WO1999016317A1

WO1999016317A1 - METHOD FOR PREVENTING INFECTION WITH $i(CALONECTRIA CROTALARIAE)

Info

Publication number: WO1999016317A1
Application number: PCT/JP1998/004380
Authority: WO
Inventors: Hiromitsu Furuya; Kaori Takahashi; Tsutomu Sato; Hiroshi Konno; Isamu Takahashi
Original assignee: Institute Of Biotechnology Applied To Soil Eumycetes
Priority date: 1997-09-30
Filing date: 1998-09-29
Publication date: 1999-04-08

Abstract

A method for growing plant seeds while protecting them from infection with Calonectria crotalariae which comprises cultivating the seeds in a soil containing the strain FERM BP-6536 or its variants.

Description

Specification

How to prevent C. clonalaria infection

Technical field

The present invention provides a method for cultivating a plant in soil containing a strain containing a strain of No. 6536 (FERM BP-6536) (hereinafter referred to as “deposited strain”) or a mutant thereof. The present invention relates to a method for preventing the infection of Calonectri acrotal arie to the plant, and an agent for preventing infection of the plant with C. crotalariae, including a strain thereof.

Background art

Black root rot, which is a typical crop disease caused by infection with the pathogen and causes browning and decay of roots and grounds, is an important disease especially in soybean perilla cultivation. Above all, soybean black root rot has spread nationwide with the increase in soybean cultivation in converted fields, and has become a serious problem in terms of the area of occurrence and damage o

The diseased root of soybean is brown or dark brown at the roots and on the ground, and is brittle, easily broken and easily pulled out. As the leaves turn yellow and adventitious roots form, orange or red ascocarps are formed on the ground or on the main roots. Black root rot fungi have a long lifespan in soil, and are susceptible to multiple outbreaks once they are continuously grown in contaminated fields. At present, there are no effective chemicals against black root rot.Therefore, as a method to control the outbreak of this disease, measures to improve drainage, avoid continuous cropping, and extract and incinerate as soon as the disease-causing strain is found, There is no other way than to cultivate varieties resistant to black root rot. Therefore, there is a need in the agricultural sector for a way to effectively control this disease.

On the other hand, in Japan, Trichoderma viable fungus (Sanyo Pharmaceutical Co., Ltd.) has been registered as a pesticide as a biological pesticide by antagonistic microorganisms, and has been put to practical use for the control of tobacco scab and scab disease. Wari disease (Kiyuu Ogawa, Tan Komada: Nihonshosho, 50, 1-9, 1984), Strawberry wilt (Nobuo Tezuka, Takahiro Makino: Plant Protection, 42, 251-254, 1988) and wilt disease of Tomato (Kenichi Yamaguchi, Masanobu Arita: Nihonbyohoho, 56, 404, 1990), etc. It has been reported that non-pathogenic Fusarium oxysporum is effective.

Biological control of soil diseases using antagonistic microorganisms has a low risk to humans and crops, and has a low impact on the ecological environment because it is selectively used by target organisms. It is not as immediate as chemical pesticides, but has the advantage of being able to expect sustained effects. However, there have been no reports of antagonistic microorganisms having a disease-suppressing effect that is effective in preventing the infection of plants with C. crotalarie pathogens.

Disclosure of the invention

An object of the present invention is to provide a method for preventing infection of plants with C. crotalaria by an antagonistic microorganism.

Means for Solving the Problems The present inventors searched for a strain that inhibits the infection of plants with C. crotalarie by mixing various strains into soil, cultivating plant seeds using the soil, and observing the growth. . As a result, by mixing the strain (deposited strain) deposited as Life Science Research Deposit No. 6536 (FERM BP-6536) in the soil, the presence of C. clonalaria in the presence of C. We found that the infection of Crotalariae to plants was significantly inhibited, and that the plants grew in the absence of C. crotalariae and had the same good growth as plants not infected with C. crotalariae. Was.

That is, the present invention

(1) A method for preventing infection of the plant with C. crotalariae, comprising a step of cultivating the plant in soil containing the deposited strain or a mutant thereof, (2) the plant is soybean, (1) The method described,

(3) a medicament for preventing infection of a plant with C. acrotalariae, comprising a deposited strain or a mutant thereof as an active ingredient; (4) the agent according to (3), wherein the plant is soybean;

(5) Encapsulation of at least one bacterial cell component selected from the group consisting of culture, conidia, and chlamydospores of the culture of NIKEN No. 6536 (FERM BP-6536) or its mutant. The drug according to (3),

(6) At least one cell component selected from the group consisting of cultures, conidia, and chlamydospores of the RIKEN No. 6536 (FERM BP-6536) or a mutant thereof is used as an active ingredient. The agent according to (3), which is a seed coating agent containing as

(7) Coated with at least one bacterial component selected from the group consisting of cultures, conidia, and chlamydospores of the NIKEN No. 6536 (FERM BP-6536) or its mutants Plant seeds, and

(8) the seed of (7), wherein the plant is soybean;

About.

In the method of the present invention, it is preferable to use a deposited strain, but it is also possible to use a mutant strain thereof. In order to artificially prepare the mutant strain, for example, a spore suspension of the deposited strain is irradiated with ultraviolet light, for example, ethyl methane sul fonate (EMS) and N-methyl-N '. Treatment with a mutagenic agent such as -nitro-N-nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine) ("Microbial Experiment Method", Microbial Research Method Roundtable, edited by Kodansha, p288- 306, 1990).

In addition, whether or not a plant is infected with C. crotarolarii pathogen can be easily determined by observing the growth state of the plant and the presence of a lesion. Alternatively, the presence of the pathogenic bacterium, Karonectria crotalarie, in a plant can be determined by, for example, placing the diseased tissue fragments of the roots or terrestrial stems on a potato dextroth agar (PDA) medium to isolate the pathogenic bacterium ( It can also be determined by detection using “Basic techniques for research on crop pathogens: isolation, culture, inoculation,” edited by Kanichi Ohata et al., Japan Plant Protection Association, p319-321 (1995). The soil that can be used in the method of the present invention includes sandy to loamy soil, soil sterilized by steam at about 70 ° C. or natural soil. These soils In order for the deposited strain to be contained in the soil, the strain can be contained in a suitable medium, such as cornmeal sand culture medium, Fusuma's bamiki light medium (1: 1), wheat grain medium, or enpaq grain medium. Then, the bacteria cultured at 25 ° C for 2 to 3 weeks can be mixed and inoculated into the soil as an inoculum together with the medium. Further, the amount of the strain to be contained (the ratio to the whole soil) is preferably 25% or more, more preferably 30% or more, and most preferably 35% or more. Plants to which the method of the present invention can be applied include soybean, laccase, ingen, azuki, and endu. By cultivating seeds of these plants in the soil prepared as described above, the seeds can be germinated and grown without infecting C. crotalariae. Temperature conditions for growing plants are preferably 15 ° C or higher, more preferably 20 ° C or higher, and most preferably 25 ° C to 30 ° C. The illuminance is preferably 8,0001x or more, more preferably 12,000lx or more, and most preferably <J5,0001x. The soil moisture is preferably 40 to 70% of the maximum water capacity of the soil, more preferably 45 to 65%, and most preferably 50 to 60%.

In addition, the agent for preventing infection with C. crotalariae, including the deposited strain or a mutant strain thereof, of the present invention can be prepared in a solid form together with a culture medium. The drug containing the deposited strain according to the present invention can also be capsuled using excipients such as Na alginate. Encapsulation means that the deposited bacterial cell or its conidia or chlamydospores are viable into particles using a protective coating in a viable state. The drug according to the present invention, which has been converted into capsules, can obtain an infection-preventing effect by being mixed into the soil where a plant to be infected with C. crotalariae is to be prevented. Further, the deposited bacterial strain of the present invention can also obtain an effect of preventing infection by coating the bacterial cell components on plant seeds. As a method of coating bacterial components on plant seeds, a bacterial component such as conidia or spores is kneaded with a thickening agent such as xanthum gum, coated on seeds and dried. I can show you how to do this. Chlamydospores are durable living organs of the deposited strain of the present invention, and can survive for a long time in a dried capsule coating agent. According to Therefore, it is desirable as a bacterial cell component used in the drug according to the present invention. Chlamydospores can be cultured by solid culture at around 25 ° C for 2-3 weeks, dried at 42 ° C-45 ° C, and recovered by sieving. The solid culture method is a method of culturing using cereal grains such as bran and barley grains, or cereal fine powder as a culture medium.

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing a method of determining an onset index.

FIG. 2 is a diagram showing a method of determining the degree of a lesion.

FIG. 3 is a photograph showing the growth state of soybean. A is a photograph when cultivated on soil containing pathogenic bacteria and deposited strain (776), B is when cultivated on soil containing only pathogenic bacteria (P cloudy control), and C is when cultivated on soil only (positive control) It is. BEST MODE FOR CARRYING OUT THE INVENTION

[Example 1] Screening of microorganisms for controlling soybean black root rot

As the pathogenic bacteria, those isolated from a continuous farming field of Akita Agricultural Test were used. First, the pathogenic bacteria and test bacteria were cultured in a cornmeal sand medium (cornmeal 3 g, bran 2 g, glucose 0.5 g, mountain sand 150 g, tap water 30 ml) at 25 ° C. for 2 to 3 weeks. During the culture, the mixture was shaken by hand every 2-3 days.

Using the cultured bacteria as an inoculum, a pathogen inoculum, a test inoculum, and a sterilized soil were mixed at a ratio of 1: 8 to 10:15 to 17 (W / W). However, the ratio of the pathogen inoculation source was always 1/26 of the total. For sterilized soil, sterile mountain sand, perlite and vermiculite are used in a ratio of 1: 1: 1.

(V / V). As a control, a section with only sterilized soil and a section in which the inoculum of pathogenic bacteria and the sterilized soil were mixed at a ratio of 1:25 (V / V) were provided.

The soil mixed with the pathogenic bacteria and the test bacteria was filled into a plastic container 3 cm in diameter and 11 cm in length. The control effect was determined by cultivating soybean immediately after mixing the soil. Two soybeans (variety: Tachiyutaka) were sowed on the mixed soil, and cultivated under fluorescent light of 30001x at 25 ° C for 1 week. Three plastic containers were used for one test bacterium.

Seven days after sowing, the emergence rate was examined. Embryos with more than half of the cotyledons exposed on the ground surface were considered as budding. After that, the roots were carefully extracted so that they did not cut, and the soil particles were brushed off with a brush. The disease index of each individual was examined according to the following criteria. Disease index

0: There is no browning at all.

1: Several short streaky browning or several millimeters (less than 1 cm) in length and one or two slightly wider lesions (small lesions).

2: There are 3-5 small lesions and small lesions such as streaks. Or, there are one or two lesions (medium lesions) that are around 1 cm long and surround the root.

3: 5 or more small lesions or 1 or 2 medium-sized lesions and 3 or 4 small lesions on the main root

. Some of these may fuse together to form large lesions. As a whole, 30 to 40% from the hypocotyl to the tip of the root is browned.

4: There are multiple large lesions, and more than half of the roots are browned. There are large lesions on the hypocotyl and small lesions on the main root. 4 to 8 harmful lesions from the hypocotyl to the tip of the main root are browned.

5: Both the main and lateral roots are browned overall. More than 80% from the hypocotyl to the tip of the main root is browned.

The disease index was determined only for the main root, and an example is shown in Fig. 1. Fig. 2 shows a method for determining the degree of lesion.

Growth index

un: No budding. Even if germinated, those that did not begin to develop true leaves in the aerial part were not yet sprouted.

unr: Seeds are decayed with almost no roots growing. 0: Both roots and lateral roots grow in the same manner as in the non-inoculated plot.

1: The length of the main root is almost the same as the non-inoculated group (80% or more). Growth of lateral roots is slightly inferior.

2: The length of the main root is about the same or slightly inferior to the uninoculated group (50% ~). Growth of lateral roots is rather poor.

3: The length of the main root is 50 to 100% of the uninoculated plot, and the growth of the lateral root is remarkably inferior.

4: The length of the main root is less than 50% of the uninoculated plot, and the growth of the lateral root is rather poor.

5: The length of the main root is less than 50% of that of the non-inoculated plot, and the lateral roots hardly grow.

The disease index was determined based on the above criteria, and the average value was used as the disease severity. The results are shown in Table 1 below for some test bacteria.

table 1

FIG. 3A shows a photograph of root growth in a section where the effect was particularly high (deposited strain mixed section; 776). As can be seen from Table 1 and Fig. 3, in the mixed strains with deposited strains (776 / Fig. 3A), the disease incidence was clearly lower than in the inoculated plots (180-10 / Fig. 3B), and the root growth was not increased (Fig. 3). It is not so different from Fig. 3C). The same results were again obtained when re-performing ^ using this deposited strain.

On the other hand, when the strains shown in Table 1 other than the deposited strains were used as test bacteria for screening of the microorganism for controlling the present organism, the disease could not be completely suppressed and germination was slightly inhibited (No. 744). 1246), the germination was strongly inhibited (No. 805) or the germination was slightly inhibited (〈ο · 780, 1234).

[Example 2] Identification of deposited strain The deposited strain in the present invention is a filamentous fungus strain BAUA-776 belonging to the genus Fusarium (deposit number: FERM BP-6536), which is isolated from dead leaves on the shore of Lake Towada in Kosaka-cho, Kazuno-gun, Akita Prefecture.

International deposit of BAUA-776 strain:

(a) Name of the deposited machine

Name: Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology

Address: Ibaraki, Japan <1-3, 1-3 East (postal code 305-8566)

(b) Deposit date (Original deposit date) August 20, 1997

(c) Accession No. 6536 (FERM BP-6536)

The mycological properties of this strain are as follows.

(1) Morphological characteristics under a microscope

The mycelium is colorless and has septum and is 2.5-5.0 m wide. It forms two types of conidia, small conidia and large conidia. The conidium on which the small conidium is formed is colorless, has a P wall, is 45 to 120 in length, and has a width of 3.5 to 5.0 Aim, and sometimes branches, forming a conidium in a pseudohead shape at the tip. I do. Small conidia are colorless and oval or oblong measuring 6-15 x 2.5-5 m. The large conidia are colorless, crescent-shaped, measuring 25-45x3.5-5.5 m, and have 3-4 partition walls. Spherical thick-film spores with a diameter of 5-10 μηη are formed on hypha and conidia.

(2) Growth on various media

The growth status when cultured on various agar media at 25 ° C for 7 days is shown below. The color tone was marked according to the standard color tone code number of ¹ color harmony manualj (Container Corporation of America).

1. Potato dextroth agar medium (PDA)

The size of the settlement is 54-58mm in diameter. The flora is white fluffy, with long hyphae dense in the center, but short hyphae covering the medium surface coarsely and densely toward the periphery. The central part is partially colored purple (hue 8 ec). Does not produce soluble dyes. The back is light It becomes yellow (hue 2 ca) and partially brown (hue 7 nl).

2. Malt extract agar medium (MEA)

The size of the settlement is 62-64 mm in diameter. The flora is long and white fluffy, and some parts of the center exhibit a fan-shaped reddish brown (hue 7 lc) from the center. Does not produce soluble pigments. The back side is pale yellow (hue 2 ca), and there are some parts that are fan-shaped reddish brown (hue 7 lc) from the center.

3. Tortmir agar medium (OA)

The size of the settlement is 70-74mm in diameter. The long, white fluffy flora forms a thick flora, and the area under the white hyphae is colored grayish yellow (hue 2 gc) and dark purple (hue 10 ie). Does not produce soluble dyes. The reverse side is yellowish brown (hue 3 le), brown (hue 4 lg) or brownish (hue 6 ni).

(3) Physiological properties

When cultured in a potato dextroth liquid medium, the range of pH that can grow is 2.0 to 12.0, and the range of optimal growth pH is 3.5 to 10.5. When cultured on a potato dextroth agar medium, the temperature range at which the cells can grow is 15 to 37 ° C, and the optimum growth temperature range is 20 to 30 ° C.

(4) Identification

Based on the above mycological properties, Takumi Matsuo, Tanada Komada, and Akira Matsuda, "Fusarium disease of crops" (National Rural Education Association, pp. 22-59, 1982) show that the deposited strain BAUA- Strain 776 forms a crescent-shaped conidia characteristic of the genus Fusarium and forms small conidia at the tip of a long conidiophore branched from the hyphae, so that Fusarium-solani ( Fusarium solani) and was identified ₀

[Example 3] Field test of soybean black root rot and laccase black root rot in a wooden frame

The pathogens used were those isolated from the soybean continuous crop field of Akita Noriken. Cultivation is carried out by adding 2.3 L of 2% sucrose improved medium to a 12 L stainless steel tray (30 X 12 X 55 cm) and 3.8 L (1 Kg) of vermiculum at 120 ° C. After autoclaving at a pressure of 20 minutes, the cells were inoculated according to a conventional method and cultured at 25 ° C for one month. The medium was aseptically stirred once every two to three days to supply the enzyme. At the end of the culture, a sclerotium of about 100 xm was formed. The pathogen culture was added to sieved mountain red soil at 1%, 3%, 5% and 10% by weight, soybean and laccase were seeded, and the disease status of black root rot was observed. The black root rot stably appeared in the 3% pathogen-added group.

The antagonistic antibacterial agent (FERM BP-6536) of the present invention was subjected to shaking culture at 25 ° C for 3 days in a potato dextros medium, and conidia were collected by centrifugation. Recovered conidia 3 ° /. Alginate Na at 3 ° / diatomaceous earth. The conidia were mixed with the added material, and the mixture was dropped into 5% salted calcium by a syringe to encapsulate the antagonist. During 1 capsule contains conidia 1 0 ^7.

2 g of such encapsulated antagonistic conidia were mixed into 100 g of soil containing 3% pathogenic bacterium red soil, which was able to produce a stable disease, and soybean and laccasei were sown. On the 60th day after sowing, the roots were carefully removed without cutting off the roots, the soil particles were brushed off with a brush, and the disease index of each individual was examined according to the criteria described above.

The results are shown in Tables 2 and 3. In the group to which the antibacterial capsule was added, both soybean and laccase had a high disease-suppressing effect.

Table 2

Soybean

Lakkasey

Inoculation-free zone Pathogen Bacteria Pathogen Bacteria Anti-bacterial disease 1% 3% 5% 10% Capsule

ΪΒ Main root Side root Main root i-side root Main root Side root Main root Side root Main root i-side root Main root Side root

0.00 0.00 0.40 0.59 0.90 1.23 1.89 2.35 2.21 2.55 0.00 0.00 Industrial applicability

According to the present invention, there has been provided a method for preventing infection of C. crotalariae in plants. In this method, by cultivating plant seeds in soil containing the deposited strain or a mutant thereof, the seeds can be grown without being infected with C. crotalarie.

Claims

The scope of the claims

Prevent infection of Calonectri a crotalariae to the plant, including the step of cultivating the plant on soil containing the RIKEN No. 6536 (FERM BP-6536) or its mutant. Method.

The method according to claim 1, wherein the plant is soybean.

A drug for preventing infection of plants with C. crotalariae (Calonectria crotal ariae), which contains, as an active ingredient, Life Science Article No. 6536 (FERM BP-6536) or a mutant thereof.

4. The agent according to claim 3, wherein the plant is soybean.

A request encapsulating at least one cell component selected from the group consisting of cultures, conidia, and chlamydospores of the culture of Life Sciences Research No. 6536 (FERM BP-6536) or its mutants. Item 3. The drug according to Item 3.

Contains at least one bacterial cell component selected from the group consisting of cultures, conidia, and chlamydospores of NIKEN No. 6536 (FERM BP-6536) or its mutants as active ingredients 4. The agent according to claim 3, which is a seed coating agent.

A seed of a plant coated with at least one cell component selected from the group consisting of a culture, conidia, and chlamydospores of a culture of Life Science Article No. 6536 (FERM BP-6536) or a mutant thereof.

8. The seed according to claim 7, wherein the plant is soybean.