WO2014057539A1

WO2014057539A1 - METHOD FOR INHIBITING PROLIFERATION OF PLANT PATHOGENIC BACTERIUM USING BACTERIUM BELONGING TO GENUS Trichoderma

Info

Publication number: WO2014057539A1
Application number: PCT/JP2012/076198
Authority: WO
Inventors: 阿野貴司; 廣瀬陽一郎
Original assignee: 十和田グリーンタフ・アグロサイエンス株式会社; 学校法人近畿大学
Priority date: 2012-10-10
Filing date: 2012-10-10
Publication date: 2014-04-17
Also published as: JP6210560B2; JPWO2014057539A1

Abstract

Provided is a novel method for inhibiting the proliferation of a plant pathogenic bacterium using a bacterium belonging to the genus Trichoderma. A method for inhibiting the proliferation of a plant pathogenic bacterium, said method being characterized by comprising culturing a strain HNT-01 microorganism that is capable of inhibiting the proliferation of plant pathogenic bacteria and belongs to the genus Trichoderma and then seeding the cultured microorganism in plant soil.

Description

Method for inhibiting the growth of plant pathogens using Trichoderma spp.

The present invention relates to a method for suppressing the growth of phytopathogenic fungi using microorganisms, and more particularly to a method for suppressing the growth of phytopathogenic fungi using Trichoderma spp.

Green tuff (hereinafter sometimes referred to as “Towada stone”) mined in Hinai-cho, Odate City, Akita Prefecture, is officially called “quartz andesitic plutonic tuff” and is known as “Green Tough” .

This tuff was formed by the accumulation and solidification of submarine volcanic ash about 10 million years ago when the Japanese archipelago was formed. When observed with an electron microscope, a porous structure in which extremely fine particles are linked together is observed. The main constituent minerals are feldspar, chlorite, quartz, and silicate minerals with about 30% amorphous crystals.

The composition of the Towada stone is shown below.

Towada stone has been cut and commercialized as a building stone and bathroom floor material from about 40 years ago because of its beautiful blue-green color and non-slip heat and moisture retention.

The crushed stone particles and chips generated during the lumbering process are used as extenders that are the cores of chemical fertilizers, and as proposed in Patent Document 1, environmentally useful microorganisms are added to the tuff grains. The technique of using as a soil improvement material by making it carry | support is known.

In Patent Document 1, it is said that the caustic substances having an average particle diameter of 1 mm to 20 mm are decomposed to cause environmental pollution such as bad odor and contribute to environmental conservation. The genus Bacillus, Nitrosomonas (Nitrosomonas) An invention for impregnating one or more microorganisms selected from the group of microorganisms belonging to the genus genus), the genus Nitrobactor, the genus Thiobacillus, and the genus Pseudomonas has been proposed.

JP 2006-143974 A JP 11-225745 A JP 2012-95570 A

The particle size obtained after adding polyaluminum chloride flocculant (PAC) to a reservoir containing wastewater containing processed fine powder generated from the Towada stone quarry, coagulating and precipitating the fine powder, collecting it and drying it. Was found and collected in a fine powder (commonly known as Towada stone cake) in the range of 1 μm to 80 μm, which suppresses the growth of Rhizoctonia solani, a typical plant disease fungus.

Next, a microorganism that inhibits the growth of Rhizoctonia solani and antagonizes Fusarium oxysporum was identified from the group of microorganisms.

As a result of identifying the specified microorganism at Techno Suruga Lab Co., Ltd., it was found that it was Trichoderma atroviride, a kind of ascomycetous fungus. Furthermore, since this strain has characteristics that have not been discovered in the past, as a new microorganism, as of September 7, 2012, the National Institute of Technology and Evaluation of the National Institute of Technology and Evaluation (Kazusakami, Kisarazu City, Chiba Prefecture, Japan) Deposited at feet 2-5-8). (Accession number NITE P-1419).

The chemical composition of the Towada stone cake where the above microorganisms were found is described below. Some elements have almost the same composition as the rock composition of Towada stone, although there are minor differences.

As an isolation source and carrier of the genus Trichoderma, in Patent Document 2, a strain (SKT-1) isolated from turf (Noshiba) rhizosphere and a strain (SKT-2) isolated from Sardana rhizosphere Is disclosed.

Patent Document 3 discloses a method of supporting Trichoderma bacteria on wood, charcoal or pulverized charcoal.

There are many other literature on Trichoderma spp., But to summarize, this species is common in soil and is also isolated from wood, decayed wood, mushrooms, mushroom compost, bark beetles, corals, etc. It is what is done. There are also reports of separation from rocks such as granite, marble, sandstone, gneiss and quartz as the genus Trichoderma.

However, there has never been an example isolated from the dried fine powder that has been processed from tuff, such as the Towada stone cake used as a fertilizer base.

Therefore, an object of the present invention is to provide a novel method for suppressing the growth of phytopathogenic bacteria using Trichoderma bacteria.

In order to solve the above problems, the following inventions are proposed.

The invention of claim 1
It is a microorganism belonging to the genus Trichoderma that has the ability to suppress the growth of plant pathogens.

The invention of claim 2
The microorganism according to claim 1, wherein the microorganism is isolated from green tuff.

The invention of claim 3
The microorganism according to claim 2, wherein the microorganism has a deposit number of NITE P-1419.

The invention of claim 4
A method for inhibiting the growth of phytopathogenic fungi, comprising culturing the microorganism according to any one of claims 1 to 3 and inoculating the cultured microorganism in plant soil.

The invention of claim 5
5. The method for inhibiting the growth of phytopathogenic fungi according to claim 4, wherein the phytopathogenic fungi are filamentous fungi belonging to the genus Rhizoctonia or the genus Fusarium.

According to the present invention, it is possible to provide a novel method for suppressing the growth of phytopathogenic fungi using Trichoderma spp.

(A), (b) is a figure showing the mycelium of mold | fungi seen in the Towada stone mining mine. It is the figure showing the suppression observation with respect to Rhizoctonia solani (Rhizoctonia solani) of unsterilized Towada stone, (a) State after inoculation, (b) State on the first day of culture, (c) Day 2 of culture The state of the eyes, (d) the state of the third day of culture, (e) the state of the fourth day of culture. It is the figure showing the suppression observation with respect to Rhizoctonia solani of the sterilized Towada stone, (a) State after inoculation, (b) State on the first day of culture, (c) Day 2 on the culture (D) State on the third day of culture, (e) State on the fourth day of culture. It is the figure showing the suppression observation with respect to Rhizoctonia solani (Rhizoctonia solani) of seven bacteria isolate | separated with 1/10 TSA culture medium, Comprising: (a) State of 1st strain, (b) State of 2nd strain, (c ) 3rd stock, (d) 4th to 7th stock. It is the figure showing the suppression observation with respect to Rhizoctonia solani of 2 bacteria isolate | separated with the TSA culture medium, Comprising: (a) State of 1st strain, (b) State of 2nd strain. It is the figure which observed the fungus which showed the suppression with respect to Rhizoctonia solani (Rhizoctonia solani) among 4 fungi strains isolated with 1/10 TSA culture medium, (a) The state isolated from Towada stone, (b) Rhizoctonia・ A state where Rhizoctonia solani is suppressed. It is the figure showing the suppression observation with respect to three Rhizoctonia solani (Rhizoctonia solani) among 4 fungi strains isolated with 1/10 TSA culture medium, (a) State of 1st strain, (b) 2nd strain (C) Third stock. (A) Diagram showing suppression observation of Rhizoctonia solani of HNT-01 strain, (b) Diagram showing suppression observation of Collimonas ナス sp. D-25 of HNT-01 strain, (c) FIG. 4 is a diagram showing the observation of inhibition of Bacillus subtilis RB14 in the HNT-01 strain, and FIG. 4D is a diagram showing inhibition observation of Fusarium oxysporum in the HNT-01 strain. This is a molecular phylogenetic tree using the 28S rDNA-D1 / D2 nucleotide sequence of HNT-01 strain. This is a molecular phylogenetic tree using ITS-5. 8S rDNA base sequence of HNT-01 strain. It is a figure showing the macroscopic observation image of HNT-01 strain, (a) State on PDA medium, (b) State on MA medium, (c) State on OA medium, (d) On CMA medium State. It is a figure showing the microscopic observation image of HNT-01 stock | strain. It is also a figure showing the microscopic observation image of HNT-01 stock. It is also a figure showing the microscopic observation image of HNT-01 stock. It is also a figure showing the microscopic observation image of HNT-01 stock. It is a figure showing conidia among the microscopic observation images of HNT-01 strain. It is a figure showing a thick film spore among the microscopic observation images of HNT-01 strain.

Hereinafter, an embodiment of the present invention will be described with reference to the accompanying drawings.

(Experimental example 1)
In order to confirm the existence of microorganisms that suppress plant pathogens in Towada stone, we conducted a suppression experiment on Towada stone against the plant pathogen Rhizoctonia solani.

<Materials used and strains used>
Unsterilized Towada stone powder Rhizoctonia solani

<Experiment method>
Sample A 0.7 g of unsterilized Towada stone, 300 μL of sterilized water
Sample B Sterile Towada stone 0.7g, Sterile water 300μL

After inoculating Rhizoctonia solani on Samples A and B, the cells were allowed to stand at room temperature for 2 days, and 3 mL of sterilized water was added to each sample and stirred using a vortex. The stirred culture solution was counted using 1/10 TSA medium as an inhibitory effect on lysoctonia, which is a phytopathogenic fungus.

<Bacteria count calculation result>
Sample A 2.1 × 10 ⁷ cfu / mL
Sample B N.D.

<Suppression observation for plant pathogens>
The culture solution of each sample prepared above was inoculated into 10 μLTSA medium and 1/10 TSA medium, and at the same time, agar slices of Rhizoctonia solani were inoculated. The culture was performed at room temperature and observed. Observation was performed for 1 week, and the observation results up to the 4th day are shown in FIGS.

As shown in FIG. 2 and FIG. 3, when an experiment of the inhibitory effect was performed without sterilizing Towada stone, a considerably large inhibition circle was formed (FIG. 2).

Even when visually confirmed, several types of bacteria and molds are present in Towada stone, and the boundary of the inhibition circle has been confirmed to be something like inhibition of mycelial growth. It was suggested.

(Experimental example 2)
In Experimental Example 1, it was suggested that microorganisms resident in Towada stone suppressed the growth of Rhizoctonia solani, so that microorganisms were searched in this experiment.

<Materials used and culture media>
Towada stone powder PDA medium 1/10 TSA medium TSA medium

<Method for isolating microorganisms>
After weighing 0.1 g of Towada stone powder, 1 mL of sterile water or sterile physiological saline was added and suspended. Using this suspension as a stock solution, a dilution series was prepared up to 10 ⁻⁶ , and 20 μL was inoculated on each agar medium.

Thereafter, the suspension using sterilized water was spread evenly with 1/10 TSA or TSA congeal rod and cultured at 24 ° C. or 30 ° C. for 4 to 7 days.

After the culture, single colonies grown on the agar medium were picked and isolated by simple streak culture. In the case of fungi, the tip of the grown hyphae was caught and inoculated into a new medium. This operation was repeated several times to purify the bacteria.

After isolation, the growth inhibitory effect against Rhizoctonia solani was confirmed in several microorganisms.

<Result>
From the 1/10 TSA medium, a total of 13 microorganisms were obtained: 7 bacteria, 4 fungi, and 2 bacteria from the TSA medium. No bacteria have been isolated from the PDA medium so far. These microorganisms were tested for inhibition against Rhizoctonia solani, and the results are shown in FIGS.

FIG. 4 shows the results of observation of inhibition of 7 strains of bacteria isolated on 1/10 TSA medium against Rhizoctonia solani. None of the bacteria inhibited Rhizoctonia solani.

FIG. 5 shows the results of observation of inhibition of two strains of bacteria isolated on TSA medium against Rhizoctonia solani. None of the bacteria inhibited Rhizoctonia solani.

FIG. 6 shows fungi that showed inhibition against Rhizoctonia solani among 4 fungi strains isolated in 1/10 TSA medium.

FIG. 7 shows the results of observation of inhibition of 3 strains of Rhizoctonia solani among 4 fungi strains isolated with 1/10 TSA medium. None of the fungi controlled against Rhizoctonia solani.

Among the 13 strains (9 bacterial strains and 4 fungal strains) isolated from Towada stone in this experiment, it was 1 fungal strain that was observed to inhibit Rhizoctonia solani. The other 12 strains of microorganisms are usually presumed to be a variety of bacteria that inhabit the environment, judging from the Towada stone mining site and product usage.

Also, since no accidents such as poisoning caused by Towada stone have occurred in the past, it is unlikely that these bacteria are harmful microorganisms to the human body and the environment.

(Experimental example 3)
When unsterilized Towada stone powder was used in Experimental Example 1, the presence of microorganisms that inhibit Rhizoctonia solani was confirmed, and the suppression of microorganisms that seemed to be filamentous fungi was isolated in Experimental Example 2. Towada stone powder resident bacteria, Collimonas sp. D-25 (Accession Number NITE P-1104), Bacillus subtilis RB14, Fusarium oxysporum and Rhizoctonia solani Antibacterial activity test was conducted on a total of 4 strains.

<Used strain>
Rhizoctonia solani
Collimonas sp. D-25 (Accession number NITE P-1104)
Bacillus subtilis RB14
Fusarium oxysporum

<Medium used>
1/10 TSA medium

<Experiment method>
Each microorganism precultured on an agar medium was fished with a toothpick and inoculated into a new 1/10 TSA medium. The isolated Towada stone powder resident bacteria (hereinafter referred to as “HNT-01 strain”) that were confirmed to be isolated were inoculated so as to be symmetrical with each of the above bacteria. Then, it was confirmed whether the HNT-01 strain suppresses or suppresses each microorganism, and the results are shown in FIG.

FIG. 8 shows the observation results on the 10th day of culture. FIG. 8 (a) shows the observation results of HNT-01 strain and Rhizoctonia solani. As in Experimental Example 1, a large blocking circle is formed.

FIG. 8 (b) shows the observation results of HNT-01 strain and Collimonas sp. D-25. Although Collimonas sp. D-25 was not covered by HNT-01 strain, it was not clearly suppressed. It can be seen that the inoculum of Collimonas sp. D-25 is thinly invaded on the right side of Fig. 8 (b).

FIG. 8 (c) shows the observation results of HNT-01 strain and Bacillus subtilis RB14. The HNT-01 strain was suppressed by Bacillus subtilis RB14, and no further hyphal growth was observed.

FIG. 8 (d) shows the observation results of HNT-01 strain and Fusarium oxysporum. Although it was not possible to suppress Fusarium oxysporum, Fusarium oxysporum and HNT-01 were in equilibrium and did not cover either.

From this experiment, it was found that the Towada stone resident HNT-01 strain suppresses Rhizoctonia solani, but Fusarium oxysporum has a degree of inhibition (antagonism). .

It was also found that Bacillus subtilis RB14 suppressed HNT-01 strain, but Collimonas sp D-25 was not suppressed, and conversely it was not covered.

(Identification test of isolate)
In order to estimate the attribution classification of HNT-01 strains isolated from Towada stone, the following identification test was conducted.

<Culture conditions>
The HNT-01 strain was cultured under the following conditions to obtain test cells.
・ Medium potato dextrose agar (PDA)
2% malt agar medium (MA)
Oatmeal agar (OA)
Cornmeal agar medium (CMA)
・ Cultivation temperature 25 ℃ and room temperature ・ Culture period 1 to 6 weeks

<28S rDNA-D1 / D2 and ITS-5. 8S rDNA nucleotide sequence analysis>
The operations from extraction to cycle sequence are based on each protocol. In the identification test, the HNT-01 strain was assigned a SIID11835 number, and the nucleotide sequence was analyzed.
・ DNA extraction Physical disruption and modification of Marmur (1961) ・ PCR PrimeSTAR HS DNA Polymerase (Takara Bio)
Cycle sequence BigDye Terminator v3.1 Kit (Applied Biosystems)
・ Primers used NL1 and NL4
ITS5 and ITS4
・ Sequence ABI PRISM 3130xl Genetic Analyzer System (Applied Biosystems)
・ Sequencing ChromasPro 1.5 (Technelysium Pty Ltd.)
・ Homology search and simple molecular phylogenetic analysis software Apollon 2.0 (Techno Suruga Lab)
Database Apollon DB-FU5.0 (Techno Suruga Lab)
International nucleotide sequence database (GenBank / DDBJ / EMBL)

<Macroscopic observation of HNT-01 strain>
Each culture plate was macroscopically observed after 1 week of culture, and the colony diameter, color tone (colony surface and back surface), surface properties, presence or absence of soluble pigment production, etc. were recorded.

As a result of observation, the characteristics shown in Table 7 below were observed on each culture plate of the HNT-01 strain after 9 days of culture. Moreover, the macroscopic observation image of each culture plate is shown in FIG.

<Microscopic observation of HNT-01 strain>
FIGS. 12 to 17 show microscopic observation images of each culture plate of HNT-01 strain.

-Vegetative mycelium Mycelium was formed on or in the agar surface, and colorless and bulky hyphae formation was observed. In addition, it was observed that spherical and subspherical colorless thick membrane spores were formed on the vegetative mycelium (FIG. 17).

Asexual reproductive organ Conidia and conidia-forming cells The conidia are upright from the vegetative mycelium, branch off regularly or irregularly, and gather together to form a wool-like lump (tuft) (Fig. 12, Fig. 12). 13).

The fialide, which is a conidia-forming cell, was ampoule-shaped and formed at the tip of the conidia pattern, and some gathered to form a green conidia mass (FIGS. 14 and 15).

2. Conidia are fialo-type conidia, colorless to green, subspherical to oval, 1 cell, and smooth on the surface (FIG. 16).

-Sexual reproductive organs Formation of sexual reproductive organs could not be confirmed in cultured specimens of about 6 weeks.

<Discussion>
The 28S rDNA-D1 / D2 nucleotide sequence obtained from the HNT-01 strain was found to be 100% homologous with the nucleotide sequence of Tricoderma atroviride CBS351.93, which is a kind of ascomycetous fungi, as a result of BLAST homology search against apollon DB-FU. (Table 3).

As a result of the homology search against the international nucleotide sequence database, the 28S rDNA-D1 / D2 nucleotide sequence of the HNT-01 strain is 100% homologous with the multiple nucleotide sequences of Tricoderma atroviride and Hypocreaatroviridis, which are types of Ascomycetes Homology was shown (Table 4).

HypocreaHatroviridis and Tricoderma atroviride found in the homology search results are known to have a relationship between teleomorph (sexual age) and anamorph (Dodd etal, 2003).

In the phylogenetic tree created on the basis of the upper sequence obtained by the homology search, the HNT-01 strain formed the same phylogenetic tree as Tricoderma atroviride and Hypocrea atroviridis (FIG. 9).

As a result of BLAST homology search against Apollon DB-FU, ITS-5. 8S rDNA base sequence of HNT-01 strain shows 100% homology with multiple base sequences of Tricoderma 菌 atroviride, a kind of ascomycete (Table 5).

As a result of homology search against the international nucleotide sequence database, the ITS-5. 8S rDNA nucleotide sequence of HNT-01 strain is 100% homologous with the multiple nucleotide sequences of Tricoderma atroviride and Hypocreaatroviridis, which are types of Ascomycota (Table 6).

In the phylogenetic tree created based on the upper sequence obtained by the homology search, the HNT-01 strain formed the same phylogenetic tree as Tricoderma atroviride and Hypocrea atroviridis (FIG. 10).

As a result of colony properties and morphological observation, the HNT-01 strain grows quickly and forms a yellowish green to greenish wooly to velvety colony (Table 7, FIG. 11), and conidial pattern is regular or irregular. In branched and mature colonies, conidial patterns gather to form a wool-like lump (bundle).

Further, morphological features (FIGS. 12 to 17) such as forming a green conidial mass at the tip of the phialide and forming a thick membrane spore on the vegetative mycelium were observed.

These characteristics are in good agreement with the characteristics of the genus Tricoderma, the Ascomycota anamorph fungus. From the results of 28S rDNA-D1 / D2 and ITS-5. 8S rDNA nucleotide sequence analysis, the HNT-01 strain was assigned. Is thought to be similar to the characteristics of Tricoderma atroviride.

On the other hand, the formation of sexual reproductive organs characteristic of the genus Hypocrea was not observed.

Based on the above, it is estimated that the HNT-01 strain belongs to Tricoderma atroviride.

The HNT-01 strain was deposited as a new microorganism on September 7, 2012 at the National Institute for Product Evaluation Technology Patent Microorganism Depositary Center (2-5-8, Kazusa Kamashichi, Kisarazu City, Chiba Prefecture, Japan). (Accession number NITE P-1419).

There has never been an example isolated from the Towada stone cake used as a fertilizer base or from the processed fine dry powder of tuff like the HNT-01 strain obtained in the present invention.
In Experimental Example 1, the growth of Rhizoctonia solani was suppressed without sterilizing the Towada stone cake, and the genus Tricoderma floating in the air from the environment of the quarry was Since it does not appear to have adhered to the Towada stone cake that happens to be dried, HNT-01 shares the fine powder wastewater generated during the process of Towada stone being cut out and processed, and precipitates and collects the wastewater. It is considered that the life cycle was maintained during the drying process, and thick film spores were formed on the dried Towada stone cake as the final product.

The Towada stone mining mine is near 10m underground and has a humidity of nearly 90%, but the temperature is about 10 ° C throughout the year and is low for mold growth. In spite of such an environment, as shown in FIG. 1, a plurality of fungal hyphae may be found in the tunnel. The growth of mycelium is remarkable in the area where wood chips or mineral oil is scattered nearby, but any organic matter is extremely small from the Towada stone bedrock that surrounds it, and the mycelium is free of wood chips and oil. Even on the Towada stone bedrock, it can be observed to grow vigorously.

Since the Towada stone has been excised, there is almost no carbon source that can keep the life cycle of the HNT-01 strain, so the HNT-01 strain nourishes trace amounts of organic matter such as wood chips and machine oil in the tunnel. On the other hand, there is a strong suggestion that it may be a “low-temperature and oligotrophic environment-resistant microorganism” that can maintain the life cycle even on Towada stone, which is an inorganic mineral.

As described above, Trichoderma spp. Have been put into practical use because they have a life cycle on the dry fine powder of green tuff used as a fertilizer base (core) and have low-temperature resistance. However, the HNT-01 strain is expected to be able to proceed with the development of a plant control agent using a new type of microorganism that is low in cost and has a wide range of applications.

Claims

A microorganism belonging to the genus Trichoderma that has the ability to suppress the growth of plant pathogens.
The microorganism according to claim 1, wherein the microorganism is isolated from green tuff.
The microorganism according to claim 2, wherein the microorganism has a deposit number of NITE P-1419.
A method for inhibiting the growth of phytopathogenic fungi, comprising culturing the microorganism according to any one of claims 1 to 3 and inoculating the cultured microorganism in plant soil.
The method for inhibiting the growth of phytopathogenic fungi according to claim 4, wherein the phytopathogenic fungi are filamentous fungi belonging to the genus Rhizoctonia or Fusarium.