Inhibition of Magnaporthe oryzae by Culture Filtrates of Fungi Isolated from Wild Mushrooms

Fungal strains isolated from the fruiting bodies of wild mushrooms were evaluated for fungicidal activity against Magnaporthe oryzae, the causal agent of the rice blast disease. Fungal isolates (n = 105) were obtained from 46 samples of wild mushrooms. Infection behaviors of M. oryzae were assessed in the presence of culture filtrates from 90 fungal isolates, of which 20 inhibited spore germination. Heat-treated culture filtrates of these isolates were classified into 3 groups according to biological activity. Blast lesion formation by M. oryzae was significantly inhibited by pretreatment with culture filtrates from 4 fungal isolates. ITS region sequence analysis indicated that these isolates shared similarities with species of the genera Annulohypoxylon, Nigrospora, and Penicillium. Studies of symbiotic and parasitic fungi from wild mushrooms may yield potential control agents for plant diseases such as the rice blast disease.


Introduction
Rice is one of the world's most important food crops and the rice blast disease caused by Magnaporthe oryzae (Hebert) Barr is the most destructive fungal disease affecting global rice production.Also, M. oryzae causes blast disease on several grass species including the cereals wheat and barley.
Resistant rice cultivars and chemical fungicides are important in blast disease control.However, the durability of genetic resistance in improved rice cultivars is often short-lived in the field because the pathogen rapidly evolves to overcome resistance [1].The key to chemical control of rice blast disease is to overcome fungicide-resistant pathogens [2] [3].Therefore, a search for antifungal compounds is required to develop new fungicides.Antifungal compounds of microbial origin play an important role in the biological and chemical control of plant diseases [4]- [6].
Some phylloplane and endophytic fungi protect plants from pathogens.These fungi produce antimicrobial compounds [7]- [9].Kawamata et al. (2004) [10] reported that fungal species isolated from the phylloplane of rice plants inhibit not only M. oryzae conidium germination and mycelial growth but also blast lesion formation.Furthermore, Pseudomonas aeruginosa (Schroeter) Migula isolated from spent mushroom substrate significantly inhibits Pyricularia grisea (Cooke) Sacc, the causal agent of gray leaf spot in perennial ryegrass [11].
The number of mushroom species in the world is estimated to be about 140,000, but only 10% have been identified [12].There are no reports on the existence of symbiotic and parasitic fungi in wild mushrooms and their utility in pathogen control.The objectives of this study were to: 1) isolate symbiotic and parasitic fungi from the fruiting bodies of wild mushrooms; 2) investigate the antifungal activity of culture filtrates of these fungi against M. oryzae; and 3) investigate the protective effect of the culture filtrates of the fungal isolates on lesion formation by M. oryzae in barley leaves.

Isolation of Fungi from Fresh Fruiting Bodies of Wild Mushrooms
Fungi were isolated from fresh fruiting bodies of 46 wild mushroom samples collected from a paddy field in Fukui, Hiroshima, Kumamoto, Osaka, Shimane, and Tottori prefectures in 2009-2011.Each fresh fruiting body was cut with a blade.Segments (approx.5 × 5 mm) from each fresh fruiting body were removed and surface-sterilized in 80% ethanol for 2 min and 1% sodium hypochlorite solution for 3 min, then rinsed twice in sterile distilled water for 2 min.Rinsed segments were placed on sterile filter paper in petri dishes to remove excess surface water.After drying the segments, each segment was placed on potato sucrose agar (PSA) medium containing chloramphenicol (200 µg/mL) in petri dishes (9 cm diameter) and incubated for 14 days at 27˚C.Fungal isolates that grew on the segments were transferred to fresh PSA medium and fungal isolates obtains by single spore or colony was maintained on PSA slants until use.

Preparation of the Culture Filtrate
Mycelial disks (7 mm diameter) prepared from a single colony of each isolate grown on PSA medium for 7 days were incubated in a test tube containing 20 mL potato sucrose broth (PSB).The culture was kept in the dark condition at 27˚C and shaken at 120 rpm for 10 days using rotary shaker.Culture filtrates were collected through a 0.2-µm membrane filter.

Infection-Inhibiting Activity of the Culture Filtrates
Magnaporthe oryzae (strain Naga 69 -150, race 007) was grown on rice bran agar at 26˚C for 14 days, washed with running water to remove aerial hyphae, and kept at 26˚C under near-ultraviolet radiation (FL20s BL-B; Panasonic, Osaka, Japan) for 2 days to induce sporulation.
M. oryzae spores (1 × 10 5 spores/mL) were suspended in the fungal culture filtrates, distilled water or PSB were dropped onto glass slides and kept in a moist chamber at 26˚C.After 24 h incubation, the percentage of spore germination was determined by light microscopy.Data analyses were performed by using the Scheffe's test.

Heat Pretreatment
Selected culture filtrates were heated at 121˚C for 20 min using the autoclave.The treated and untreated filtrates were inoculated together with a spore suspension (1 × 10 5 spores/mL) of M. oryzae onto glass slides and kept in a moist chamber at 26˚C.After 24 h incubation, the percentages of spore germination and appressorium formation were determined by light microscopy.Data analyses were performed by using the Scheffe's test.

Culture Filtrate Pretreatment and Fungal Inoculation
To investigate the effects of culture filtrates on the induction of blast resistance, barley seedlings (Hordeumvul-gare L., cv.Minoriomugi) at the 1-to 2-leaf stage were sprayed with culture filtrates.PSB and distilled water spray were used as a control.The pretreated barley plants were kept under light condition in a moist chamber at 26˚C for 24 h, and then sprayed with a suspension of M. oryzae spores (1 × 10 5 spores/mL).Inoculated plants were incubated in a moist chamber at 26˚C.Blast lesions on barley leaves were counted 4 days after inoculation.

DNA Extraction and PCR Amplification and Sequencing
To identify the active isolate, the sequence of the internal transcribed spacer (ITS) region (including 5.8S rDNA) was determined by PCR with the ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCT TATTGATATGC-3') primers.Fungal genomic DNA was extracted from the mycelia as described by Suzuki et al. (2006) [13], and used as the PCR template.PCR amplification of the ITS region was performed as follows: an initial step of 5 min at 95˚C; followed by 30 cycles of denaturation at 94˚C, for 1 min, annealing at 62˚C, for 1 min, and elongation at 72˚C, for 1 min; and a final step for 5 min at 72˚C.PCR-amplified fragments were purified by using the HiYield Gel/PCR DNA fragment extraction kit (RBC Bioscience, Taipei, Taiwan).DNA sequencing was performed with a Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA).DNA sequence analysis was performed on an ABI PRIZM 3130xl Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA).Sequence homology was determined by searching with the BLAST suite of programs (DNA Data Bank, Japan).

Statistical Analysis
Data are reported as mean ± standard deviation.Significant differences from the values were determined using a Scheffe's test (p < 0.05).
Penicillium spp.are known biocontrol agents with inhibitory activities against various plant pathogens.Peni- cillium spp.produce antifungal compounds such as mycophenolic acid, patulin, 3-omethylfunicone, and decumbenone A, which directly or indirectly inhibit infection by plant pathogens [16] [17].In addition, phomalactone isolated from Nigrospora sphaerica (Sacc.)E.W. Mason inhibits mycelial growth and affects the infection behavior of plant pathogens [18].Nigrosporin A and B, which are phytotoxic metabolites, are produced from a culture filtrate of Nigrospora oryzae (Berk.& Br.) Petch [19].However, in this study, the culture filtrate of Nigrospora sp.showed no phytotoxic activity in barley.There are no reports on the utility of culture filtrates of Annulohypoxylon for control of plant pathogens.Annulohypoxylon is members of the Xylariaceae family and can  survive as endophytes.Xylariaceae is a large family (Xylariales, Ascomycotina) of more than 36 genera.Whalley (1995) [20] reported that secondary metabolites produced by representatives at least one-third of these genera have been isolated and identified.Further studies are essential to identify the active antifungal compound(s) in the culture filtrates of these fungi.Studies of symbiotic and parasitic fungi from wild mushrooms may yield potential control agents for plant diseases such as the rice blast disease.

Figure 1 .
Figure 1.Culture filtrates of fungi isolated from wild mushrooms influence the infection behaviors of Magnaporthe oryzae.Data were obtained from the results of 6 experiments of 3 replicates and expressed as mean ± SD.Means followed by different letters are significantly different according to the Scheffe's test (p < 0.05).

Figure 2 .
Figure 2. Inhibition by heat-treated culture isolates on the infection behaviors of Magnaporthe oryzae.(a) and appressorium formation (b) were determined after 24 h incubation.(+): culture filtrates with ethyl acetate soluble (+) and insoluble (−) antifungal compound against M. oryzae spore germination and appressorium formation.Data were obtained from the results of 6 experiments of 3 replicates and expressed as mean ± SD.Means followed by different letters are significantly different according to the Scheffe's test (p < 0.05).