JP4310466B2 - Composition and method for biological control of soybean black root rot - Google Patents

Description

  The present invention relates to a composition for biological control of plant diseases, particularly soybean black root rot, and a method for controlling plant diseases using the same.

  Soybean has been one of the important crops produced in Japan since ancient times, but since it has been mainly cultivated using paddy field conversion fields in recent years, black root rot has occurred and spread due to continuous cropping, causing significant damage. Has produced.

  Soybean black root rot causing soybean black root rot is a kind of filamentous fungus that is a polycytic pathogen belonging to the Ascomycota fungus. In addition to soybean, many legumes such as azuki bean, groundnut, kidney bean, pea, lupine, etc. Invade or Cha. The microscopic sclerotia formed in the diseased tissue is the primary source of infection, and is a soil-borne disease that can survive in the soil for several years.

  There is no pesticide effective for controlling soybean black root rot, no chemical control method has been established, and the current situation is that pest control is carried out. The occurrence of this disease is often in places with high soil moisture, so in addition to Takatsuki to increase the drainage capacity of the field, it is supported by cultivation techniques such as installation of alum, extraction of diseased strains and field rotation, etc. Not enough. In addition, even if agrochemicals effective in controlling soybean black root rot are developed, there is a great demand for development of biological control technology as a low environmental load disease control technology due to increasing public interest in the environment.

  In addition, as a biological control technique of soybean black root rot, Carenectria crotalaria (Calontriaria) including the process cultivated in the soil containing the life research article 6536 (FERM BP-6536) which belongs to Fusarium fungi, or its variant. Although there is a method (Patent Document 1) for preventing infection of the plant with C. (Calatalariae), the microorganism as an active ingredient is completely different from that used in the present invention.

International publication WO99 / 16317 pamphlet

  An object of the present invention is to provide a biological control technique using the antagonism of microorganisms as a low environmental load control technique against black root rot in plants.

  The present inventors have attempted to select promising antagonistic bacteria that can be used in biological control technology using the antagonistic action of microorganisms from the soil, and have completed the present invention.

The present invention includes the following inventions.
(1) A composition for controlling black root rot in plants, comprising, as an active ingredient, a microorganism belonging to the genus Trichoderma having the ability to control black root rot in plants. In the present invention, the “control composition” is a concept including “control agent” and “control material”.
(2) The composition according to (1), wherein the plant is soybean.
(3) The composition according to (1) or (2), wherein the microorganism belonging to the genus Trichoderma is Trichoderma harzianum T-29 strain (reception number NITE AP-40) or a mutant strain thereof.
(4) A method for controlling black root rot in a plant, comprising applying the composition according to any one of (1) to (3) to the plant.
(5) The method according to (4), wherein the plant is soybean.
(6) Trichoderma harzianum T-29 strain (reception number NITE AP-40).

  According to the present invention, black root rot caused by plants can be biologically controlled.

  The black root rot to be controlled according to the present invention is the filamentous fungus Caronectria ilicicola (synonymous with: Caronectria crotalarie) (this may be referred to as “black root rot fungus” in the present specification). It is caused by infection.

  Black root rot fungus invades many legumes such as soybean, azuki bean, peanut, kidney bean, pea, lupine and tea, and causes black root rot. The composition of the present invention is suitable for controlling black root rot in these plants, and in particular for controlling black root rot in soybean.

  The microorganism belonging to the genus Trichoderma that can be used in the present invention is not particularly limited as long as it can control black root rot, but typically, Trichoderma harzianum, Trichoderma hamatum ( Trichoderma hamatum), Trichoderma virens (synonymous: Glyocladium virens syn. Gliocladium virens), Trichoderma polysporum (Trichoderma polysporum), Trihaderma Is preferred. In particular, Trichoderma harzianum T-29 strain (reception number NITE AP-40, date of receipt November 25, 2004), which has been applied for deposit at the National Institute of Technology and Evaluation for Microorganisms of the National Institute of Technology and Evaluation, is particularly preferred. In the present invention, the mutant strain of the Trichoderma haldianum T-29 strain subjected to mutagenesis treatment can also be used. The mutagenesis treatment can be performed using any appropriate mutagen. Here, the term “mutagen” should be understood in a broad sense to include not only a drug having a mutagenic effect but also a treatment having a mutagenic effect such as UV irradiation. Examples of suitable mutagens include ethyl methanesulfonate, UV irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, nucleotide base analogs such as bromouracil, and acridines, but any other effect A typical mutagen can also be used.

  Trichoderma species used in the present invention are usually used for liquid culture (including fluid culture of solid-containing liquid) such as reciprocal shaking culture, jar fermenter culture, culture tank culture, and solid culture such as shelf culture. The culture method can be used. A culture medium for culturing Trichoderma is not particularly limited as long as the bacterium can efficiently propagate and sporulate. As such a medium, for example, barley grain medium (6 walnuts 20 g, water 20 g) or potato broth medium (PSA medium or PDA medium: potato 200 g, saccharose or dextrose 15 to 20 g, agar 15 to 15) 20g), sugars such as glucose, sucrose, starch, dextrin, brown sugar, bran, rice bran, etc. as carbon sources, ammonium salts such as ammonium sulfate, ammonium chloride, ammonium nitrate, inorganic nitrogen sources such as nitrates as nitrogen sources, or , Yeast extract, corn steep liquor, meat extract, wheat germ, polypeptone, sugarcane squeezed potato, beer lees, soybean meal, rice bran, fish meal, etc., organic mineral sources such as monopotassium phosphate, magnesium sulfate, sulfuric acid Manganese and ferrous sulfate Of phosphorus, potassium, manganese, magnesium, salts containing iron, and a medium of synthetic or natural, each containing.

  Trichoderma genus used in the present invention is inoculated with fungus, wheat straw, rice bran, rice bran, corn straw, oatmeal, wheat grains, soybean meal, corn meal, food residue, etc. For example, it can also be cultured under dark conditions at 24-26 ° C. Moreover, what added inorganic substances, such as bentonite, diatomaceous earth, talc, pearlite, vermiculite suitably, to these organic substance powder can also be used as a culture medium. A culture thus obtained or a product obtained by appropriately drying it is preferable because it can be suitably used as a plant disease control material. Since Trichoderma spp. Exhibit excellent spore-forming ability when the above organic powder is used as a medium, such a control material is easily mass-produced.

  As the control composition of the present invention, the culture of the genus Trichoderma can be used as it is. The culture preferably contains various spores such as conidia spores, ascospores and thick film spores (in the present specification, these may be collectively referred to simply as “spores”). The mycelium or a fragment thereof may be further contained. Such a culture can be appropriately dried before use.

  As the control composition of the present invention, it is also possible to use a product obtained by separating or increasing the concentration of spores formed from the above culture by a conventional method or a dried product thereof. Again, mycelium or fragments thereof may be included. Examples of the method for separation or concentration increase include the following method in addition to a method for obtaining a pellet-like or suspension-like composition by a method such as filtration or centrifugation. That is, in fluid culture of a solid-containing liquid, the mycelium is first grown, the liquid is removed by filtration, the whole remaining solid is dried, the spore formation is promoted, and the spore is formed sufficiently. It is also possible to divide the spore, or after spore formation in this step, to treat with water containing a surfactant, collect the spore suspension by filtration and dry it to obtain spores. In solid culture, spores can be collected by sieving together with the medium after spore formation on the solid surface, or by pulverizing and sieving the medium after drying.

  The control composition of the present invention also adsorbs the culture solution of the above Trichoderma spp. To a porous adsorbent such as activated carbon powder, diatomaceous earth, or talc. The dried product can also be used.

  In any of the above, the drying method may be a normal method, for example, natural drying or ventilation drying at a low temperature, as well as freeze drying and drying under reduced pressure. These dried products may be further pulverized appropriately by a pulverizing means such as a ball mill after drying.

  The cultures of Trichoderma genus described so far, separated or highly concentrated spores, adsorbents of culture solution or dried products thereof (hereinafter referred to as “cultures etc.”) may be used alone or in appropriate combination. It can be used as a control composition of the invention. Cultures etc. can also be used in the form of compost-like materials combined with organic matter such as various soils, wheat grains, wood pieces, sugarcane squeezed rice cake, beer rice cake, soybean meal, rice bran, fish meal, various composts, food residues, etc. . Cultures and the like can also be formulated in the same form as normal microbial preparations (for example, powders, wettable powders, emulsions, solutions, flowables, coatings, etc.) in combination with other optional components. The wettable form is particularly preferred. This is because the use of a wettable powder enables application by soil irrigation and spraying at the time of sowing and growth of plants, and it can be used for a wide range of uses not only in the facility but also in the open ground. Examples of optional components used in combination in these materials and preparations include solid carriers and adjuvants. Examples of the solid carrier include bentonite, diatomaceous earth, talc, perlite, vermiculite, sodium carboxymethylcellulose, beer lees, sugar cane pomace, okara, bran, chitin, rice bran, flour, etc., and auxiliary agents include, for example, gelatin, gum arabic , Sugars, gellan gum and other sticking agents and thickeners.

  The method for applying the control composition of the present invention to a plant is appropriately selected according to various conditions such as the occurrence of plant diseases, the type of plant to be applied, and the dosage form of Trichoderma sp. It can be performed by each treatment such as spraying, in-facility application, soil mixing application, soil irrigation application, surface treatment (seed dressing treatment, seed application treatment). More specific application methods include powdering and applying various kinds of Trichoderma-containing compositions on plant seeds, irrigating plant seedling culture soil and cultivated soil, plant seedling culture soil and cultivated soil. The process which mixes, the process sprayed on the foliage of a plant, and the process made to contact the wound part of a plant are mentioned. These processes may be performed singly or in combination. The application time may be any time such as sowing or growing. In soybeans such as Tamba black soybeans, seedling transplantation cultivation is carried out by mixing the control composition of the present invention with the seedling culture soil, or using seedling soil (that is, seedling material) that has been bred with Trichoderma spp. As a result, seedlings immediately after emergence can be protected from infection with black root rot fungi, and the initial growth can be made healthy.

  Furthermore, when applying the above Trichoderma spp. To plants, other active ingredients usually used as necessary, such as insecticides, nematicides, acaricides, herbicides, fungicides, bactericides An agent, an antiviral agent, a fertilizer, a soil improver, etc. can be mixed and applied, or can be applied alternately or simultaneously without mixing.

  The application amount of the control composition of the present invention to a plant is appropriately determined according to various conditions such as the occurrence of plant diseases, the type of plant to be applied, and the dosage form of Trichoderma. For example, in a bran / vermiculite medium culture, a high black root rot control effect is produced by mixing about 1 ton per 10a of the entire soil.

  Hereinafter, the present invention will be described based on examples, but the present invention is not limited to these examples.

Trichoderma spp. Isolation method Trichoderma spp. Were isolated from soybean root areas in various fields, fields with a history of soybean cultivation, or soil from soybean black root rot occurrence fields. 10 g of soil in each field was placed in a 300 ml Kolben containing 90 ml of sterilized water, and the suspension obtained by reciprocal shaking for 20 minutes (124 reciprocations / min) with a shaking incubator was diluted with sterilized water at a dilution step and then 1000 Martin's Rose Bengal medium (Martin, JP (1950) Use of acid rose bengal and streptomycin in the plate method sizing. Soil: 1 ml). 215-232.) The mixture was mixed with 9 ml and cultured under dark conditions at 25 ° C. for 3 to 5 days. The resulting flora of Trichoderma spp. Each of the isolated strains was cultured on an elementary agar medium and then made into a single mycelium isolate by a conventional method. The trichoderma microbial flora grows quickly in rose bengal medium, forms a flora with a characteristic mycelium, and forms white to blue-green spore clumps, which makes it easy to distinguish.

Microorganisms of Trichoderma spp. Cultured for 3 days in the dark at 25 ° C. in the PDA medium on the opposite side on the culture medium of isolated Trichoderma spp., And black root rot fungus cultivated in the dark for 5 days at the autoclave medium A flora disc (diameter 6 mm) was cut out from the periphery of the flora, placed on a PDA plate medium at a distance of 4.5 cm, and cultured in an opposing manner under dark conditions at 25 ° C. Opposite aspects were observed after 3 and 7 days of culture. As a result, the Trichoderma spp. And the black root rot fungus were cultivated oppositely. As a result, the Trichoderma spp. Three types of antagonists were observed, in which black root rot fungus invades the Trichoderma spp.

Selection of promising strains under glass chamber conditions In order to obtain Trichoderma spp. Promising for the biocontrol of soybean black root rot fungi, screening was performed under glass chamber conditions using a small pot. Trichoderma spp. And black root rot fungi were mixed together with the medium in soil sterilized with a high-pressure sterilizer and packed in a plastic pot (280 ml), and then 5 soybean seed varieties Tachinagaha were sown per pot. At this time, Trichoderma spp. Were cultured in a bran / vermiculite medium at 25 ° C. in the dark for 10 days. Similarly, soybean black root rot fungus (Agricultural Research Center isolate S-14) was cultured in the bran / vermiculite medium at 25 ° C. in the dark. What was cultured for 20 days was used. The mixed Trichoderma spp. And black root rot fungi were each 2 g per pot. The test was performed in triplicate using 6 pots per strain. The pot was kept in a glass greenhouse, and after 20 days after sowing, the pre-emergence seedling mortality rate was examined, the soybean was pulled out, the diseased stalk rate after budding was examined, and the disease incidence rate was determined based on the following criteria.

  And based on the disease incidence, the control value based on the disease incidence was calculated by the following formula.

In addition, 20 days after sowing, roots were removed and the roots were observed to determine the severity of the disease. The severity of the disease was calculated using the following formula after dividing the disease severity of the root into the following five stages, counting the number of strains showing each disease severity.
0: No disease (healthy), 1: Browning is observed at the root part or the border part, 2: Browning surrounds the main root or the whole border part, 3: Browning progresses mainly around the border part, 4: The main root decayed and the amount of root decreased, 5: withered.

  Moreover, the control value based on the disease severity was calculated according to the following formula based on the disease severity.

  The results are shown in Table 1. The numerical values are shown in terms of control values because of the notation including the results of different test times.

  As a result of the seedling test, strains having high control values were found in strains T-5, T-13, T-28 and the like. Seventeen strains shown in Table 1 were obtained by seedling tests using many strains.

Among the 17 strains, the mycological properties of the T-29 strain are as follows.
(Observation with a microscope)
Both the conidial pattern branching pattern and the phialide settlement pattern on a 2% Marz medium are regular, and the phialide grows monotonically. The conidia have dark green spheres to subspheres, and the size is 2.1 to 2.4 × 2.4 to 2.6 μm, and thus the present bacterium is classified as a refinery (Rifai, MA). (1969) A reversion of the genus of Trichoderma. Mycologic Papers 116: 1-56.) Was identified as Trichoderma harzianum.

(Culture properties)
The PSA medium grew in the range of 10 ° C. to 35 ° C., and the optimum growth temperature (mycelial growth rate 20.1 mm / day) was obtained at 30 ° C. The bacterial flora after 2 days of culturing at 25 ° C. in the PSA medium had a pale yellow color at the center of the medium, the mycelium expanded white and smoothly, and there were few aerial mycelia. Mycelium cells had an average of 2.2 nuclei. In addition, conidia formation was good.

  From the above properties, the T-29 strain was identified as a new strain. This strain was filed as a Trichoderma harzianum T-29 (Trichoderma harzianum T-29) at the National Institute of Technology and Evaluation Patent Microorganism Depositary (Receipt Number NITE AP-40).

The mycological properties of the T-27 strain are as follows.
(Observation with a microscope)
It typically exhibits hyphal elongation without the formation of conidia or phialide (Sterile hyphal elongations) on a 2% Marz medium. A short conidial pattern and a pearless type of phialide grow densely at the base of the Steylil hyphae where no conidia pattern or phialide is formed. The conidia have dark green fallen egg shape to cocoon shape, and the size is 3.6 to 4.6 × 2.4 to 3.0 μm. Therefore, the present bacterium is classified as a refinement (Rifai, MA). (1969) A reversion of the genus of Trichoderma. Myological Papers 116: 1-56.).

(Culture properties)
The PSA medium grew in the range of 10 ° C. to 35 ° C., and had an optimum growth temperature (mycelial growth rate of 19.9 mm / day) at 30 ° C. The mycelium after 2 days of culture at 25 ° C. in the PSA medium had white mycelia that grew smoothly, few aerial mycelia, and no coloration of the medium. Mycelium cells had an average of 3.8 nuclei.

  In addition, the seed | species was identified about the following among the strains of Table 1. T-5: Trichoderma harzianam, T-13: Trichoderma harzianam, T-21: Trichoderma vilence, T-26: Trichoderma harzianam, T-27: Trichoderma hamatumum, T-28: Trichoderma harzianam, T- 29: Trichoderma harzianam ,: Trichoderma harzianam, T-30: Trichoderma hamatum, T-31: Trichoderma polisporum.

Using 17 kinds of Trichoderma spp. Including Trichoderma harzianum T-29 strain selected in Example 1, the biocontrol effect on soybean black root rot was examined under the outdoor installation pot conditions. In addition, the bran / vermiculite medium used in this example was mixed with 200 g of bran, 200 g of vermiculite and 600 g of water uniformly using a tub etc., and then dispensed 500 g per 1 L beaker and autoclaved (120 ° C., 15 min). And made it. Three pots of concrete frame pots (50 cm x 50 cm x 50 cm) installed in the field filled with kuroboku soil were used per treatment, soil disinfection (30 L / 10a) with chloropicrin agent, and the polyfilm mulch was left as it was. Three months later (May 27th), 140 g of a culture obtained by culturing black root rot fungus (Caronectinia iricicola, Agricultural Research Center isolate S-14) in bran / vermiculite medium at 25 ° C. in the dark for 40 days It was mixed with surface soil (between 0 and 10 cm) and inoculated with black rot fungus. The pots did nothing in particular until treatment of Trichoderma spp. After inoculation. Eight days later, on June 4, 140 g of each Trichoderma spp. Cultured in bran / vermiculite medium at 25 ° C. under dark conditions for 10 days was mixed with the surface soil (between 0 and 10 cm) of each pot. As an untreated control, 140 g of a bran / vermiculite medium without any culture was similarly mixed with the surface soil of the pot. Then, two seeds of soybean variety Tachinagaha were sown at 9 spots per pot. After sowing, each pot was supplied with tap water. On the 20th day after sowing, the pre-emergence seedling mortality was examined. Further, the post-emergence post-emergence rate was examined at the time of harvest (December 2).
The sum of the pre-emergence seedling mortality (%) and postemergence mortality (%) was defined as the disease incidence (%).

そして、発病率に基づき下記式により防除価（１）を算出した。

And control value (1) was computed by the following formula based on the disease incidence.

At the time of harvest (December 2), roots were removed and the roots were observed to determine the severity of the disease. The disease severity was calculated using the following formula, dividing the disease severity at the root into the following 5 levels, counting the number of strains showing each disease severity.
0: Healthy. 1: Browning and decay are observed in a part (about 1/4) of the root including the main root. 2: About half of the roots including the main root are browned or decayed. 3: Many side roots decay and fall off, and the main roots are also severely decayed. 4: Died by harvest, and in many cases, the side roots are all decayed and the main roots are burdock roots. See also FIG. 1 for disease severity.

  Moreover, the control value (2) was calculated by the following formula based on the disease severity.

  The results are shown in Table 1. T-29 showed particularly high values for both control values (1) and (2).

It is a photograph which shows the classification of the onset degree.

Claims (6)

  A composition for controlling black root rot in plants, comprising, as an active ingredient, a microorganism belonging to the genus Trichoderma having the ability to control black root rot in plants.   The composition according to claim 1, wherein the plant is soybean.   The composition according to claim 1 or 2, wherein the microorganism belonging to the genus Trichoderma is Trichoderma harzianum T-29 (accession number NITE AP-40) or a mutant thereof.   The control method of the black root rot in a plant including applying the composition of any one of Claims 1-3 to a plant.   The method according to claim 4, wherein the plant is soybean.   Trichoderma harzianum T-29 strain (reception number NITE AP-40).

Images