J. Pestic. Sci., 32(3), 222–228 (2007) DOI: 10.1584/jpestics.G06-35
Original Article
Mode of action of Trichoderma asperellum SKT-1, a biocontrol agent against Gibberella fujikuroi Satoshi WATANABE,* Kazuo KUMAKURA, Norihiko IZAWA, Kozo NAGAYAMA, Takashi MITACHI,† Masaki KANAMORI,†† Tohru TERAOKA††† and Tsutomu ARIE††† Kumiai Chemical Industry Co., LTD. Life Science Research Institute, Kikugawa, Shizuoka 439–0031, Japan † Department of Bioregulation and Biointeration, Graduate School of Agriculture, Tokyo University of Agriculture and Technology (TUAT), Fuchu, Tokyo 183–8509, Japan †† United Graduate School of Agricultural Sciences, Tokyo University of Agriculture and Technology (TUAT), Fuchu, Tokyo 183–8509, Japan ††† Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology (TUAT), Fuchu, Tokyo 183–8509, Japan (Received September 29, 2006; Accepted February 9, 2007)
Trichoderma asperellum SKT-1 and Gibberella fujikuroi, known as causal agents of “Bakanae” disease, were both transformed with genes encoding green fluorescent protein (GFP) and hygromycin B (hygB) by restriction enzyme- mediated integration (REMI). Rice seeds inoculated with GFP-tagged G. fujikuroi showed “Bakanae” symptoms. GFP-tagged SKT-1 maintained biocontrol activity against the pathogen by soaking seeds in SKT-1 spore suspension. Then, we monitored in situ interactions between SKT-1 and G. fujikuroi on rice seeds using GFP-tagged transformations under confocal scanning laser stereomicroscopy. G. fujikuroi disappeared from the embryo of rice seeds after treatment with SKT-1, whereas SKT-1 was observed on the embryo 24 hr after initiation of germination. In addition, the hyphae of G. fujikuroi were penetrated by the hyphae of SKT-1, and degradation of the cell walls of G. fujikuroi was observed under SEM in co-culture. The cell wall of G. fujikuroi on the embryo of rice seeds was lysed, suggesting that mycoparasitism is the mode of action of T. asperellum SKT-1. © Pesticide Science Society of Japan
Keywords: Trichoderma asperellum, biocontrol, transformed with GFP, In planta observation, “Bakanae” disease.
Introduction Trichoderma spp. have been used as active ingredients in several commercial biopesticides for the control of a range of economically important soilborne and seedborne plant pathogens.1–11) Although the mode of control by Trichoderma spp. may includes antibiosis,12) mycoparasitism,13) competition for nutrients and/or space,14,15) and the induction of resistance in plants,16) the complexity of three-way interactions among pathogen-plant-biocontrol agents makes understanding the mode difficult. Effective monitoring of biocontrol agents and pathogens in planta will provide bases for the development and improvement of biopesticides. * To whom correspondence should be addressed. E-mail:
[email protected] Published online June 27, 2007 © Pesticide Science Society of Japan
Genetic engineering of biocontrol agents with marker or reporter genes can be a useful tool for the detection and monitoring of introduced biocontrol agents in the environment.17,18) For example, the selectable hygromycin B phosphotransferase (hygB) gene, coding resistance to this antibiotic, has been used for the specific detection of fungal pathogens or biocontrol agents in the rhizosphere and phyllosphere.18,19) Green fluorescent protein (GFP) from a jellyfish, Aequorea victoria, also has been used for monitoring the fate and behavior of bacterial and fungal pathogens or biocontrol agents in situ.20–29) GFP, unlike other biomarkers,30) does not require any substrate or additional cofactors to fluoresce. Even a single cell expressing GFP can be easily detected among many cells under an epifluorescence microscope or a confocal scanning laser microscope (CSLM).27,30,31) Orr et al.32) confirmed that GFP is a useful tool to distinguish active hyphal biomass from inactive propagules. Bae et al. (2000) suggested that cotransformation with GFP and
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GUS (b -glucuronidase) can provide a valuable tool for the detection and monitoring of specific strains of T. harzianum released into the soil.20) Lu et al. (2004) generated a GFPtagged T. atroviride and monitored the biocontrol agent visually in co-cultures with Rhizoctonia solani, on plant surfaces, and in soil under CSLM and fluorescence stereomicroscopy. These results suggested that GFP-tagged fungi can provide valuable tools for the detection and monitoring of specific interactions between a pathogen and biocontrol agent on seeds, on plants and in the soil. T. asperellum SKT-1 (SKT-1) is a valuable fungal biocontrol agent against various seed-born diseases of rice including “Bakanae” disease. We have commercialized spores of SKT-1 with the brand name of Eco-hope (Registration No. 21009) and Eco-hope Dry wettable powder (Registration No. 21434) in Japan. Understanding the mode of action of SKT-1 against target phytopathogens will be an important step in fulfilling the potential of T. asperellum SKT-1. Our objective in this study was to study the in vitro and in situ interactions between G. fujikuroi, the “Bakanae” disease pathogen, and T. asperellum SKT-1, a biocontrol agent, using GFP-tagged mutants.
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Fig. 1. A plasmid pMK412 carrying the engineered GFP (EGFP) derived from Aureobasidium pullulans translation elongation factor promoter (pTEF) and Aspergillus awamori glucoamylase terminator (tgpd1).
T. asperellum33) SKT-1 (FERM P-16510; International Patent Organism Depository, Ibaraki, Japan)3) and G. fujikuroi N-68 (Kumiai Chemical Industry Co., LTD. Life Science Research Institute, Shizuoka, Japan) known as a biocontrol agent and a causal agent of “Bakanae” disease, respectively, were used throughout this study. Conidia of T. asperellum SKT-1 were harvested from solid culture on potato dextrose agar (PDA) plates incubated at 27°C for seven days in the dark. Bud cells of G. fujikuroi N68 were harvested from the culture on potato dextrose broth (PDB, Difco Laboratories, Detroit, USA) at 27°C for four days by shaking at 120 rpm.
inoculated with the fungus at an initial concentration of ca. 1106 spores/ml and incubated at 27°C for 24 hr by shaking at 120 rpm, respectively. The resultant hyphae were filtrated and washed with 1.2 M MgSO4. Fresh hyphae were resuspended in 10 ml of 1.2 M MgSO4 and transferred to a 100-ml flask containing 20 ml of sterile-filtered (f 0.45 m m) enzymatic solution consisting of 20 mg/ml lysing enzyme (Sigma, Missouri, USA), 20 mg/ml driselase (Kyowa Hakko Kogyo, Tokyo, Japan), and 50 m g/ml chitinase (Wako Pure Chemical, Osaka, Japan) in 1.2 M MgSO4. The flask was incubated at 28°C for 3 hr by shaking at 80 rpm. Protoplasts were separated from mycelial debris by filtering through sterilized double-layer tissue paper. The protoplast solution was overlayed with 0.6 M mannitol in a 50-ml centrifuge tube. The tube was centrifuged at 6000 g for 20 min, and the protoplasts gathered in the middle layer were transferred to a new 50-ml centrifuge tube. The protoplasts were washed with 0.8 M mannitol by centrifugation at 3000 g for 20 min, suspended in 600 m l STC solution [1.2 M sorbitol, 10 mM Tris–HCl (pH 7.5), 50 mM CaCl2 · 2H2O] and used for transformation.
2.
4.
Materials and Methods 1.
Fungal strain
Transformation vectors and preparation of plasmid DNA
A plasmid pMK412 (Fig. 1) carrying the engineered GFP (EGFP) gene (Clontech, California, USA) derived from Aureobasidium pullulans translation elongation factor promoter (pTEF) and Aspergillus awamori glucoamylase terminator (tgpd1) was constructed from pTEFEGFP and pCSN43.34) The plasmid was propagated in E. coli strain HB101, purified by QIAGEN QIAprep Spin Miniprep kit (QIAGEN, Tokyo, Japan). The purified pMK412 (1 m g/m l) was linearized with 750 U Sac I in 50 m l TE (Tris-EDTA) at 37°C for 24 hr and used to transform fungi without inactivating the enzyme.
3.
Fungal protoplast preparation
T. asperellum SKT-1 and G. fujikuroi N-68 were cultured on PDB. The medium (100 ml) in a 500-ml Erlenmeyer flask was
Fungal transformation
Sac I-digested pMK412 (40 m g in 50 m l) was mixed into 180 m l of protoplast suspension (1107/ml) in a 15-ml centrifuge tube. The mixture was incubated for 20 min on ice. Polyethylene glycol (PEG) solution [1.2 ml, 60% (w/v) PEG 4000, 10 mM Tris–HCl (pH 7.5), 10 mM CaCl2 · 2H2O] was added in a dropwise manner into the mixture. The mixture was incubated again for 20 min on ice, and 6 ml of ice-cold STC solution was added to the suspension, which was mixed with 100 ml of molten regeneration medium [RM, 34.2% (w/v) sucrose, 0.1% (w/v) yeast extract, 1% (w/v) agar] and plated in plastic Petri dishes. After incubation at 27°C for 18 hr, the solidified plate was overlaid with 10 ml of molten agar (1%) containing 125 m g/ml of hygromycin B. After incubation at 27°C for three to four days, the emerged colonies were transferred to new RM plates containing 150 m g/ml of hygromycin B.
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Transformants that showed GFP under a fluorescence microscope using UV and U-MWIB-2/GFP filter (Olympus, Tokyo, Japan) were selected. To establish stable GFP-expressing strains, several conidia from each transformant were transplanted on PDA containing 150 m g/ml hygromycin B and the colonies expressing GFP isolated (single-spore isolation).
5.
Evaluation of phytopathogenicity of GFP-tagged G. fujikuroi N-68 and biocontrol activity of GFP-tagged T. asperellum SKT-1 against “Bakanae” disease
Rice seeds (Oryza sativa L. cv. Tangin-bouzu) infested with G. fujikuroi N-68 or GFP-tagged N-68 were prepared by the method of Kumakura et al. (2003)3) Three grams of infested seeds (ca. 100 seeds) were soaked in a 10-ml spore suspension (1104, 1105 and 1106 spores/ml) of T. asperellum SKT-1 or GFP-tagged SKT-1 and kept at 15°C. Ten milliliters of 250 mg/l ipconazol-150 mg/l copper hydroxide (200 times dilution of Teclead C flowable, Kumiai Chemical Industry, Tokyo, Japan) solution and sterile water were also used as standards. After 24 hr, seeds were removed from the spore suspension and incubated in 20 ml sterilized water at 15°C for five days, and then the seeds were decanted onto a gauze cloth and incubated for 24 hr at 32°C to promote germination. Germinated seeds were sown in soil (Ube-Baido, Ube Industries, Yamaguchi, Japan) in a plastic cup (5 cm diameter) and incubated for three days at 30°C in 100% RH in the dark. Plants were then maintained for 18 days in the greenhouse, and the percentage of diseased plants having leaves several centimeters longer, thinner, and more yellow than the healthy control plants was calculated for each treatment. Three replications were set up for each treatment. The mean disease incidence for each experiment was statistically analyzed by Tukey’s multiple range tests.
6.
Interactions between G. fujikuroi and T. asperellum SKT-1 on PDA
G. fujikuroi N-68 or GFP-tagged N-68 was cocultured with T. asperellum SKT-1 or GFP-tagged SKT-1 on a PDA plate to observe their interactions. Bud-cells of G. fujikuroi were washed in sterile water by centrifugation at 6000 g for 10 min, and 1106 cells were dispersed into 100 ml of molten PDA, and plated in f 90 mm Petri dishes (15 ml/dish). After incubation at 27°C for 24 hr, a sterile paper disk (f 9 mm diameter) carrying 1106 conidia of T. asperellum SKT-1 or GFPtagged SKT-1 was put on the center of the solidified plate. The plate was incubated at 27°C for 48 hr. Observations were conducted under CSLM (Model TCS; Leica, Heidelberg, Germany) with excitation wavelengths of 488 nm (Ar) and 633 nm (He, Ne). Emission light was collected in the range from 510 to 560 nm for GFP and in the range from 620 to 660 nm for background fluorescence. Images were obtained using Leica confocal software (version 2.477). Scanning electron microscope (SEM) observation was car-
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ried out as follows: agar blocks (ca. 442 mm) cut from the culture plate were fixed in 2% (v/v) glutaraldehyde aqueous solution at room temperature for about 16 hr and postfixed in 2% (v/v) osmium tetroxide aqueous solution for 3 hr. The specimens were dehydrated, dried and gold-coated as previously described.33) The distinction of T. asperellum SKT-1 or G. fujikuroi N-68 was judged by measuring the width of the hyphae on a PDA.
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Interactions between G. fujikuroi and T. asperellum SKT-1 on rice seeds
Rice seeds infested with G. fujikuroi N-68 were rinsed with sterilized water, soaked in 70% (v/v) ethanol for 2 min, and rinsed with sterilized water three times. The seeds were transferred onto sterile filter paper to absorb excess water and soaked in 2% (w/v) sodium hypochlorite for 2 min for surface sterilization. The seeds were then rinsed with sterilized water five times, and excess water was removed by blotting them with sterilized filter paper under aseptic conditions. Fifty surface-sterilized infested seeds were soaked in 5 ml spore suspension (1106 spores/ml) of GFP-tagged SKT-1 and maintained at 15°C for 24 hr. Sterile water were also used as a control. The seeds were removed from the spore suspension, incubated in 10 ml sterilized water at 15°C for five days, put onto a gauze cloth and incubated at 32°C for 24 hr. Germinated seeds were sown in soil in plastic cups and incubated for three days at 30°C and 100% RH in the dark. Plants were then maintained in the greenhouse (22°C, 30,000 m E m2 s1) for five days. At 0, 1 and 3 days after sowing, several rice seedlings were arbitrarily removed from each treatment. The rice seedlings were sliced to a thickness of 0.5–1 mm in order to observe under CSLM. One drop of sterilized water was added to each slice before a coverslip was placed, and the preparation was examined by CSLM. The distinction of T. asperellum SKT-1 or G. fujikuroi N-68 was also judged by measuring the width of the hyphae on a rice seed.
Results 1.
Fungal transformation and stability
Regeneration of untransformed protoplasts was completely inhibited at 150 m g/ml hygromycin B (data not shown); therefore, growth at this concentration was used as the selection criterion for hygromycin B-tolerant transformants. To select a monokaryon from each fungal transformant, several colonies developed from a single conidium of each transformant were chosen and tested for GFP-express strains. Seven transformants of each fungus showed the ability to express GFP. To test expression stability in transformants, they were subcultured successively on PDA without 150 m g/ml hygromycin B, up to five times, and then one strain was selected as a stable GFP-expressing strain from among seven strains of each fungus.
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In planta observation of biocontrol by T. asperellum SKT-1
Table 1. Biocontrol activity of Trichoderma asperellum SKT1 and GFP-tagged SKT-1 against “Bakanae” disease caused by Gibberella fujikuroi
Treatment
Concentration
Diseased plantsa)
(spores/ml)
(%)
G. fujikuroi N-68 10.0 ab)
No treatment 6
GFP-tagged SKT-1
110
SKT-1 (wild type)
0.0 c
5
110
0.7 c
1104
2.0 b
1106
0.0 c
1105
0.3 c
4
3.0 b
110 Ipconazol-copper
250 mg/l ipconazol– 150 mg/l Cu(OH)2
0.0 c
c)
7.4 ab)
GFP-tagged SKT-1
SKT-1 (wild type)
6
110
0.0 c
1105
0.3 c
1104
3.0 b
1106
0.0 c
1105
0.3 c
4
2.0 b
110 Ipconazol-copper
250 mg/l ipconazol– 150 mg/l Cu(OH)2
0.0 c
c)
a)
Rice seeds (cv. Tangin-bouzu) infested with G. fujikuroi N-68 or GFP-tagged N-68 were prepared following Kumakura et al. (2003).3) Plants were grown for 18 days after sowing germinated seeds, and then the percentage infection of diseased plants leaves of which were several centimeters longer, thinner and yellower than healthy plants, was calculated. b) The same letter indicates that a significant difference was not recognized in Tukey’s multiple range test (p0.05). There were three replications for each treatment. c) 200 times dilution of Teclead C flowable, Kumiai Chemical Industry, Tokyo, Japan.
2.
Interactions between G. fujikuroi and T. asperellum SKT-1 on PDA
The distinction of hyphae between T. asperellum SKT-1 and G. fujikuroi N-68 was clear because the former was about 10–25 m m, while the latter was about 3–5 m m. CSLM observations of co-culture of GFP-tagged G. fujikuroi and GFPtagged SKT-1 on PDA are shown in Fig. 2. GFP in the hyphae of G. fujikuroi sometimes disappeared when cocultured with GFP-tagged SKT-1. GFP in hyphae of SKT-1 was observed constitutively. SEM observations of coculture of G. fujikuroi and T. asperellum SKT-1 on PDA are shown in Fig. 3. The wide hyphae of G. fujikuroi were penetrated by narrow hyphae of SKT-1 (Fig. 3A), and degradation of the cell walls was observed (Fig. 3B). Conidia of SKT-1 were formed on the degradated hyphae of G. fujikuroi (Fig. 3B).
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GFP-tagged G. fujikuroi N-68 No treatment
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Interactions between G. fujikuroi and T. asperellum SKT-1 on rice seeds
When SKT-1 was not treated, GFP-tagged G. fujikuroi extended hyphae on the embryos of rice seeds 16 hr after initiation of germination (Fig. 4A). At five days after sowing, hyphae of G. fujikuroi were observed in coleoptile (Fig. 4B). When GFP-tagged SKT-1 was treated, both GFP-tagged N68 and GFP-tagged SKT-1 extended hyphae on the embryo of rice seeds 16 hr after initiation of germination (Fig. 5). Although GFP was observed constitutively in the hyphae of SKT-1, it disappeared from several cells of the hyphae of N68 24 hr after initiation of germination (Fig. 6A–B). At five days after sowing, hyphae of GFP-tagged N-68 were not observed in coleoptile, instead, hyphae of SKT-1 were frequently observed on this surface (Fig. 6C).
Discussion The mode of action (MOA) of biocontrol by Trichoderma spp. against fungal plant pathogens has been studied in vitro and in situ using a reporter gene-expressing mutant of Trichoderma.32) Lu et al. (2004) monitored in situ interactions be-
Phytopathogenicity (“Bakanae” disease of rice) of GFP-tagged G. fujikuroi strain N-68 and biocontrol activity of GFP-tagged T. asperellum strain SKT-1
The phytopathogenicity of G. fujikuroi N-68 or GFP-tagged N-68 and the biocontrol activity of T. asperellum SKT-1 or GFP-tagged SKT-1 are shown in Table 1. Seeds infested with N-68 and GFP-tagged N-68 showed “Bakanae” symptoms without significant difference. T. asperellum SKT-1 and GFPtagged SKT-1 were equal in the control against “Bakanae” disease.
Fig. 2. CSLM images of co-culture of GFP-tagged G. fujikuroi and GFP-tagged T. asperellum SKT-1. The white arrow indicates SKT-1 hyphae. The red arrow indicates disappeared GFP activity in G. fujikuroi hyphae. Bars represent 50 m m.
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Fig. 3. SEM images of co-culture of G. fujikuroi and T. asperellum SKT-1. A, The wide hyphae of G. fujikuroi were penetrated by the narrow hyphae of SKT-1 (arrow). B, The red arrow indicates SKT-1 degrading the cell walls of G. fujikuroi. The white arrow indicates SKT-1 forming conidia on the degraded hyphae of G. fujikuroi. Bars represent 10 m m.
tween a GFP-tagged biocontrol strain of T. atroviride and soilborne plant pathogens that were grown in co-culture and on cucumber seeds. Our objective in this study was to study in vitro and in situ interactions between T. asperellum SKT-1 as a biocontrol agent and G. fujikuroi as a “Bakanae” disease pathogen using GFP-tagged transformants. The phytopathogenicity of the GFP-tagged mutant of G. fujikuroi N-68 and the biocontrol activity of GFP-tagged mutant of T. asperellum SKT-1 were the same as those of wild-type strains, indicating that the transformation and expression of egfp did not affect phytopathogenicity and biocontrol activity, and GFP-tagged fungi can provide valuable tools for the detection and monitoring of specific interactions between pathogen-biocontrol agents in/on plant tissues and in natural soil. All cells in the hyphae of G. fujikuroi expressed GFP under CSML observation when GFP-tagged G. fujikuroi was cultured on PDA without T. asperellum SKT-1; however, in coculture experiments, GFP disappeared from several cells in hyphae of G. fujikuroi, indicating that GFP was inexpressed or degraded in cells where hyphae of G. fujikuroi contacted hyphae of SKT-1. SEM observation indicated that hyphae of
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Fig. 4. CSLM images of GFP-tagged G. fujikuroi on infested rice seedlings without T. asperellum SKT-1 treatment. A, Hyphae of G. fujikuroi expanded on embryos of rice (cv. Tangin-bouzu) 16 hr after initiation of germination. B, Hyphae of G. fujikuroi were observed in coleoptiles at 5 days after sowing. Bars represent 300 m m.
Fig. 5. CSLM images of GFP-tagged G. fujikuroi N-68 on infested rice seedlings with T. asperellum SKT-1 treatment. Hyphae of SKT-1 and G. fujikuroi occurred on embryos of rice seeds (cv. Tanginbouzu) 16 hr after initiation of germination. SKT-1 and G. fujikuroi have not come into contact yet. The wide hyphae of G. fujikuroi are shown by the red arrow, and the narrow hyphae of SKT-1 by the white arrow. Bars represent 200 m m.
G. fujikuroi were penetrated by SKT-1 hyphae and the cell wall of G. fujikuroi was degraded, suggesting that mycoparasitism is one of the MOAs of T. asperellum SKT-1 against G. fujikuroi. To ascertain the MOA of SKT-1 against G. fujikuroi, in situ observation of their interactions was performed. On infested
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Fig. 6. CSLM images of interaction between GFP-tagged T. asperellum SKT-1 and GFP-tagged G. fujikuroi on embryos and coleoptile of rice seedlings. A, Hyphae of SKT-1 and G. fujikuroi on rice seed (cv. Tangin-bouzu) embryo 24 hr after initiation of germination. Bars represent 200 m m. B, Close-up of A. Though GFP of SKT1 with narrower hyphae was observed (red arrow), GFP sometimes disappeared (white arrow) from G. fujikuroi. Bars represent 100 m m. C, Hyphae of SKT-1 occurred on the coleoptile surface of rice seeds at 5 days after sowing. Bars represent 100 m m.
and/or treated rice seeds, GFP-tagged SKT-1 and GFP-tagged G. fujikuroi extended their hyphae on the embryo when seeds were incubated for 24 hr at 32°C. Infested seedlings of G. fujikuroi extended hyphae from the embryo to invade the coleoptile. On the embryos of treated rice seeds, though GFP in GFP-tagged SKT-1 hyphae were observed, GFP in GFPtagged G. fujikuroi hyphae were absent. This might have occurred because G. fujikuroi was parasitized by SKT-1 on the embryo. Then, in treated seedlings, SKT-1 obstructed hyphal elongation and/or invasion of G. fujikuroi. Mycoparasitism by T. asperellum SKT-1 may be due to degradation of the cell wall by glycosidases, such as chitinase and b -1,3-glu-
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canase.35–42) The activity of cell wall-degrading enzymes is likely to be influenced by ambient temperatures, which affect both relative growth rates of fungi and enzymatic activity.33) It is noteworthy that the optimum growth temperature (27°–30°C) of SKT-1 is almost the same as the optimum temperature for rice seed germination and seedling growth, suggesting that SKT-1 is an ideal biocontrol agent.43) In our experiments, SEM observation showed direct mycoparasitic interaction between T. asperellum SKT-1 and G. fujikuroi on the seed surface, and degradation of the cell wall of G. fujikuroi by hyphae of SKT-1. Inbar et al. (1996)44) showed hyphae of T. harzianum strain BAFC Cult. No. 72 coiling along Sclerotinia sclerotiorum hyphae in co-culture. Whereas hyphae of SKT-1 did not show any coiling around G. fujikuroi hyphae, hyphae of SKT-1 showed mycoparasitic action against G. fujikuroi hyphae. This suggests that type of mycoparasitism may be different among the Trichoderma-biocontrol agents. In addition, MOA of most Trichoderma harzianum reported that the synergism of hydrolytic enzymes and antibiotics may have an important role in antagonistic action against fungal phytopathogens,45–48) whereas T. asperellum has not been reported to produce antibiotics. In this study, whereas we showed that mycoparasitism is one of the MOAs of T. asperellum SKT-1 against G. fujikuroi, T. asperellum SKT-1 is also highly effective against bacterial seedling blight caused by Burkholderia plantarii, bacterial grain rot caused by B. glumae and bacterial blight caused by Acidovorax avenae subsp. avenae.3) The MOA of SKT-1 against these bacterial phytopathogens does not rule out the additional possible involvement of other antagonistic mechanisms (e.g., antibiosis, competition for nutrients or space, induction of resistance in plants, etc.). The MOA of T. asperellum T203 against the leaf pathogen Pseudomonas syringae pv. lachrymans modulates the expression of genes involved in the jasmonate/ethylene signaling pathways of ISR (induced systemic resistance) and accumulation of phytoalexins in cucumber plants.9,49,50) Research into the ability of T. asperellum SKT-1 will become a new target MOA in the future. On the basis of our CSLM and SEM study, we concluded that colonization on rice plants and direct mycoparasitism by SKT-1 might be the main role in the biocontrol of G. fujikuroi. Acknowledgments We are grateful to Dr. John H. Andrews and Dan Cullen for providing pTEFEGFP. We also thank Dr. Takashi Tsuge for fruitful advice on the work. This research was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese government.
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