OXYPORUS LATEMARGINATUS STRAIN HAVING ANTAGONISTIC ACTIVITYAGAINST PLANT DISEASES

FIELD OF THE INVENTION

The present invention relates to an Oxyporus latemarginatus strain having antagonistic activity against plant diseases; a microbial agent for controlling plant diseases comprising same; and a method for biologically controlling plant diseases using same.

BACKGROUND OF THE INVENTION

The use of biopesticides based on microorganisms and biochemical materials has been suggested as a way to solve various problems associated with conventional chemical pesticides. Microorganisms used as biopesticides exhibit antagonistic activity against plant pathogens. The antagonistic effect can be achieved by the action of volatile or nonvolatile compounds produced by the microorganisms. The volatile materials produced by microorganisms can be used as an environmental-friendly biopesticides which can replace harmful methyl bromide conventionally used as a fumigant for soil. Biofumigant can be effectively used to control infectious soil diseases and storage molds to protect fruits and vegetables during storage and transportation.

The possibility for controlling soil diseases and storage molds of fruits using Muscodor albus, a mold producing volatile compounds, has been proposed by AgraQuest, Inc. (Mercier and Manker, 2002, Phytopathology 92:S55). Muscodor albus is an endophyte isolated from Cinnamomum zeylanicum and secretes 28 volatile materials which are effective in controlling various kinds of molds, oomycetes and bacteria. The major components of the volatile compounds produced by Muscodor albus have been identified to be 2- methyl- 1-buthanol and isobutyric acid (Atmosukarta et al., 2005, Plant Science 169:854-861; Lacey and Neven, 2006, Journal of Invertebrate Pathology 91 :195- 198). However, cultures or formulations of Muscodor albus can not be stored over a long period of time even at a low temperature.

Thus, there has been a need to develop microorganisms which exhibit antagonistic activity against plant pathogens and have long-term stability.

The present inventors have endeavored to identify a microorganism producing a volatile antifungal compound among endophytes isolated from various plants, and have found that a fungus, Oxyporus latemarginatus EF069 isolated from pepper plants shows antagonistic activity against several kinds of plant diseases and superior storage survivability due to its basidiospore formation.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a strain of Oxyporus latemarginatus having antagonistic activity against plant diseases.

It is another object of the present invention to provide a microbial agent for controlling plant diseases comprising the strain.

It is a further object of the present invention to provide a method for controlling plant diseases using the microbial agent.

In accordance with one aspect of the present invention, there is provided a strain of Oxyporus latemarginatus EF069(KCTC 11038BP) having antagonistic activity for controlling plant diseases.

In accordance with another aspect of the present invention, there is provided a microbial agent for controlling plant diseases comprising the strain or, a culture, an extract, a spore or a mycelium thereof. In accordance with a further aspect of the present invention, there is provided a method for controlling plant diseases comprising the step of applying an effective amount of the microbial agent to plants.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, which respectively show:

Fig. 1 : a photograph showing inhibitory activity of Oxyporus latemarginatus EF069 on growth of Botrytis cinerea, comparing with the control group; Fig. 2: a photograph of Oxyporus latemarginatus EF069 cultured in a potato dextrose agar medium;

Fig. 3: a photograph showing disease controlling activity of Oxyporus latemarginatus EF069 against gray mold of apple;

Fig. 4: a photograph showing disease controlling activity of Oxyporus latemarginatus EF069 against Rhizoctonia disease of Phalenopsis;

Fig. 5: a chromatogram (A) of gas chromatography (GC) for sterilized solid culture; and GC chromatogram (B) and mass spectrum (C) of a volatile material produced by Oxyporus latemarginatus EF069.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a strain of Oxyporus latemarginatus EF069(KCTC 11038BP), exhibiting broad-spectrum antagonistic activity against plant diseases. The strain of the present invention is isolated from pepper plants, preferably stems thereof. Morphological study and ITS1-5.8S-ITS2 nucleotide sequencing analysis reveal that this strain belongs to Oxyporus latemarginatus. This novel strain was designated Oxyporus latemarginatus EF069 and was deposited on November 28, 2006 with the Korean Collection for Type Cultures (KCTC) (Address: Korea Research Institute of Bioscience and Biotechnology (KRIBB), #52, Oun-dong, Yusong-ku, Daejon, 305-333, Republic of Korea) under accession number KCTC 11038BP, in accordance with the terms of Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure. Oxyporus latemarginatus EF069 of the present invention shows the following morphological and biochemical characteristics: it forms white aerial hyphae on a potato dextrose agar medium, and ITS1-5.8S-ITS2 nucleotide sequencing analysis shows that Oxyporus latemarginatus EF069 has the

nucleotide sequence of SEQ ID No: 1 which has 98% sequence homology with that of Oxyporus latemarginatus AF232721.

Oxyporus latemarginatus EF069 thus characterized shows broad and high activity for controlling plant diseases including Alternaria leaf spot of apple tree, anthracnose diseases, Fusarium wilt diseases, rice blast, clubroot of crucifers, Rhizoctonia diseases, and Botrytis diseases.

Thus, Oxyporus latemarginatus EF069 of the present invention can be employed as a microbial agent for controlling plant diseases. The microbial agent may be prepared by mixing suitable carriers with the strain alone, a whole culture of the strain, a solvent extract obtained therefrom, a spore or a hypha thereof, and then formulating the mixture into powders, pellets, granules or solutions. The carriers employable in the inventive formulation include, but are not limited to, water, white carbon and kaolin. The culture may be of a liquid or solid form. The microbial agent of the present invention may be advantageously used for preventing growth inhibition or withering of plants caused by plant pathogens by applying an effective amount thereof to soil where plants grow, to the leaves of plants, or to the space where plants grow or are stored.

Oxyporus latemarginatus EF069 may be applied to target plant leaves at a cell concentration of 1.0 X 10 ⁶ to LO X 10 ¹² cfu/mC, preferably 3.7 X 10 ⁷ to 3.3 X 10 ⁹ cfu/nώ. When applied to soil, a solid culture of the microbial agent may be applied to the soil around target plants in an amount of 0.1 to 10% percent as solid cultures by weight based on the soil volume. Further, when fumigation is desired, it may be applied to target plants in an amount of 1 X 10 ³ to 1 X 10 ⁵ g/m ³ as solid cultures.

The following Examples are intended to further illustrate the present invention without limiting its scope.

Example 1: Isolation of endophytic fungi from red pepper plants

Whole red pepper plants were collected from 8 fields located in Chungcheongbuk-do, South Korea (3 plants from each field), and the leaves,

stems and roots thereof were separated to be divided into 24 samples. The samples were subjected to surface sterilization so that only endophytic fungi could be isolated: Each pepper leaf sample was soaked in 2% NaOCl for 10 sec to sterilize the surface thereof; washed 2 times with sterile distilled water; cut to pieces; and embedded onto a cormeal malt extract agar medium (CMA medium; 17.0 g of corn powder, 20.0 g of malt extract, 2.0 g of yeast extract and 1.0 I of distilled water) supplemented with 50 mg/E chloramphenicol (Sigma). Each of the stem and root sample was briefly soaked in ethanol, subjected to flame sterilization, and then embedded onto the same medium mentioned above. Each of the media thus treated was incubated at 25 ^° C until hyphae were formed. The hyphae were separated from the medium, inoculated onto a PDA medium and subjected to several subculture cycles. From the cultures obtained by repeating the above procedure, 89 fungal strains were isolated. The hyphal tips were separated from the isolated strains grown on PDA media for 7 days; put into solutions of sterilized distilled water and 6% dimethylsulfoxide solution; and stored at room temperature and -70 ^° C, respectively.

Example 2: Screening for an endophytic fungus producing a volatile antifungal compound

The antifungal activity of volatile antifungal compounds produced by each of the 89 strains isolated in Example 1 was examined in a mycofumigation plate assay for Botrytis cinerea, which causes gray mold (Strobel et al, 2001, Microbiology 147:2943-2950). Specifically, a PDA medium (90 ^χ 15 mm; Becton and Dickinson Co., Farnklins Lakes, NJ, USA) was placed in a Petri dish and divided into 2 parts by removing a 1 cm -wide center strip therefrom. Each of the 89 strains isolated in Example 1 was inoculated to one part of the PDA medium in a Petri dish. Each of the Petri dish was sealed with a parafilm and incubated at 25 ^° C . After 3 days, the Petri dish was unsealed, and Botrytis cinerea was inoculated to the other part of the medium. The Petri dish was sealed with a parafilm again and incubated at 25 °C .

The inhibitory activity of each of the 89 strains on growth of Botrytis

cinerea was screened by analyzing the resulting culture, and one strain was found to be particularly effective in controlling the growth of Botrytis cinerea as shown in Fig. 1.

Example 3: Identification of the active fungal strain

The active fungal strain found in Example 2 was identified based on characterization of its morphological features and ITS1-5.8S-ITS2 nucleotide sequencing.

(1) Morphological characteristics

The morphology of the active strain cultured on a PDA medium was tinged with white as shown in Fig. 2, which did not change with time.

(2) ITS1-5.8S-ITS2 nucleotide sequencing

The active strain was inoculated onto a PDA medium covered with a cellophane paper, and incubated at 25 "C for 4 days, followed by DNA extraction therefrom. ITS1-5.8S-ITS2 nucleotide sequence of nuclear rDNA was amplified by PCR employing ITS4 and ITSl primers (White et al., 1990, PCR protocols: a guide to methods and application, pp.315-322). After examining the purity of the amplified product, nucleotide sequencing analysis was conducted by using ABI3700 automated DNA sequencer (Applied Biosystems).

The result revealed that the ITS1-5.8S-ITS2 nucleotide sequence of the isolated strain had the nucleotide sequence of SEQ E) No: 1 which showed 98% sequence homology with ITS1-5.8S-ITS2 sequence of Oxyporus latemarginatus (GenBank accession number AF232721). These results demonstrated that the active strain is a strain of Oxyporus latemarginatus, which has been designated Oxyporus latemarginatus EF069 and deposited on November 28, 2006 with the Korean Collection for Type Cultures (KCTC) (Address: Korea Research Institute of Bioscience and

Biotechnology (KRIBB), #52, Oun-dong, Yusong-ku, Daejon, 305-333, Republic of Korea) under accession number KCTC 11038BP.

Example 4: Test for antifungal activity of a volatile compound produced by Oxyporus latemarginatus EF069 against plant pathogens

Antifungal activity of Oxyporus latemarginatus EF069 (hereinafter, referred to as "EF069") against plant pathogens was examined in vitro by using a method similar to that of Example 2. Specific plant pathogens; Rhizoctonia solani, Magnaporthe grisea,

Fusarium oxysporum, Colletotrichum gloeosporioides, Botrytis cinerea and Alternaria alternata subsp. mali, which cause rice sheath blight, rice blast, Fusarium wilt, red pepper anthracnose, gray mold disease and Alternaria leaf spot of apple tree, respectively; were obtained from Kyung Nong Co., Ltd. As a control group, each of pathogens was inoculated onto one side of the medium and another side of the medium was left untreated. The extent of the pathogen growth (growth length) was measured and percentage fungal control value based on the untreated control (control value) was calculated using Formula 1. The results are shown in Table 1.

<Formula 1>

Control value (%) = (GLU _CG -GL _TE G) í GL _UCG X 100 wherein, GLU _CG is the growth length of the untreated control group, and GL _TE G _* the growth length of the treated experimental group.

<Table 1>

As shown in Table 1, the volatile compound produced by EF069 exhibits high antifungal activities against various plant pathogens.

Example 5: Test for in vivo antifungal activity of Oxyporus latemarginatus EF069 against apple gray mold

5 pieces of EF069 mycelia having a 8 mm-diameter were inoculated into a wheat bran-rice hull medium (200 mi of wheat bran, 100 ml of rice hull and 100 m£ of distilled water) and incubated at 25 ^° C for 14 days. Then, 25 g and 50 g of the solid culture of EF069 were respectively placed in each plastic box (7 I ) with apples whose surface were artificially cut and infected with Botrytis cinerea. As a control group, apples infected with Botrytis cinerea were placed in a plastic box without the solid culture of EF069. The boxes were sealed with a wrap and placed in a 25 ^° C -incubator under dark condition. 6 apples were employed per each box, and the bottom of each box was covered with a water-wetted paper towel. After 5-days incubation, incidence rate was assessed by checking the change of the lesion size on the surface of each apple. Percent disease control value based on the untreated control was calculated using Formula 2.

<Formula 2>

Control value (%) = (LD _UCG -LD _TE G) íLD _UC G>< 100 wherein, LD _UCG is the lesion diameter of the untreated control group, and LD _TEG , the lesion diameter of the treated experimental group.

As shown in Fig. 3, while the control group exhibited intense progress of gray mold at the infected site, the experimental groups treated with 25 g and 50 g of the solid culture of EF069 showed the disease control rates of 91% and 100% against gray mold, respectively.

Example 6: Test for in vivo antifungal activity of Oxyporus latemarginatus EF069 against damping-off of radish

Antifungal activity of EF069 for damping-off of radish was examined as follows.

The solid culture of EF069 medium was prepared using a wheat bran-rice hull medium as made in Example 5, and 5% and 10% by weight of the culture were mixed with normal soil, based on the volume of the soil, respectively (experimental group). In non-treated control group and a comparative group, sterilized wheat bran-rice hull medium only were employed instead of the solid culture of EF069. In a comparative group, 10 mi of a commercial pesticide called flutolanil (Kyung Nong Co., Ltd.) (100 βg/ml) was added to a pot. Each soil mixture was placed in a 4.4 cm-diameter pot, and seeded with a radish (Hungnong Seed Co., Ltd.). The pots were placed in a greenhouse for a week. Then, soil around the plants roots was removed using sterilized distilled water, and the plants were transferred to diseased soil, which was prepared by incubating a rye culture with Rhizoctonia solani (Korea Research Institute of Chemical Technology) causing damping-off on radish for 2 weeks, and mixing 1 g of the rye culture with 100 n^ of soil. After 10 days, the degree of plant withering was examined for each pot. Percent disease control value based on the untreated control was calculated using Formula 3. The results are summarized in Table 2.

<Formula 3>

Control value (%) = (NW _UC G-NWTEG) íNW _UC G ^X 100 wherein, NW _UCG is the number of withered plants of the untreated control group, and NW _TEG , the number of withered plants of the treated experimental group.

<Table 2>

As shown in Table 2, EF069 showed superior antifungal activity as compared with the commercial pesticide, flutolanil, despite the removal of the soil mixture containing EF069 in the transfer to the diseased soil. It suggests that EF069 penetrated into the plant root and grew endogenously therein.

Example 7: Test for in vivo antifungal activity of Oxyporus latemarginatus EF069 against Rhizoctonia disease of Phalenopsis

EF069 was cultured in a wheat bran-rice hull medium in the same manner of Example 5 for 2 weeks, and the resulting culture was shifted to 4.4 cm-diameter pots (5 g per each pot). Then, the pots were put under other pots in which young Phalenopsis sp. plants in 2-3 leaf stage naturally infected with Rhizoctonia solani were planted, and the circumferential gap between the two stacked ports was sealed with a parafilm (experimental group). In a comparative group, the same plants as employed in the experimental group were placed in 4.4 cm-diameter pots and drenched with 10 ml of flutolanil solution (100 μg/M). In a control group, the plant pots were just watered. Conventional farming was employed to grow the plants of the three experimental, comparative and control groups for 2 months, and, then, fresh EF069 culture, flutolanil solution and water were reapplied to the three groups, respectively, in the same manner. After next 2 months, the number of diseased plants and the average number of leaves per plant were measured. Percent disease control value based on the untreated control was calculated using Formula 4. The results are summarized in Table 3.

<Formula 4>

Control value (%) = (ND _UCG -ND _T E _G ) íND _UCG X 100 wherein, ND _UCG is the number of diseased plants of the untreated control group, and ND _TEG , the number of diseased plants of the treated experimental group.

<Table 3>

As can be seen in Table 3, the antifungal activity of EF069 was higher than that of flutolanil, and the growth of Phalenopsis sp. in the experimental group showed fair traits as compared with the control group. Fig. 4 also demonstrates the better growth of Phalenopsis sp. in the experimental group.

Example 8: Test for antifungal activity of Oxyporus latemarsinatus EF069 against clubroot disease of Chinese cabbage

The solid culture of EF069 prepared in Example 5 was mixed with normal soil in a ratio of about 10% by volume per volume of soil. Then, the mixture was placed in a 4.4 cm-diameter pot and sowed with seeds of a Chinese cabbage(Hungnong Seed Co., Ltd.). The pot was placed in a greenhouse for 3 weeks. Seedlings of Chinese cabbages sprouted from the seeds were transplanted to soil infected with Plasmodiophora brassicae. In a comparative group, 0.5% of a commercial pesticide called Furoncide ^® (Dongbu HyTec) was added to the infected soil before transplanting the cabbage. In a control group, nothing was treated to the soil.

Farming field was divided into 3 cells for each group, and 20 Chinese cabbages were placed per cell. After 4 weeks, the control value was measured and the results are summarized in Table 4.

<Table 4>

As illustrated in Table 4, EF069 showed high antifungal activity against clubroot disease of Chinese cabbage in the field.

Example 9: Analysis for the volatile compound produced by Oxyyorus latemarsinatus EF069

In order to analyze the volatile compound produced by EF069, a wheat bran-rice hull medium culture of EF069 (cultured for 2 weeks) was subjected to modified methods of Strobel et al. (Mirobiology, 147:2943-2950, 2001), and Ezra and Strobel (Plant Science, 88:239-247, 2003). The volatile material was collected using an SPME (solid-phase micro-extraction syringe; Supelco Inc., Bellefonte, PA, USA) packed with a fibre material, 50/30 divinylbenzene/ carburen on polydimethylsiloxane. The needle of the syringe was inserted through a septum attached to the flask in which the solid culture of EF069 had been kept, to allow the syringe packing to absorb the volatile material for 45 minutes, and the volatile compound absorbed in the syringe packing was injected to a device for gas chromatography (JEOL QC5050) equipped with a mass-selective detector and a fused silica capillary column SPB-5(30 m ^χ θ.25 mm, film thickness: 0.25 μm; Supelco Inc., Bellefonte, PA, USA). The temperature of the syringe injector was 240 ^° C, and the column temperature was programmed from 30 ^° C (2 minutes) to 220 °C (9 minutes) at an initial column head pressure of 50 KPa using high purity helium carrier gas.

As can be seen in Fig. 5, a single pick was observed after 20.3 minutes (Fig. 5(b)), which showed a molecular weight of 166 faced on the mass spectrum result (Fig. 5(c)). No peak was observed for the sterilized solid culture which did not contain EF069.

While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.