JP5052873B2 - Control agent and method for biological control of soybean stem blight - Google Patents

Description

  The present invention relates to a control agent for biological control of plant diseases, particularly soybean stem blight, 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 in recent years it has mainly been cultivated using paddy field conversion fields. There is a lot of damage.

  Registered pesticides for soybean pesticidal diseases include oxazil and copper wettable powder, but the production of this drug was discontinued due to the problem of resistant bacteria, but the effect of the newly registered Bordeaux agent as an alternative pesticide was weak and impractical . For this reason, this disease control mainly focuses on cultivating measures. The occurrence of this disease is common in places with high soil moisture, and in addition to Takatsuki to increase drainage capacity in the field, it is supported by cultivation techniques such as the installation of alum and extraction of diseased strains and field rotation. The effect is not enough.

  In addition, low environmental impact disease control technology is strongly demanded as a modern agricultural technology due to the increasing public interest in the environment.

Sadao Tsuchiya, Yayoi Nakagawa Primary crops, pests and pests 2nd edition Wheat, soybeans, azuki bean, feed crops, etc. Agricultural cooperatives. Tokyo. pp219-226.

  As described above, a promising biological control material (BCA) has not been clarified or established as a biological control technique for soybean stem blight. Moreover, the effect is not sufficient only by cultivating control technology. Even if pesticides can be used by promoting registration, there is a great demand for the development of biological control technology as a low environmental load disease control technology.

  Then, this invention aims at selecting the promising antagonistic bacteria which can be utilized for the biological control technique using the antagonism which microorganisms have as a low environmental load type control technique with respect to a soybean stem rot.

  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 pesticidal control agent in soybean, which contains, as an active ingredient, a microorganism belonging to the genus Trichoderma having the ability to control pesticidal disease in soybean.
(2) The control agent according to (1), wherein the microorganism belonging to the genus Trichoderma belongs to Trichoderma viride or Trichoderma virens.
(3) The microorganism belonging to the genus Trichoderma is Trichoderma viride T-3 strain (reception number NITE AP-279) or its mutant strain, Trichoderma virens T04398 strain (reception number NITE AP-280) or its mutant strain, Trichoderma virence The control agent according to (2), which is T04401 strain (reception number NITE AP-281) or a mutant thereof, or Trichoderma virens T04464 strain (reception number NITE AP-282) or a mutant thereof.
(4) A method for controlling stem blight in soybean, comprising applying the control agent according to any one of (1) to (3) to soybean.
(5) Trichoderma viride T-3 strain (reception number NITE AP-279).
(6) Trichoderma virens strain T04398 (reception number NITE AP-280).
(7) Trichoderma virens strain T04401 (reception number NITE AP-281).
(8) Trichoderma virens strain T04464 (reception number NITE AP-282).

  The present invention provides a means for biologically controlling stem blight in soybean.

  Phytophthora sojae is a microorganism (soybean stem rot) that causes stem rot that is subject to control according to the present invention.

  Pathogenic bacteria invade only soybeans and cause stem blight. The control agent of the present invention is suitable for controlling stem blight 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 stem rot, but typically, Trichoderma viride or Trichoderma vilens (Trichoderma virens). Among them, Trichoderma viride T-3 strain (reception number NITE AP-279, date of receipt November 14, 2006), Trichoderma virens strain T04398 (deposited with the National Institute of Technology and Evaluation) Receipt number NITE AP-280, receipt date November 14, 2006), Trichoderma virens strain T04401 (reception number NITE AP-281, receipt date November 14, 2006), or Trichoderma virens strain T04464 (receipt number NITE) AP-282, date of receipt, November 14, 2006) is preferred.

  In the present invention, mutant strains obtained by mutagenesis treatment of the above-mentioned four specific strains 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. Examples of such a medium include a barley grain medium (6 g raw walnuts, 20 g water) and a potato broth medium (PSA medium or PDA medium: potato 200 g, saccharose or dextrose 15-20 g, agar 15-20 g). ), Sugars such as glucose, sucrose, starch, dextrin, brown sugar, bran and rice bran as a carbon source, ammonium salts such as ammonium sulfate, ammonium chloride and ammonium nitrate as nitrogen sources, inorganic nitrogen sources such as nitrates, or Organic nitrogen sources such as yeast extract, corn steep liaker, meat extract, wheat germ, polypeptone, sugarcane squeezed potato, beer lees, soybean meal, rice bran, fish meal, etc., inorganic potassium salt of potassium phosphate, magnesium sulfate, manganese sulfate , Ferrous sulfate Of phosphorus, potassium, manganese, magnesium, salts containing iron, and a medium of synthetic or natural, each containing.

  Trichoderma bacterium used in the present invention is inoculated with organic powder such as bran, wheat straw, rice bran, rice bran, corn straw, oatmeal, wheat grain, soybean cake, 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 sporulation ability when the organic powder is used as a medium, such a control material is easily mass-produced.

  As the control agent of the present invention, the above Trichoderma spp. Culture 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 agent 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. As a method for separation or concentration enhancement, for example, the following method may be mentioned in addition to a method of 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.

  In addition, as the control agent of the present invention, the Trichoderma spp. Can be adsorbed on a porous adsorbent such as activated carbon powder, diatomaceous earth, or talc when dried by liquid culture (including fluid culture of solid-containing liquid) and dried. You can also use the

  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 agent 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 of applying the control agent 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 in-facility application, soil admix application, soil irrigation application, surface treatment (seed dressing treatment, seed application treatment). More specific application methods include treatment of applying various forms of Trichoderma spp. Control agent on plant seeds, irrigation of plant seedling cultivation soil and cultivation soil, plant seedling cultivation soil and cultivation 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 adding the control agent of the present invention to the seedling culture soil or by raising seedlings using the seedling soil (that is, the seedling material) on which Trichoderma spp. The seedlings immediately after emergence can be protected from the infection of the stem rot fungus, and the initial growth can be improved.

  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 agent 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 the bran / vermiculite medium culture, a high effect of controlling pesticidal diseases can be obtained by mixing the whole soil with 250 kg to about 2 t per 10a.

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

Reference example 1: Selection of pots in a greenhouse (stalk fungus)
Phytophthora soja S-2 (Hyogo Agricultural Technology Center isolate) was used as a stem rot fungus. This stem blight fungus was cultured in a dark bran and vermiculite medium at 25 ° C. for 20 days. The bran / vermiculite medium used was obtained by mixing wheat bran: vermiculite: water in a weight ratio of 1: 1: 3, and then placing in a container and autoclaving.
(Candidate strain)
220 strains isolated from various soils (such as fields) were used as candidate strains. Each candidate strain was cultured on a bran / vermiculite medium in the dark at 25 ° C. for 10 days.
(plant)
Soybean variety Tachinagaha (Ibaraki Prefectural Economical Purchasing Seeds) was used as a test plant.
(soil)
Sterilized Kuroboku soil (high-pressure sterilization) was used as the soil for cultivating the soybean.
(Test method)
2 g of each cultured culture of candidate Trichoderma fungus and pesticidal fungus were mixed simultaneously with 200 ml of sterilized soil. The mixed soil was placed in a plastic pot (280 ml), and 5 soybean seeds were sown per pot. After sowing, it was kept in a glass greenhouse (25 ° C, 70% Rh). The test was performed in triplicate using 6 pots per treatment.

Soybean was extracted 20 days after sowing, and the disease severity was determined according to the following formula.
Disease severity = {Σ (number of diseased stems by index × index) / (number of surveyed stems × 4)} × 100

  In the above formula, “index” means a class index according to the severity of disease. The index was evaluated according to the index shown in FIG. Specific evaluation criteria are as follows. 0: No disease (healthy), 1: Slight lesions are observed on the ground stem (no wilt), 2: Spots are observed on the ground, and some leaves are wilted. 3: Causes lesions on the border and causes wilt throughout the leaves. 4: Death.

The control value based on the severity was determined according to the following formula.
Control value based on disease severity = 100 × (morbidity of untreated area−morbidity of treated area) / (morbidity of untreated area)

(result)
220 candidate strains were divided into 20 strains and tested. Table 1 shows the results of one experiment.

  From this experiment, it was shown that 9 strains, such as T04398 strain, T04401 strain, T04464 strain, T-3 strain, etc. have the control effect with respect to stem rot.

Reference example 2: Selection under the condition of outdoor installation concrete frame pot (pot)
A field-installed concrete frame pot (50 cm x 50 cm x 50 cm) was used. Three pots were tested per treatment. As the soil, Kuroboku soil sterilized with chloropicrin (30L / 10a) was used.

(Pesticide)
Phytopthora soja S-2 (Hyogo Agricultural Technology Center isolate) was used as a stem rot fungus. The stem rot fungus was cultured in a bran / vermiculite medium at 25 ° C. in the dark for 40 days. 500 g of the cultivated pesticidal fungus thus cultured was finely loosened and mixed with the surface layer (between 0-10 cm) of each pot soil and inoculated.

(Test method)
500 g of 10 strains cultured in bran / vermiculite medium for 9 days selected in the greenhouse pot test were mixed in the soil surface of each pot (between 0-10 cm), and 3 soybeans (variety Tachinagaha) in one place, 15 seeds were sown per pot.

20 days after sowing, the germination inhibition rate (pre-emergence disease incidence rate) was examined, the degree of disease occurrence was examined at the time of harvest, and the disease incidence was determined according to the following formula.
Disease severity = {Σ (number of diseased stems by index × index) / (number of surveyed stems × 4)} × 100

In the above formula, “index” means a class index according to the severity of disease. The evaluation criteria for the index are as follows. 0: No disease (healthy) 1: Slight lesions are observed on the ground stem (no wilt), 2: Withering or wilt occurs in 1/3 or less of the entire strain. 3: Causes 1/3 to 2/3 of the entire strain to wither and wilt. 4: Withering and wilt of 2/3 or more of the whole strain occurs.
Control value based on disease severity = 100 × (morbidity of untreated area−morbidity of treated area) / (morbidity of untreated area)

(result)
The result is shown in figure 2. In FIG. 2, the T04398, T04401, T04464 and T-3 strains marked with an asterisk showed particularly high control values.

Reference Example 3: Identification of 4 strains (observation with a microscope)
In the T-3 strain, 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 are dark green spheres to subspheres, and are rough with small protrusions on the surface. The size is 3.0 to 4.5 × 2.8 to 3.8 μm.

  On the other hand, the strains T04398, T04401 and T04464 form rod-like conidia on a 2% Marz medium and form a conidial spore mass on the most advanced phialide. The conidia pattern is colorless and upright, simple or branched. It has several septa. The conidia are dark green spheres, sub-spheres to fallen eggs, and have a size of 3.3 to 5.8 × 3.0 to 4.0 μm.

(Culture properties)
The T-3 strain grows in the range of 10 ° C to 30 ° C in the PSA medium, has an optimum growth temperature (mycelium growth rate 219.8 mm / day) at 25 ° C, and the mycelial growth rate at 10 ° C is 3.9 mm / It was a day. At 30 ° C., the mycelium growth rate was 4.4 mm / day. The bacterial flora after 2 days of culture at 25 ° C. in PSA medium expanded smoothly and white, and there were few aerial hyphae. Mycelium cells had an average of 3.0 nuclei. Green conidia were formed well.

  On the other hand, the strains T04398, T04401, and T04464 grow on PSA medium in the range of 10 ° C to 35 ° C and have an optimum growth temperature (mycelium growth rate 219.8 mm / day) at 30 ° C. Little growth was observed. At 30 ° C., the mycelial growth rate was 20.8 to 31.4 mm / day. In addition, the mycelial growth rate was 9.4 to 14.6 mm / day at 35 ° C. The medium after 2 days of PSA culture at 25 ° C is colored from pale yellow to brown. The flora was white and extended smoothly, and there were few aerial hyphae. Mycelium cells had an average of 2.0-2.6 nuclei. In addition, green conidia were formed and conidia formation was good.

  From the above properties, the T-3 strain was identified as a new strain. This strain was identified as Trichoderma viride according to the classification of Riffai (Rifai 1969), and was filed as a deposit of Trichoderma viride T-3 strain at the National Institute of Technology and Evaluation Microbiology (Receipt Number NITE). AP-279). Similarly, T04398 strain, T04401 strain, and T04464 strain were also identified as new strains. These strains were identified as Trichoderma virens according to the Bisst classification (Bisset 1991), and Trichoderma virens strain T04398 (reception number NITE AP-280), T04401 strain (reception number NITE AP-281), T04464 strain ( Application No. NITE AP-282) was filed with the National Institute for Product Evaluation Technology Patent Microorganism Depositary.

Example 1: A field test was carried out using four species of the genus Trichoderma selected in the field test reference example.
(place)
The test was conducted at a farmer's field in Sasayama City, Hyogo Prefecture. This field is a field where stalk disease occurs naturally and is a paddy field conversion field.
(Test area)
The size of 1 ward is 1.5m x 5.4m, and 1 shoot and 12 soybeans per ward were used.
(Test Trichoderma spp.)
Four strains T04398, T04401, T04464 and T-3 were tested.

  These strains were all cultured in a bran / vermiculite medium at 25 ° C. in the dark for 30 days. The cultured strain was mixed at a rate of 250 kg / 10a on the soil surface (between 0-10 cm) with a small management machine.

(Soybean variety)
Tamba black soybean was used as a soybean variety.
Soybeans were directly planted on the field at 3 centimeters per paddy at 150 cm wide and 45 cm apart. One stand was established 15 days after sowing.
(Method)
On June 8, 2005, soybean was sown in soil mixed with Trichoderma sp.

  The disease was investigated three times on August 25, 2005, September 2, and September 29, when the outbreak of stem plague was observed.

15 days after sowing, the budding inhibition rate (pre-emergence morbidity rate) was examined, and the morbidity was examined over time, and the morbidity was determined according to the following formula.
Disease severity = {Σ (number of diseased stems by index × index) / (number of surveyed stems × 4)} × 100

  In the above formula, “index” means a class index according to the severity of disease. The evaluation criteria for the index are as follows. 0: No disease (healthy) 1: Slight lesions are observed on the stem of the ground (no wilt), 2: Withering / wilt occurs in 1/3 or less of the entire strain. 3: Causes 1/3 to 2/3 of the entire strain to wither and wilt. 4: Withering and wilt of 2/3 or more of the whole strain occurs.

Moreover, the control value based on the disease severity was calculated in the same manner as in Reference Examples 1 and 2.
The results of the final survey on September 29, 2005 are shown in Figure 3. In FIG. 3, the disease rate in the untreated area was 16.9%, and the disease severity was 12.7. All four strains were found to have a high control effect on stem rot.

Example 2: A field-installed concrete frame pot (50 cm x 50 cm x 50 cm) was used in the test for controlling the stalk disease effect of Trichoderma material by raising seedlings . Three pots were tested per treatment. As the soil, Kuroboku soil sterilized with chloropicrin (30L / 10a) was used.

(Pesticide)
The soybean stem fungus was inoculated on June 20, 2005 using the S-2 strain (Hyogo Agricultural Technology Center isolate). Stem fungus is cultured in bran / vermiculite medium at 25 ° C in the dark for 40 days, and 500g per 1 pot (50cm x 50cm) of cultured soybean stem fungus S-2 is cultured on soil surface soil (between 10cm ).

(Trichoderma spp.)
Trichoderma spp .: 4 strains T04464, T04401, T04398, T-3. T04464, T04401 and T04398 strains were made into materials (Idemitsu Kosan Prototype) and mixed with the seedling culture soil at a rate of 0.5 to 4.0 / paper tube at the time of sowing. On the other hand, since the sporulation of T-3 strain was worse than that of T04464, T04401 and T04398 strains, bran culture was used instead of the material, and the same amount as the material was used.

(Cultivation overview)
June 17, 2005 Soybean (variety Tachinagaha) was sown in a paper tube, seeded in a greenhouse for 10 days, and on June 27 (Monday), 9 strains were transplanted into a concrete frame. The survey was conducted on October 28 to determine the severity of the disease, and the severity was determined according to the following formula.
Disease severity = {Σ (number of diseased stems by index × index) / (number of surveyed stems × 4)} × 100

  In the above formula, “index” means a class index according to the severity of disease. The evaluation criteria for the index are as follows. 0: No disease (healthy) 1: Slight lesions are observed on the stem of the ground (no wilt), 2: Withering / wilt occurs in 1/3 or less of the entire strain. 3: Causes 1/3 to 2/3 of the entire strain to wither and wilt. 4: Withering and wilt of 2/3 or more of the whole strain occurs.

Moreover, the control value based on the disease severity was calculated in the same manner as in Reference Examples 1 and 2.
The results are shown in Table 2. All four strains showed high control values. The T04464 strain is a useful strain with a particularly high control value.

Figure 1 shows the survey criteria for soybean stalk plague (index by disease severity). FIG. 2 shows the effects of various Trichoderma strains on the control of soybean scab. FIG. 3 shows the control effect of soybean scab on various Trichoderma strains under local field conditions.

Claims (6)

A control agent for stalk disease in soybean, containing as an active ingredient a microorganism belonging to the genus Trichoderma having the ability to control pesticidal disease in soybean , wherein the microorganism belonging to the genus Trichoderma is Trichoderma viride T-3 strain (Accession No. NITE) P-279) or a mutant strain thereof, Trichoderma virens strain T04398 (Accession No. NITE P-280) or a mutant thereof, Trichoderma virens strain T04401 (Accession No. NITE P-281) or a mutant thereof, or Trichoderma virens T04464 The said control agent which is a strain (Accession number NITE P-282) or its variant . A method for controlling stem blight in soybean, comprising applying the control agent according to claim 1 to soybean. Trichoderma Viride T-3 strain (consignment number NITE P -279). Trichoderma Virensu T04398 strain (consignment number NITE P -280). Trichoderma Virensu T04401 strain (consignment number NITE P -281). Trichoderma Virensu T04464 strain (consignment number NITE P -282).

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