healthy seedlings, would probably suppress infection by FOC during the set and bulb stages. Conversely, some soil samples contained volatile compounds strongly (M7, M11, C2 and C10) and moderately (M1, M2, C9 and S4) inhibitory for AN spore germination, but did not contain large numbers of fungi strongly antagonistic for this pathogen. Since both mechanisms have an important role in fungistasis toward AN, it appears that a single mechanism in these soil samples will not be enough for suppression of infection by this pathogen during the set and bulb stages. Soil samples, M13, S1 and S3, where damping off was not observed at the seedling stage, did not show both types of fungistasis. This result indicates that these soils may not be able to decrease the inoculum potential of the pathogens during the next growth stages. The physicochemical properties of the soils did not appeared to have any effect on antagonistic fungus populations. It is assumed that antagonistic fungi have different characteristics for using nutrients from soil, which Lockwood (1986) referred to as substrate antagonism in the soil. In conclusion, our data provide evidence that some onion-growing soils have fungus populations strongly antagonistic toward AN and FOC, as well as volatile compounds which inhibit AN spore germination. This study suggests the possible role of two fungistasis mechanisms on the suppression of black mould and basal rot diseases caused by AN and FOC, respectively. However, identity of the volatile compounds inhibiting spore germination remains unknown. Considering that few fungi have been previously reported to display antagonism against AN and FOC, our results also open the way to new avenues of investigation toward achieving biocontrol of basal rot and black mould diseases of onion using antagonist fungi. 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