Biocontrol efficiency of Fusarium wilt diseases by a root-colonizing fungus Penicillium sp.

Soil Science and Plant Nutrition ISSN: 0038-0768 (Print) 1747-0765 (Online) Journal homepage: http://www.tandfonline.com/loi/tssp20 Biocontrol efficiency of Fusarium wilt diseases by a root-colonizing fungus Penicillium sp. Syed Sartaj Alam, Kazunori Sakamoto & Kazuyuki Inubushi To cite this article: Syed Sartaj Alam, Kazunori Sakamoto & Kazuyuki Inubushi (2011) Biocontrol efficiency of Fusarium wilt diseases by a root-colonizing fungus Penicillium sp., Soil Science and Plant Nutrition, 57:2, 204-212, DOI: 10.1080/00380768.2011.564996 To link to this article: https://doi.org/10.1080/00380768.2011.564996 View supplementary material Published online: 26 Apr 2011. Submit your article to this journal Article views: 1534 View related articles Citing articles: 5 View citing articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalinformation?journalcode=tssp20

Soil Science & Plant Nutrition Soil Science and Plant Nutrition (2011), 57, 204 212 doi: 10.1080/00380768.2011.564996 ORIGINAL ARTICLE Biocontrol efficiency of Fusarium wilt diseases by a root-colonizing fungus Penicillium sp. Syed Sartaj ALAM, Kazunori SAKAMOTO and Kazuyuki INUBUSHI Graduate School of Horticulture, Chiba University, Matsudo, Chiba 271-8510, Japan Abstract Soil-inhabiting fungal pathogen Fusarium oxysporum often causes severe yield losses in many crops. We investigated the effect of a plant growth-promoting fungus, Penicillium sp. EU0013 on Fusarium wilt disease. In dual culture experiments, EU0013 inhibited the growth of Fusarium wilt pathogens by producing an inhibition zone. In experiments using sterile potting medium under controlled conditions, EU0013 significantly reduced the severity of Fusarium wilt on tomato (Solanum lycopersicum L.) and cabbage (Brassica oleracea L. var. capitata). In non-sterile soil, benomyl-resistant mutants of EU0013 were selected by exposing the conidial solution of EU0013 to ultraviolet light. The selected mutant EU0013_90S isolate did not show any distinct differences from EU0013 in colony characteristics, growth rate or antifungal activity against Fusarium wilt pathogens in dual culture. The effect of EU0013_90S on tomato wilt was studied under greenhouse conditions using non-sterile soil. Two-weeks old tomato seedlings were dipped in four different concentrations of EU0013_90S conidial suspension (1 10 3,1 10 4,1 10 5, and 1 10 6 conidia ml 1 ). Seedlings were then planted in soil inoculated with either F. oxysporum f. sp. lycopersici race 1 CU1 or race 2 JCM 12575 (1 10 6 bud-cells g 1 ). We found the greatest disease suppression occurred when seedlings were dipped in the highest concentration of EU0013_90S conidia. This same inoculum concentration of EU0013_90S also resulted in the highest disease reduction in soil infested with JCM 12575. Higher root colonization with EU0013_90S showed a significant reduction in Fusarium wilt disease, suggesting that colonization by Penicillium sp. EU0013_90S is important for efficient biocontrol of these diseases. Key words: biocontrol, Fusarium wilt, plant growth promoting fungi, tomato. INTRODUCTION Fusarium wilt disease caused by Fusarium oxysporum f. sp. lycopersici is one of the most destructive and economically damaging diseases of tomato (Solanum lycopersicum L.). This disease, characterized by stunted seedlings with leaf yellowing and abscission, frequently results in the death of infected plants (Jones et al. 1991). Cabbage (Brassica oleracea L. var. capitata) wilt disease was first reported in 1952 spreading rapidly throughout cabbage fields in Japan (Nomura et al. 1976). Endophytic growth of the pathogen and persistence in Correspondence: K. SAKAMOTO, Graduate School of Horticulture, Chiba University, Matsudo, Chiba 271-8510, Japan. Email: ksakamoto@faculty.chiba-u.jp Received 22 November 2010. Accepted for publication 19 January 2011. soil are the main problems in the management of Fusarium wilt (Alström 2001). Soil fumigation with methyl bromide has been the major control method of soil-borne wilt diseases; however, this soil fumigant is environmentally hazardous (Ristaino and Thomas 1997). Breeding resistant cultivars to soil-borne diseases is cost-effective and environmentally friendly. However, new races of wilt pathogens continue to appear, resulting in a loss of host resistance to these pathogens (Tello and Lacasa 1988; Ligoxigakis and Vakalounakis 1992; Dobinson et al. 1996). Thus, biocontrol of wilt diseases either individually or in integrated disease management strategy offer a more effective alternative to hazardous chemicals (Mao et al. 1998; Fravel et al. 2003). Numerous biocontrol agents have been used for protecting tomato plants against wilt disease, which include various fungal and bacterial species (Caron et al. 1985; Linderman 1992; Ren et al. 2010). ß 2011 Japanese Society of Soil Science and Plant Nutrition

Biocontrol of Fusarium wilts by Penicillium sp. 205 Non-pathogenic isolates of Fusarium oxysporum and F. solani have been reported as effective biocontrol agents, providing significant reductions in disease incidence (Larkin and Fravel 1998). Numerous studies using Penicillium oxalicum and non-pathogenic F. oxysporum (e.g. Duijff et al. 1998; Larena et al. 2003; Shishido et al. 2005), have demonstrated significant reductions in the incidence and severity of tomato Fusarium wilt disease. Plant growth promoting fungi (PGPF) have also been reported to suppress plant diseases. Some PGPF have lead to systemic disease resistance in plants after colonizing the root system (Dewan and Sivasithamparam 1989; Narita and Suzui 1991; Meera et al. 1994, 1995). For example, P. simplicissimum GP17-2, isolated from zoysiagrass significantly enhanced the growth of a variety of crops and effectively controlled soil-borne diseases (Hyakumachi 1994; Shivanna et al. 1994). A PGPF, EU0013, was isolated from eucalyptus roots and identified as Penicillium sp. from morphological features of the conidiophores and sequence data on the internal transcribed spacer (ITS) region of rdna (Teshima and Sakamoto 2006). The isolate EU0013 colonized cabbage seeds and roots, significantly increasing seed germination and seedling growth. However, no published data were available on the potential of EU0013 as a biocontrol of Fusarium wilt disease. Therefore, we sought to investigate the effect of Penicillium sp. EU0013 on soil-borne Fusarium wilts under different environmental conditions using various inoculum concentrations. For studies in non-sterile soil, we selected a fungicide resistance mutant isolate EU0013_90S. MATERIALS AND METHODS Plant materials We selected a commercial tomato cultivar Oogatafukuju, which is susceptible to Fusarium and Verticillium tomato wilts, and a cabbage cultivar Shikidori susceptible to Fusarium wilt. Seeds were surface sterilized with 1% sodium hypochlorite (NaOCl) for 2 min, then triple rinsed with sterilized distilled water. Pathogenic fungi The pathogenic fungi used in this study are the causal agents of Fusarium tomato wilt (Fusarium oxysporum f. sp. lycopersici race 1 CU1, MAFF 305121 and race 2 JCM 12575), cabbage (F. oxysporum f. sp. conglutinans K124F), and spinach (F. oxysporum f. sp. spinaciae GF1). In vitro bio-efficacy of Penicillium sp. EU0013 Initial screening of Penicillium sp. EU0013 as biocontrol agent was studied in dual culture experiments. In the test, inocula of Fusarium oxysporum, pathogens of cabbage (K124F), spinach (GF1) and tomato wilt (CU1, MAFF 305121, JCM 12575) and EU0013 were equidistantly placed on potato dextrose agar (PDA) in a Petri dish (9 cm diameter) and incubated in the dark at 25 C for 10 days. The antagonistic interaction of EU0013 with the pathogens were recorded either in the formation of an inhibition zone or overgrowth of the pathogen (Baker and Cook 1974). Effect of Penicillium sp. EU0013 on the development of Fusarium wilt in sterile potting medium The effect of EU0013 on tomato and cabbage wilts was studied under controlled conditions using sterile potting medium. Tomato and cabbage seed were grown in 5cm 5cm5cm plastic pots. Each pot contained 100 g of sterile potting medium (peat and vermiculite mixed; 1:1 v/v), in which one seed was placed. Pots were then placed in the growth chamber (200 mmol m 2 s 1 fluorescent light) at 25 C and 18 C (16 h day/8 h night). Five different concentrations of EU0013 (1 10 2, 1 10 3, 1 10 4,110 5, and 1 10 6 conidia g 1 ) were applied to the potting medium one week after sowing. Two weeks after seed sowing each of CU1 and K124F races were applied (1 10 5 bud-cells g 1 ). The inoculum was applied as a water drench to the base of the seedlings. Seedlings were treated with filter-sterilized liquid fertilizer @2 ml L 1 water (Hyponex Japan Co., Japan, 5:10:5 NPK, 0.1% v/v) on a weekly basis until the end of the bioassay. After 10 days of the pathogen inoculation, seedlings were monitored regularly for external symptoms of Fusarium wilt until the end of the experiment. Disease severity was evaluated using the percentage of leaves showing yellowing or wilting symptoms, to the total number of leaves. A total of eight treatments comprising of different pathogen and EU0013 inoculum concentrations were arranged in a completely randomized design with nine replicate plants. At the end of the experiment, plant roots grown in the potting medium inoculated with EU0013, or the pathogen with different EU0013 combinations were washed with tap water for 10 min and then with sterile water. The roots were surface disinfected with 0.5% NaOCl, then 70% ethanol and cut into 5 mm segments. Twenty root segments from three replicates were placed on PDA containing chloramphenicol (200 mg L 1 ) and incubated at 25 C in the dark for one week. Colonies of EU0013,

206 S. S. Alam et al. CU1 and K124F growing from root segments were examined. Identification of Penicillium sp. EU0013, cabbage and tomato wilt races (K124F and CU1, respectively), were determined by comparison with the original cultures on PDA. Root colonization was scored positive, when a typical colony was developed. The isolation frequency was determined by counting the number of colonized roots segments out of 20 root segments tested. Selection of benomyl-resistant mutant isolates of Penicillium sp. EU0013 Benomyl-resistant mutant isolates of EU0013 were selected using published methods (Postma and Luttikholt 1993). EU0013 conidia were harvested from PDA plates and conidial suspensions were prepared by adding conidia to sterilized distilled water. The conidial suspensions were spread on PDA plates containing benomyl (10 mg L 1 ). Plates were exposed to ultraviolet (UV) light for different lengths of time (30 90 s), and then incubated at 25 C in the dark. The conidia forming colonies were selected, followed by single spore culture preparation. The mutant isolates were selected initially for their growth on PDA with 10 mg L 1 benomyl, then cultured on PDA. Mutant isolates with similar growth rate and colony morphology as the original EU0013 were selected. Test for antagonism of Penicillium sp. EU0013 mutant isolates The selected mutant isolates of EU0013 were tested for their antifungal activity in dual cultures. Mutant isolates having the same antifungal activity as EU0013 were selected for further testing in greenhouse studies using non-sterile soil. Inocula of Fusarium oxysporum isolates (CU1, MAFF 305121, JCM 12575, K124F and GF1) were placed equidistantly around the periphery of a Petri dish (9 cm in diameter) and the EU0013 mutant isolates selected were placed in the center, then incubated for 10 days at 25 C in the dark. The antagonistic interaction of the EU0013 isolates was recorded with the formation of an inhibition zone or overgrowth of the pathogen. We selected the mutant EU0013_90S. Inoculum production of Penicillium sp. EU0013_90S The EU0013_90S was cultured on PDA for seven days at 25 C. After incubation, pieces of mycelia were placed in 500 ml flask containing 250 ml of potato dextrose broth on a rotary shaker at 25 C for seven days. The suspension was filtered to remove large mycelial fragments with the conidial concentration measured using a hemacytometer. Inoculum production of Fusarium oxysporum CU1 and JCM 12575 The bud-cell suspension of CU1 and JCM 12575 was prepared by culturing these two races for seven days in Czapek-Dox broth (NaNO 3 3g; K 2 HPO 4 1g; MgSO 4 7H 2 O 0.5 g; KCl 0.5 g; FeSO 4 7H 2 O 0.01 g; glucose 30 g and distilled H 2 O 1000 ml, ph 7.3) on a rotary shaker at 25 C. The resulting bud-cell suspension was filtered to remove mycelial fragments and washed by centrifugation (6000 g for 15 min) and resuspended in sterile distilled water. The bud-cell suspension concentration was determined using a hemacytometer. We then adjusted concentrations by diluting the original suspension. Effect of Penicillium sp. EU0013_90S on the development of tomato Fusarium wilt in nonsterile soil The effect of EU0013_90S on the development of tomato Fusarium wilt was studied under greenhouse conditions in non-sterile soil. Tomato seeds were sown in plastic trays containing autoclaved vermiculite and placed in a growth chamber at 25 C (16 h day/8 h night cycle). Two weeks after emergence, the seedlings were removed and the roots were washed then dipped in four different concentrations of conidial suspension of EU0013_90S (1 10 3,110 4,110 5, and 1 10 6 conidia ml 1 ) for 10 min. We used air-dried sieved Andosol (5 mm mesh size) soil mixed with vermiculite (7:3 v/v). The soil was collected from Chiba University experimental farm containing the following properties; 58.0% sand, 35.2% silt, and 6.8% clay bulk density, 0.80 kg L 1, ph 5.5, electrical conductivity (EC) 48.8 ms m 1, total carbon (C) 20.9 g kg 1 soil, total nitrogen (N) 2.1 g kg 1 soil (Sakamoto and Hodono 2000). We inoculated the soil with CU1 or JCM 12575 (1 10 6 bud-cells g 1 soil). Seedlings treated with different concentration of EU0013_90S conidial suspension were then planted in 300 ml plastic pots containing 280 g per pot of pathogen infected soil, one seedling per pot. Similarly, seedlings not treated with EU0013_90S were grown as control in CU1 or JCM 12575 infected soil. A total of 10 treatments comprising different combinations of tomato wilt pathogens and EU0013_90S were arranged in a completely randomized design with three replicates and three plants per replicate. Seedlings in the greenhouse were treated with filtersterilized liquid fertilizer @2 ml L 1 water (Hyponex Japan Co., Japan, 5 : 10 : 5 NPK, 0.1 % v/v) per week

Biocontrol of Fusarium wilts by Penicillium sp. 207 Table 1 Effect of Penicillium sp. EU0013 and its mutant isolate EU0013_90S on the growth of Fusarium wilt pathogens on potato dextrose agar (PDA) after 10 days of incubation Width of inhibition zone (mm) Pathogen Host EU0013 EU0013_90S Fusarium oxysporum f. sp. lycopersici race 1 CU1 Tomato 3.8 0.03 3.9 0.07 F. oxysporum f. sp. lycopersici race 1 MAFF 305121 Tomato 2.0 0.05 2.1 0.07 F. oxysporum f. sp. lycopersici race 2 JCM 12575 Tomato 0.5 0.02 0.5 0.02 F. oxysporum. f. sp. conglutinans K124F Cabbage 2.5 0.06 2.7 0.10 F. oxysporum f. sp. spinaciae GF1 Spinach 4.5 0.07 4.7 0.06 Note: The values are expressed as mean standard error (SE) (n ¼ 5). until the end of the bioassay. Between seven and 40 days after transplantation, seedlings were monitored for external symptoms of Fusarium wilt. Disease severity was evaluated as percentage of leaves with symptoms (yellowing or wilting) to the total number of leaves. EU0013_90S root colonization was examined using 5 mm root segments from 30 root segments across three replicates. Each segment was washed with running tap water for 10 min and then with sterile water. After washing, the root segments were surface disinfected with 0.5% NaOCl, followed by immersion in 70% ethanol for a few seconds and rinsed in sterile water. Root segments were placed on PDA containing chloramphenicol (200 mg L 1 ) and benomyl (10 mg L 1 ) and incubated at 25 C in the dark for one week. Root colonization was scored positive when a typical EU0013_90S colony developed. The isolation frequency was determined by counting the number of the root segments colonized by EU0013_90S out of 30 root segments from each treatment. The greenhouse experiments occurred from May to June 2010. RESULTS Effect of Penicillium sp. EU0013 on the growth of Fusarium wilt pathogens In dual culture test, EU0013 formed an inhibition zone near all the Fusarium wilt pathogens without physical contact. The width of inhibition zones varied depending on the specific EU0013 isolates and Fusarium wilt pathogen interaction (Table 1). The widest inhibition zone was formed with GF1 (4.5 mm), followed by 3.8 mm to CU1. Effect of Penicillium sp. EU0013 on the development of tomato and cabbage wilts in sterile potting medium Under controlled conditions, disease severity in tomato and cabbage seedlings grown in sterile potting medium varied significantly (P < 0.05), depending on the inoculum concentration of EU0013 (Table 2). The disease severity in pathogen-inoculated plants was 72.4% and 77.3% in tomato and cabbage, respectively. However, application of EU0013 significantly reduced the disease severity in these plant species (Fig. 1). Disease suppression by EU0013 was observed in tomato and cabbage wilts but the rate of suppression varied. When disease suppression was expressed as a reduction relative to the pathogen-only control, EU0013 suppressed disease severity from 46% to 78% in tomatoes, and in cabbage from 32 to 74%. Application of CU1 and K124F only, or the lower dose of EU0013 resulted in higher mean disease severity. Root colonization data indicated a negative correlation between EU0013 and CU1 recovery on tomato seedlings. The same pattern was observed in cabbage. Increasing inoculum concentrations of EU0013 decreased CU1 and K124F root colonization (see supporting supplementary material, Table S1). No EU0013 was detected on untreated roots. Selection of benomyl-resistant mutant isolates of Penicillium sp. EU0013 Exposure of EU0013 to UV light resulted in the production of various mutant isolates. Mutant isolates produced from the different UV exposure times were selected based on their growth on benomyl-containing PDA. These isolates were sub-cultured on the PDA until a stable colony growth was established. We selected mutant isolate EU0013_90S which showed similar morphology and growth rate to EU0013. EU0013_90S only produced an inhibition zone against Fusarium pathogens in dual culture, compared with other isolates which showed overgrowth patterns to the pathogen (see supporting supplementary material Fig. S1). In gel electrophoresis, the band pattern of EU0013_90S was identical to EU0013 (see supporting supplementary material Table S2). The ITS sequence analysis of EU0013_90S revealed three base differences compared with EU0013, with a genetic similarity of 99%.

208 S. S. Alam et al. Table 2 Effect of inoculum concentration of Penicillium sp. EU0013 on the disease severity y of tomato and cabbage wilt in growth chamber using sterile potting medium EU0013:pathogen (conidia g 1 potting medium) z 0:0 10 4 :0 0:10 5 10 2 :10 5 10 3 :10 5 10 4 :10 5 10 5 :10 5 10 6 :10 5 Tomato 0 0 72.4 (e)* 39.0 (d) (46) 33.1(cd) (54) 26.4(bc) (64) 21.1 ab) (71) 16.9(a) (78) Cabbage 0 0 77.3 (e) 52.8 (d) (32) 44.1 (c) (43) 31.1(b) (60) 22.3 (a) (70) 19.9(a) (74) Fisher s least significant difference for tomato wilt at P < 0.05 ¼ 8.30 Fisher s least significant difference for cabbage wilt at P < 0.05 ¼ 9.97 y Disease severity was assessed as proportion of leaves with symptoms (yellowing and wilting) compared with the total number of leaves (n ¼ 9) at 40 days postpathogen inoculation. Numbers in parenthesis indicate percent of disease reduction compared to pathogen inoculated plants alone. z Inocula of EU0013, CU1 and K124F fungi were added as a water drench. EU0013 inoculum was applied one week after seeds were sown. CU1 and K124F were applied two weeks after sowing the seeds. *Statistical comparisons between treatments were performed using analysis of variance (ANOVA). Letters in common in parenthesis indicate a non-significant difference, P < 0.05 (comparisons are valid within each row). Figure 2 In vitro inhibition of Fusarium wilt pathogens by Penicillium sp. EU0013_90S isolate on potato dextrose agar after 10 days of incubation. CU1, Fusarium oxysporum f. sp. lycopersici race1 CU1; MAFF, F. oxysporum f. sp. lycopersici race1 MAFF 305121; JCM, F. oxysporum f. sp. lycopersici race 2 JCM 12575; K124F, F. oxysporum f. sp. conglutinans K124F; GF1, F. oxysporum f. sp spinaciae GF1. Figure 1 Effect of Penicillium sp. EU0013 on the development of tomato and cabbage wilts in sterile potting medium under growth chamber conditions. (A) Tomato (cv. Oogatafukuju) grown in potting medium inoculated with Fusarium oxysporum f. sp. lycopersici CU1 (1 10 5 bud-cells g 1 potting medium). (B) Cabbage (cv. Shikidori) grown in potting medium inoculated with Fusarium oxysporum. f. sp. conglutinans K124F (1 10 5 bud-cells g 1 potting medium). Disease progress curves of EU0013 for treated and non-treated seedlings. T1, (non- EU0013 treated seedlings); T2, (1 10 2 conidia g 1 potting medium); T3, (1 10 3 conidia g 1 potting medium); T4, (1 10 4 conidia g 1 potting medium); T5, (1 10 5 conidia g 1 potting medium); T6, (1 10 6 conidia g 1 potting medium). Bars indicate standard errors of means. Antagonism of EU0013_90S to Fusarium wilt pathogens We found that EU0013_90S formed an inhibition zone with the five Fusarium wilt pathogens (Fig. 2). However, the width of the inhibition zone produced varied with each pathogen (Table 1). Effect of Penicillium sp. EU0013_90S on tomato wilt development in non-sterile soil In our greenhouse experiment using non-sterile soil, treatment of seedling roots with EU0013_90S

Biocontrol of Fusarium wilts by Penicillium sp. 209 significantly (P < 0.05) reduced the development of tomato Fusarium wilt (Fig. 3). Seedlings grown in soil infected with either CU1 or JCM 12575, exhibited severe yellowing and wilting. Seedlings not treated with EU0013_90S conidia, showed the highest disease severity of 78.0% in CU1 and 83.3% in JCM 12575 (Table 3). The highest disease reduction in CU1 infected soil (48.7%) was observed when seedlings were treated with EU0013_90S 1 10 6 conidia ml 1 conidial suspension. In JCM 12575 infected soil the same inoculum concentration of EU0013_90S showed a 43.6% reduction in disease. Root colonization by Penicillium sp. EU0013_90S Under greenhouse conditions, the number of tomato root segments positive for isolate EU0013_90S from CU1 and JCM 12575 infected soils varied. In CU1 infected soil the amount of EU0013_90S detected in root segments varied from 21.0% to 59.3% (Fig. 4A). In JCM 12575 infected soil, the amount of EU0013_90S detected in root segments varied from 16.7% to 48.7% (Fig. 4B). There was a positive correlation between root colonization and EU0013_90S spore concentration. No EU0013_90S isolate was detected in untreated roots. DISCUSSION In this study, a PGPF Penicillium sp. EU0013 was tested as a biocontrol of Fusarium wilt disease. In a dual culture test, EU0013 formed inhibition zones with different Fusarium wilt pathogen. The width of inhibition zone produced was different for each pathogen and to different races of the same pathogen. Inhibition zone production indicates that antifungal compounds may be produced by EU0013. However, variation in the size of the inhibition zone shows difference in the efficiency of EU0013 against each pathogen. To our knowledge, the nature and chemical composition of antifungal compounds produced by EU0013 has not been studied. The genus Penicillium produces both antibacterial (Kumagai et al. 1990; Matsukuma et al. 1992; Jackson et al. 1993), and antifungal compounds (Yang et al. 2008). In environmentally controlled experiments using sterile potting medium, EU0013 significantly suppressed Fusarium wilt in tomato and cabbage. In sterile conditions, the efficacy of EU0013 to colonize tomato and cabbage roots showed a positive association with increasing inoculum concentration. The efficacy of EU0013 also increased when more roots were colonized by the Penicillium sp. Results from this study suggested that root colonization was important for EU0013 to suppress the effect of Fusarium wilt pathogens. Figure 3 Effect of Penicillium sp. EU0013_90S on the development of tomato Fusarium wilt under greenhouse conditions in non-sterile soil. (A) Tomato (cv. Oogatafukuju) transplanted in soil containing Fusarium oxysporum f. sp. lycopersici race 1 CU1 (1 10 6 bud-cells g 1 soil). (B) Tomato (cv. Oogatafukuju) transplanted in soil containing F. oxysporum f. sp. lycopersici race 2 JCM 12575 (1 10 6 bud-cells g 1 soil). Disease progress curve in seedlings treated with EU0013_90S conidial suspension (1 10 6 conidia ml 1 ) are marked (þ). Seedlings not treated with EU0013_90S conidial suspension ( ). Statistically different mean values at each time point (P < 0.01) are indicated by an asterisk (*). Bars indicate standard errors of means. Root colonization by PGPF like Penicillium simplicissimum GP17-2 (Hossain et al. 2007) and non-pathogenic Fusarium oxysporum (Duijff et al. 1998) have been reported to be responsible for induced resistance in host plants. Exposure of Penicillium sp. EU0013 conidial to UV light for different durations resulted in the production of numerous mutant isolates. EU0013_90S was selected for further study because it exhibited a similar morphology, growth rate and antifungal activity as EU0013. EU0013_90S formed an inhibition zone to all the Fusarium wilt pathogens tested. Interestingly, the width

210 S. S. Alam et al. Table 3 Effect of inoculum concentration of Penicillium sp. EU0013_90S on the disease severity y of tomato wilt grown in non-sterile soil under greenhouse conditions Inoculum concentration of EU0013_90S z (conidia ml 1 ) 0 1 10 3 1 10 4 1 10 5 1 10 6 Race 1 CU1 78.0 a* 75.1 a (3.71) 60.3 b (22.7) 45.0 c (42.3) 40.0 c (48.7) Race 2 JCM 83.3 a 70.3 b (15.6) 61.4 bc (26.3) 56.2c (32.5) 47.0 cd (43.6) Least significant difference for CU1 (P < 0.05) ¼ 8.69 Least significant difference for JCM (P < 0.05) ¼ 11.1 y Disease severity was assessed as proportion of leaves with symptoms (yellowing and wilting) compared with the total number of leaves seven days after transplanting. Numbers in parenthesis indicate percent of disease reduction compared to pathogen inoculated plants alone. z Bud-cells suspension of Fusarium oxysporum f.sp. lycopersici, race 1 CU1 and race 2 JCM 12575 were mixed with soil-based medium (1 10 6 bud-cells g 1 soil) and two week old tomato seedlings were dipped in EU0013_90S conidial suspension (1 10 3,1 10 4,1 10 5, and 1 10 6 conidia ml 1 ) for 10 min before transplanting in pathogen infected soil. *Statistical comparisons between treatments were performed using analysis of variance (ANOVA). The same letter in parenthesis indicate a non-significant difference at P < 0.05 (comparisons are valid within each row). Figure 4 Effect of inoculum concentrations on the vascular root colonization of tomato by Penicillium sp. EU0013_90S at 40 days post-inoculation in greenhouse using non-sterile soil. (A) EU0013_90S root colonization in CU1 infested soil. (B) EU0013_90S root colonization in JCM 12575 infested soil. Bars indicate standard errors of means. of the inhibition zones produced was greater than zones produced by the original EU0013 isolate. Cook and Baker (1983) reported that fungicide resistance can increase the efficacy of biocontrol agents. In our study, EU0013_90S formed wider inhibition zone to race 1 CU1 than to race 2 JCM 12575. This suggests that the antifungal compound(s) produced by EU0013_90S have stronger suppressive effects on the CU1 race. Comparison of the ITS sequence showed 99% similarity between EU0013_90S and the EU0013. In our greenhouse experiments using non-sterile soil, we found seedlings treated with isolate EU0013_90S showed significantly less disease severity of tomato wilt. However, the degree of protection in non-sterile soil was lower compared with sterile potting medium. The highest disease reduction under greenhouse conditions was 48.7% in CU1 infected soil and 43.6% in soil infected with JCM 12575. This difference in disease reduction shows that EU0013_90S is more effective against CU1 than JCM 12575. However, this reduction was significantly different in treatments where seedlings were not treated with EU0013_90S. Our study indicated that host plant colonization was important for EU0013 and EU0013_90S. We found a positive association between EU0013 and EU0013_90S efficacy and root colonization. The degree of root colonization was highest when EU0013 was inoculated in sterile conditions. In contrast, the degree of colonization by EU0013_90S was reduced in non-sterile soil. We hypothesize that competition from indigenous soil microbes present in non-sterile soil compete with EU0013_90S, consequently decreasing the degree of protection offered by EU0013_90S. Root colonization by EU0013_90S increased depending on the EU0013_90S spore concentration. Shishido et al. (2005) have reported differences in efficiency of nonpathogenic Fusarium oxysporum in the biocontrol of tomato wilt under different environmental conditions. They reported that a biocontrol agent was more effective when used in sterile seedbeds, compared with non-sterile soil, as it decreased competition from indigenous soil microbes. Furthermore, the efficacy of the biocontrol

Biocontrol of Fusarium wilts by Penicillium sp. 211 agent is reduced under greenhouse conditions, compared with growth chamber conditions. Biocontrol agents are generally more effective when inoculated antagonist propagules exceed pathogen propagules by an order of magnitude or more (Shishido et al. 2005). Our results are in agreement with these findings. Comparing results from our experiments, it appears important to apply Penicillium sp. EU0013_90S isolate to tomato seedling roots at least two weeks before planting in natural soil. This early application would enable EU0013_90S to intensively colonize roots and suppress the Fusarium wilt, possibly by an induced resistance mechanism. Further studies on isolate EU0013_90S are required to understand other mechanisms of disease suppression. ACKNOWLEDGMENTS This study was supported by a grant to K.S. from the Japan Science and Technology Agency (Research for Promoting Technological Seeds, No. 04-069). 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