JP2007217294A - Controlling agent for plant virus diseases and method of controlling - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To control plant virus diseases by using a soil disinfectant and soil-improving agent as a substitute of methyl bromide, or to provide an effective controlling agent for plant viral diseases in order to perform a preventive action. <P>SOLUTION: This controlling agent for plant viral diseases is characterized by containing microorganisms having an antagonizing activity against bacteria mediating the plant viruses. The method for controlling the plant viral diseases of seeds, cultivating soil, etc., by the above controlling agent is also provided. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a microorganism having an antagonistic action against a bacterium that mediates plant viruses, a plant virus disease control agent containing the microorganisms, and a plant virus disease control method using the control agent.

  Melon necrotic spot disease, lettuce big bein's disease, beet necrotic leaf vein yellowing disease, pea stem rot disease etc. are diseases caused by soil infectious plant viruses, especially melon necrotic spot disease, It is a viral disease mediated by the orpidium ubiquitous in the soil and has spread and spread nationwide. The pathogenic virus is present in soil, residue / residual roots, weeds, and seeds, and is transmitted by planting and management work. In order to prevent transmission, it is considered important not to intervene bacteria and insects that carry viruses.

  As a countermeasure against such soil-borne viral diseases, conventionally, a methyl bromide agent has been controlled by soil fumigation. However, this drug was abolished in 2005 except for some uses according to international regulations. As an alternative technology, soil disinfection technology using steam and hot water has begun to be used in some areas, but virus-preserving orpidium remaining in the deep soil has risen and spread, causing disease easily.

  Technological development to prevent soil transmission of such difficult-to-control soil-borne viral diseases is indispensable. In addition, the trend toward reduced pesticides has increased due to the recent increase in awareness of environmental issues. Under such circumstances, it will be indispensable to use environmentally resistant virus diseases by using resistant varieties and biological control. Other plant virus control agents include sodium alginate and shiitake mycelium culture extract (Patent Document 1), but none is used for soil disinfection.

Japanese Patent Publication No.54-33994

  An object of the present invention is to provide a plant virus disease control agent effective as a soil disinfectant and soil improver in place of methyl bromide for controlling plant virus diseases or for preventive measures. It is another object of the present invention to provide a method for biological control of plant virus diseases that uses the plant virus disease control agent and enables environmental conservation type agriculture with reduced agricultural chemicals.

The present inventors have intensively studied to solve the above-mentioned problems, and paying attention to the fact that orpidium is the main plant virus-borne bacterium, and from the group of microorganisms present in the rhizosphere soil, organic fertilizer and compost, The present inventors have searched for microorganisms that antagonize, and found microorganisms having high antagonism against orpidium that mediates Melon necrotic spot virus (MNSV) disease, thereby completing the present invention. In particular, Bacillus sp. BS-05242 AV (Accession number: FERM P-20743) belonging to the genus Bacillus
It has been found that microorganisms belonging to the group are effective in controlling plant virus-borne bacteria and plant virus diseases.

  That is, the present invention is Bacillus sp.BS-05242 AV having an antagonistic action against bacteria that mediate plant viruses.

  The present invention also provides a plant virus disease control agent comprising a microorganism having an antagonistic action against a bacterium that mediates a plant virus.

  The present invention also relates to a method for controlling plant viral diseases, characterized by treating seeds, leaves, roots and / or cultivated soils of cultivated plants with microorganisms having an antagonistic action against bacteria that mediate plant viruses. is there.

  Further, the present invention is a method comprising treating a cultivated plant seed, leaves, roots and / or cultivated soil with a microorganism having an antagonistic action against a bacterium that mediates a plant virus, and applying a soil disinfectant to the cultivated soil. Is a method for controlling plant viral diseases.

  In the above-mentioned control agent and control method, the bacteria that mediate plant viruses are Orpidium bacteria, and the microorganisms that have an antagonistic action against bacteria that mediate plant viruses are Bacillus sp.BS-05242 AV, respectively. Furthermore, examples of the plant virus or plant virus disease include melon necrotic spot virus or melon necrotic spot disease, respectively.

  The antagonistic bacterium against the infection of the fungus that mediates the plant viral disease of the present invention significantly suppressed the infection of Orpidium, and further significantly suppressed the infection of the pathogenic virus of melon spot spot disease. Moreover, the high virus control effect was shown also in the field by the combination process of the soil disinfectant and the microorganisms. Therefore, the plant virus disease control agent of the present invention can be effectively used for control measures and preventive measures of virus diseases such as difficult-to-control plant virus disease contaminated fields. From this, a new plant virus control method that has not been heretofore established by the present invention has been established, and it can be used as a methyl bromide substitute for virus diseases, and furthermore, environmental conservation type agriculture with reduced agricultural chemicals has become possible.

The present invention will be described in detail below.
As the microorganism of the present invention, a microorganism having antagonism against a plant virus disease vector is used. In particular, microorganisms having an ability to antagonize orpidium, which is representative of plant virus disease vectors, are effectively used. Examples of the plant virus disease include melon necrotic spot disease, lettuce big bein's disease, beet necrotic leaf yellowing disease, pea stem necrosis and the like. The microorganisms include bacteria (Pseudomonas genus, Bacillus genus, Corynebacterium genus), actinomycetes (Streptomyces genus, Actinomyces genus), yeast (Saccharomyces). (Saccharomyces)), filamentous fungi (Aspergillus (Aspergillus) genus, Penicillium (genus Penicillium)), etc., any microorganism can be used as long as it has an antagonistic activity against a plant virus disease vector. Examples of Bacillus bacteria include Bacillus sp. BS-05242 AV, BS-91, and BS-275. In particular, Bacillus sp. BS-05242 AV It is used effectively because of its high antagonistic activity.
Table 1 shows the bacteriological characteristics of BS-05242 AV.

This strain is classified into the genus Bacillus because it is positive for rod-shaped Gram staining in the above-mentioned mycological characteristics, forms a spore, and grows mainly under aerobic conditions. Related species of this bacterium include Bacillus cereus (B. cereus) and Bacillus thuringiensis. However, unlike the typical characteristics of Bacillus cereus (B. cereus) in that it does not exhibit lecithinase activity, that is different from the characteristics of the Bacillus Chu phosphorus thuringiensis in that crystalline inclusions is not detected (B. thuringiensis) [ JOHN GHOLT et al., BERGEY'S MANUAL OF Systematic Bacteriology): Birge's Manual of Systematic Bacteriology Vol.

About 5 bp at the 5 'end of the 16S ribosomal RNA gene (16S rDNA) of this strain
These regions were amplified and sequenced, and their sequences were used to search for homology to the bacterial reference strain database and GenBank / DDBJ / EMBL. As a result, the 16S rDNA of this strain showed 99% or more homology with B. cereus and B. thuringiensis. The closely related species were considered to be Bacillus cereus (B. cereus) and then B. thuringiensis, and it was assumed that they were closely related to Bacillus sp. From these, this strain was determined to be equivalent to a new species of Bacillus sp. This bacterium, Bacillus (Bacillus) as sp.BS-05242 AV to the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary Center has been commissioned as of December 22, 2005 (accession number FERM P-20743).

BS-05242 Examination of culture conditions for AV fungus
BS-05242 AV strain, 1 platinum loop is added to each liquid medium (Erlenmeyer flask 100ml) and cultured with shaking in a constant temperature oven with amplitude of 100 amplitude / min and 30 ° C for 4 days, 7 days, 10 days Later, the number of bacteria was examined by a dilution plate method using YPMG agar medium. The composition of the liquid medium is (1) YPMG (yeast extract 0.3 g, peptone 0.5 g, meat extract 0.1 g, glucose 1 g distilled water 100 ml, pH 7.0), (2) YPMD (yeast extract 0.3 g, peptone 0.5 g, meat extract 0.1 g, dextrin 1g of distilled water 100ml, pH7.0), (3) soy broth (soy broth (Soy broth) was used made of Difco (Toriputo-soy broth (Tryptic Soy broth) 3g, distilled water 100ml). culture test As a result, BS-05242 AV strain (2) YPMD stably maintained the viable cell count of 10 ¹² CFU / ml in the culture for 4 to 10 days (Table 2).

  This strain can be grown in the above liquid medium. In particular, YPMD liquid medium can maintain a stable number of bacteria. The YPMD medium used can be an agar medium or a liquid medium. The culture using the YPMD liquid medium is preferably performed at 30 ° C. for 4 to 10 days.

  The microorganism of the present invention is cultured and grown using an agar medium or a liquid medium. The proliferated microorganism can be collected by a platinum loop or the like, collected by an operation such as centrifugation, collected, or used in the state of a culture solution as a plant virus disease control or plant virus disease control agent.

The microorganisms obtained by culturing and growing in this way can be used alone or in combination of two or more. In addition, this microorganism can be used as the plant virus disease control agent of the present invention only by the microorganism, but in combination with other various agents, it can be in any shape such as granules, powders, tablets, emulsions, wettable powders, etc. It can also be used. Furthermore, the plant virus disease control agent of the present invention can be combined with an inorganic fertilizer, an organic fertilizer, a herbicide, soil, and used as a fertilizer, a soil conditioner, a seedling culture medium, or the like.

  Since this microorganism has an antagonistic activity against plant virus vector bacteria, this plant virus disease control agent is treated on the seeds, leaves, roots, cultivated soil, etc. of cultivated plants to have a plant virus disease control effect. Demonstrated. This treatment method includes application to soil, addition to seedling culture soil, bacterization treatment on crop seeds, foliar application, addition to nutrient solution in hydroponics, addition to plant stock in soil culture, irrigation, etc. Can give. This treatment with microorganisms can be used not only to control plant virus-borne bacteria but also to prevent transmission of plant virus disease-contaminated fields or as a preventive measure.

The plant virus disease control method of the present invention can be treated with a plant virus agent alone as described above. However, when combined with various soil disinfection, the plant virus disease control effect is further enhanced. The soil disinfection, DD agent, although hot water can be cited, it is preferable to use a DD agent. The treatment can be carried out with this microorganism after treatment and degassing, and the treatment method can be carried out on seeds, leaves, roots, soil and the like in the same manner as the above microorganism treatment method.

The amount of the plant viral disease control agent used is, for example, 10 kg to 500 kg per 10a for cultivated soil. In the case of seed treatment , air-dried seeds are used after being immersed in a culture solution for 10 min to 24 h.

  Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to these examples.

Production of plant virus disease control agent (1)
Using a spray drier (manufactured by Yamato Lab-Tech Co., Ltd.) was added 10% skim milk in this strain BS-05 242 AV culture solution ^{(1 × 10 12 CFU / ml} ), the spray outlet temperature is 100 to 110 ° C. Was set as follows.

With the setting of the spray outlet temperature (100 ° C.), 1 × 10 ¹⁰ CFU / g dry cells were obtained with a recovery rate of 80%.

Production of plant virus disease control agent (2)
Put each sample material of organic substrate (rice bran, bran) and porous substrate (zeolite, vermiculite) in a sterile bag at a ratio of Table 4 to 2.5 kg, add 750 mL of water, mix, seal, and 121 Autoclave sterilization was carried out at 30 ° C. for 30 minutes. After sterilization, 10 mL of the BS-05242 AV strain culture solution was added and cultured at 30 ° C., and the number of bacteria after 1 month of culture was measured.
The results are shown in Table 4.

From the results in Table 4, since the number of bacteria in the mixing ratio (3) of each test material was the largest, (3) was used as a plant virus control agent.

Simple selection test for antagonistic bacteria in a pure culture system of Orpidium bacteria Bacteria possessed by the Company (mainly Bacillus bacteria) that have an antagonistic activity against Fusarium bacteria isolated from soil and compost, including this strain BS-05242 AV In this study, it was investigated whether or not it has an antagonism against orpidium. One platinum loop of each strain is added to 100 ml of YPMG liquid medium (
After inoculation in a 200 ml Erlenmeyer flask containing 3.0 g yeast extract, 5.0 g peptone, 1.0 g meat extract, 10 g dextrin, 1.0 liter distilled water, pH 7.0), then 100 amplitude / min, 30 ° C, dark condition For 10 days. Dilute the culture solution 10 times with distilled water, immerse cucumber seeds (Kintaro Takii seedlings) in the diluted solution for 30 minutes, and plant 2 artificially in conical tubes filled with vermiculite (40 ml). It was managed in a meteorological instrument at 27 ° C for 16 hours. 1.0 ml of a crude solution of cucumber roots (about 100 pieces / side root 1 cm) on which orpidium had previously grown was added to the roots of the cucumber cotyledon development stage on the 10th day after sowing. Immediately thereafter, distilled water was added so as to be about 1 cm from the surface of vermiculite, and subsequent water management was added to such an extent that the ground surface was covered. Furthermore, it was grown for 3 weeks under the same cultivation management. After the growth, one strain was taken out so as not to damage the roots, the roots were washed with water, and the number of zoospore spore and dormant spores formed in the roots were counted under an optical microscope. In addition, the counting is performed for the roots that were selected from five main roots branched from the main roots close to the subterranean area per share, and that had a large number of roots among the roots cut to a length of 1 cm from the branched part. It was. The survey area was counted 5 times per strain.

The following formula, infection rate (%) = (BS-05242 AV treatment section orpidium colonization number / untreated orpidium colonization number) × 100
As a result of calculating the infection rate, the infection rate of BS-91 , BS-05242 AV and BS-275 among the tested bacteria was lower than that of the untreated group, especially 3% in the BS-05242 AV treated group. The infection rate. The results are shown in Table 5.

From this test result, it was found that the antagonistic bacteria can significantly suppress the infection of Orpidium by treating with BS-05242 AV bacteria.

BS-05242 Melon necrotic spot virus infection suppression pot test using AV bacteria Antagonistic bacteria treatment method : 2 treatments at seeding and planting Treatment at seeding: BS-05242 AV bacterial cell concentrate ( 2.0 × 10 ¹⁰ CFU / ml) melon seeds (variety Earls Masaharu) are soaked in a cell tray (1 hole capacity: about 30 ml), and then a 100-fold diluted solution (distilled water) of the bacterial solution 20 ml was poured per cell.
Treatment at the time of planting: After planting melon seedlings in a plastic pot filled with the test soil, 50 ml of the cultured bacterial solution (1.6 × 10 ¹¹ CFU / ml) was poured into the stock.
Test soil: Diluted in three levels with the same soil that was autoclaved (120 ° C, 1.2 atmospheres, 30 minutes) using the soil where this disease frequently occurred in semi-forced cultivation (raw soil) , 1/4 raw soil ward, 1/8
The soil was used and mixed with cow manure hull compost that was autoclaved at a ratio of 1 to the main soil 4. In addition, the same soil was autoclaved and used for growing healthy plants as controls. The virus line of the contaminated soil was MNSV-NH.
Test method: Seed melon, managed in an artificial meteorograph (25 ° C constant temperature), true leaf 10 days after sowing
Planting one strain per pot in a plastic pot (diameter 9 cm) filled with test soil at the leaf stage, 25 ℃
After growing for 3 weeks in a constant temperature meteorological apparatus, the roots were washed with water and the soil was well removed. The roots immediately below the ground were collected, and 100-fold crude juice was tested by DAS-ELISA.

The following formula, positive strain rate (%) = (number of positive strains in treatment area / number of test strains) × 100
As a result of calculating the positive strain rate by the treatment area, it was 25% in the 1/4 raw soil area and 0% in the 1/8 raw soil area. In the ward, infection was suppressed. The average absorbance was the average of the DAS-ELISA values of each strain. The average virus concentration was less than half that of the treated area even in the raw soil area. The results are shown in Table 6.

From this test result, it is possible to reduce the virus concentration to some extent even in the original soil region of melon necrotic spot disease frequent soil by performing BS-05242 AV fungus treatment, and by suppressing the degree of contamination in advance, melon necrotic spots The virus infection was significantly suppressed.

BS-05242 Field test of melon necrotic spot disease control using AV fungi
Implementation period March to July 2005)
Melon seeds (Earth Masaharu) were soaked for 2 hours in a concentrated cell fluid of this strain (1 × 10 ¹⁰ CFU / ml). The seedling soil was packed in a 16-hole connection pot (5 × 5 × 5 cm), and the soaked seeds were sown. Further, a 100-fold diluted solution (distilled water) of the concentrated cell solution was poured into each pot at 20 ml. Then, after germination for 3 days in a germination room (30 ° C.), the seedlings were grown in the seedling greenhouse to the third leaf stage of the main leaf. At the time of planting, 50 ml of the bacterial solution (2 × 10 ⁹ CFU / ml) was poured into the planting hole and the plant stock after planting. The outline of cultivation was as follows: two-row planting of 200 cm (bet 130 cm, passage 50 cm), inter-strain 36 cm, and streak 60 cm. Fertilization and other cultivation management followed the customary law. For soil disinfection, after subsoiler crushing the hard disk in the greenhouse, DD-92 (30L / 10a) equivalent was treated, covered with vinyl for 8 days, and then degassed three times. After degassing, for the purpose of controlling root-knot nematodes, 20g / m ² of nematrine was mixed with the soil by a control machine. About the hot water disinfection method, after crushing the hard disk in the field, treat 300 L / m ² of hot water with a moving spray type hot water disinfection machine, remove the coating on the 8th day after treatment, and leave it for another 5 days Tillage was carried out by a management machine. Each treatment group was repeated 3 times with 14 strains per group. The investigation method was to investigate the degree of disease on the leaf lesions (small lesions and large lesions) by visual observation according to the following criteria just before harvesting, and calculate the disease severity. For lesions that were difficult to discriminate, a virus test was performed by the DAS-ELISA method.

[Index by disease severity]
0: No disease is found
1: 1 to 4 diseased leaves
2: Number of diseased leaves 5-9
3: More than 10 diseased leaves
4: Withering

The following equation, disease severity = [{Σ (number of strains by disease severity × index)} / (number of strains studied × 4)] × 100
As a result of calculating the disease severity, 8.9 in the microorganism + soil disinfectant group was lower than 16.1 in the soil disinfectant alone group, and further lower than in the hot water disinfectant group 25.6. The results are shown in Table 7.

In addition, the rate of disease strain was lower in the microorganism + soil disinfectant group than in the other groups.
From this test result, it was possible to confirm the effect even in the field by performing BS-05242 AV fungus treatment. Furthermore, it was proved that there was a significant difference depending on the combination of soil disinfection.

Claims (10)

  A plant virus disease control agent comprising a microorganism having an antagonistic action against bacteria that mediate plant viruses.   The plant virus disease control agent according to claim 1, wherein the fungus that mediates the plant virus is Orpidium.   3. The plant virus disease control agent according to claim 1 or 2, wherein the microorganism having an antagonistic action against bacteria that mediate plant viruses is Bacillus sp.BS-05242 AV.   The plant virus disease control agent according to any one of claims 1 to 3, wherein the plant virus is melon spot spot virus and the plant virus disease is melon spot spot disease.   A method for controlling a plant viral disease, comprising treating seeds, leaves, roots and / or cultivated soil of a cultivated plant with a microorganism having an antagonistic action against bacteria that mediate the plant virus.   A plant virus obtained by treating a cultivated plant seeds, leaves, roots and / or cultivated soil with a microorganism having an antagonistic action against bacteria that mediate the plant virus, and applying a soil disinfectant to the cultivated soil Disease control method.   The method for controlling a plant viral disease according to claim 5 or 6, wherein the fungus that mediates the plant virus is Orpidium.   The method for controlling a plant virus disease according to any one of claims 5 to 7, wherein the microorganism having an antagonistic action against bacteria that mediate plant viruses is Bacillus sp.BS-05242 AV.   The method for controlling a plant virus disease according to any one of claims 5 to 8, wherein the plant virus is a melon spot spot virus, and the plant virus disease is a melon spot spot disease.   Bacillus sp.BS-05242 AV which has an antagonistic action against bacteria that mediate plant viruses.