JP4892583B2 - Method for detecting antagonistic microorganism against genus Psium, antagonistic microorganism and soil disease control agent using the same - Google Patents

Description

The present invention relates to a method for detecting antagonistic microorganisms that are effective in biological control of diseases caused by Pycesium, the antagonistic microorganisms, and a soil disease control agent using the same.
According to the present invention, as a pathogenic filamentous fungus that causes spoilage of seeds immediately after sowing and causes germination failure or withering of young seedlings, it is possible to obtain an antagonistic microorganism against Pisium spp. The antagonistic microorganism obtained by the present invention can be used to provide a biological control agent for diseases caused by the genus Psium.

Many studies have been made on the biological control of seedling blight by Pythium spp. (Non-Patent Document 1 and Non-Patent Document 2). Regarding diseases other than the genus Psium, biological control of plant diseases using antagonistic microorganisms has been actively studied as a control method with a low environmental load (Non-patent Document 3).
However, there are only a few examples of the development of biopesticides and microbial materials using antagonistic microorganisms. This is because it is difficult to establish an experimental system that can test antagonistic microorganisms that are effective in a field environment.

As a disease suppression mechanism of antagonistic microorganisms, it is generally considered that the mechanism is as follows.
1) Antibiotics that inhibit the growth of other microorganisms through the production of antibiotics 2) Competition that competes for nutrients and invasion sites among microorganisms 3) Parasitism that contacts or invades other microorganisms and lyses them 4) Elicits an ecological defense mechanism for plants Induction of resistance Specific examples of antagonistic microorganisms against diseases of the genus Psium are described below. In the antibiotic of 1) above, Pseudomonas fluorescens Pf-5 strain is related to the antibiotic pyoliteorin produced by this strain, and causes the seedling wilt of cotton by Pythium ultimum. There is a suppressed report (Non-Patent Document 4). In the competition of 2) above, Pseudomonas sp. Quickly covers the seed surface after sowing and preferentially uses the exudate from the seed to suppress seedling blight caused by Psium (Non-Patent Document 5). In the parasitism of the above 3), it is mentioned as one of the mechanisms of disease control that non-pathogenic Rhizoctonia spp. Parasitize Pythium Ultimum (Non-patent Document 6). In the resistance induction of 4) above, there is a report that Pseudomonas sp. Induces resistance to cucumber and suppresses stem rot caused by Pycium aphanidermartam (Non-patent Document 7).

  Conventionally, for the detection of novel antagonistic microorganisms, counter-culture on a culture medium of a test bacterium and a pathogenic bacterium has generally been performed. This is mainly a selection method based on the antibiotic of 1) above, and there have been reports that many antagonistic microorganisms have been obtained by this method. However, although it shows a disease suppression effect at the laboratory level, it is effective at the field level. There is very little to show, and there are few things that have been commercialized as biological pesticides.

The following are registered as biological pesticides for diseases caused by plant pathogens other than Psium.
The antagonistic bacteria in the Bacillus subtilis wettable powder are said to exhibit an antagonistic action against gray mold fungus due to the competition of the growing place and nutrients (Non-patent Document 8). Moreover, it is said that the antagonistic bacteria in the non-pathogenic Erbinia carotobola wettable powder show an antagonistic action against soft rot due to nutritional competition on the leaf surface and antibacterial action (Non-patent Document 9).

  The pathogenesis suppression mechanism of these antagonistic microorganisms that have been put to practical use is not limited to antibiotics, but also exhibits a combined effect. In these successful cases, instead of the conventional screening method using anti-cultivation, a screening system close to the actual field environment is used. However, a detection system for selecting an antagonistic bacterium against the important disease Pisium spp. Has not been studied in detail so far. The genus Psium belongs to algae and causes various diseases in plants. Symptoms of plants caused by this bacterium are roughly classified into seedling wilt, seedling and underground rot, and fruit rot. All of them are characterized by producing white and fluffy hyphae in the damaged part when wet. Organs of this fungus include hyphae, zoospores, zoospores, conidia, and spore. Oospores are durable organs in the soil. Representative examples of the genus Psium include Pythium aphanidermatum and Pythium ultimum. The difference in morphological characteristics between the two is that the former forms follicular spores, but the latter does not form follicular spores and mainly grows on conidia. The host plant also invades the former at the seedling stage, while the latter invades plants that have grown to some extent. In addition, the former occurs more frequently in a moist environment, and the latter is different in that the disease occurs even under relatively dry conditions.

Tsuneo Watanabe, Encyclopedia of Plant Soil Diseases, Asakura Shoten, p. 244-245, 1998 Martinn and Looper. Critical Reviews in Plant Science, 18 (2): p. 111-181, 1999 Agriculturally useful microorganisms-utilization and prospect-, Umedani Seiji, Kato Satoshi, Yokendo, p. 95-200, 1990 Howell and Stipanovic, Physiology 70: 712-715, 1980 Nelson and Craft. Phytopathology, 79, 1009-1013 Siwek et. al. , Phytopathology, 145, 417-423. Zhou and Paulitz, J.A. Phytopathology, 142: 51-63, 1994. Biological pesticide guidebook, Japan Plant Protection Association, p. 26-33, 1999 Biological pesticide guidebook, Japan Plant Protection Association, p. 20-25, 1999

  In order to establish a method for detecting antagonistic bacteria against the genus Pythium, the inoculated Psium genus stably develops disease under conditions that are closer to the field environment, and without overlooking the antagonistic bacteria that are effective in the field environment. It has been required to establish conditions for efficient screening.

As a result of intensive research aimed at finding a means for solving the above-mentioned problems, the present inventors have succeeded in establishing conditions for efficient screening without overlooking antagonists to the genus Psium. Furthermore, a novel antagonistic microorganism against Pythium was found by such a detection method. The present invention has been completed based on these findings.
That is, the present invention relates to a method for detecting an antagonistic microorganism that exhibits a biological control effect of a disease caused by Psium,
After preparing Pisium-contaminated soil, inoculating and mixing the test bacteria on the contaminated soil, planting seeds and cultivating them, and evaluating the inhibitory effect of the test bacteria on plant diseases caused by Pycesium, A detection method based on a pot test for detecting Pisium-contaminated soil prepared so that the number of Psium species in the contaminated soil is a minimum number of bacteria necessary for causing all the seeded plants to become diseased. Use
This is a method for detecting an antagonistic microorganism.
Furthermore, the present invention is an antagonistic microorganism against the genus Psium belonging to the genus Arthrobacter sp., The genus Paecilomyces, the genus Malblancea or the genus Cunninghamella.
Furthermore, this invention is a biological control agent with respect to the soil disease by the genus Psium which contains the said antagonistic microorganism as an active ingredient.

  In the present invention, by reducing the number of Psium bacteria in the contaminated soil as much as possible and establishing conditions under which stable pathogenesis is recognized, not only pathogenic bacteria and antagonistic microorganisms but also the interrelationships between plants and soil are considered. We succeeded in developing a screening system closer to the field environment. That is, in the detection method of the present invention, the number of Pisium spp. To be inoculated is reduced to a minimum while considering the mutual relationship with plants, soil, and indigenous microorganisms, under conditions closer to the natural environment. Therefore, it has become possible to efficiently select antagonistic bacteria against the genus Psium. The antagonistic bacterium against the genus Psium obtained by the detection method of the present invention can be effective for biological control of seedling blight caused by the genus Psium.

It is the graph which showed the inhibitory effect with respect to the blight of the antagonistic bacteria detected by the method of this invention by Psium aphanidermartam. In FIG. 1, no treatment indicates no test bacteria, and no treatment / no inoculation indicates a case where neither the test bacteria nor the Psium species are present. It is the graph which showed the inhibitory effect with respect to the blight of the antagonistic filamentous fungi acquired by the method of this invention by Pycesium aphanidermartam. In FIG. 1, no treatment indicates no test bacteria, and no treatment / no inoculation indicates a case where neither the test bacteria nor the Psium species are present.

  In the present invention, after preparing Pisium-contaminated soil, inoculating and mixing the test bacteria there, plant seeds are sown and cultured to evaluate the disease inhibitory effect of the test bacteria, and detection of antagonistic microorganisms is a pot test To do.

The soil for producing the Psium-contaminated soil used in the detection method of the present invention preferably has physical and physicochemical properties that do not prevent germination of plant seeds. Moreover, it is preferable to avoid soil containing a large amount of immature organic matter because it promotes the growth of Psium. It is also preferable to avoid soil with an extremely large number of microorganisms. The water purification plant generated soil (water purification cake) satisfies the above conditions and is suitable for soil for producing Psium spp. As the water purification plant generated soil, any water purification plant generated soil may be used, and a water purification cake obtained by concentrating and dewatering sedimentary mud (purified sludge) generated in the water purification process is usually used. More specifically, as the soil generated from the water purification plant, it was precipitated by adding polyaluminum chloride or aluminum sulfate as a coagulant to the sedimentary mud (purified water sludge) generated in the water purification process, and dehydrated by limeless treatment. Things are usually used. The soil for producing Psium-contaminated soil is preferably not sterilized in order to detect the antagonistic microorganisms under conditions that also take into account the effects of indigenous microorganisms. In addition, it is desirable to confirm in advance that no disease will occur in the test plant.
In the detection method of the present invention, the soil used for the production of Psium-contaminated soil is preferably a soil that is as close to the field environment as possible. Therefore, the soil contains the same amount of microorganisms contained in normal soil. Is desirable. More specifically, for example, it is preferable to use soil having a filamentous fungus count of 100 to 10,000 cfu / g and a bacterial count of 100,000 to 10,000,000 cfu / g dry soil. By using this, it is possible to detect antagonistic microorganisms against Pythium bacteria at a pot level under conditions close to the field environment.

For the production of Psium-contaminated soil, which is the soil for detecting antagonistic microorganisms from the test bacteria, the above-mentioned soil is usually mixed with the spore or spore cage of Psium so that the appropriate number of bacteria is obtained. To do. Oospores or spores are preferably formed on a plant body. For example, a plant body immediately after germination is affected with Pythium spp., And then the plant body is removed and air-dried and pulverized is used as an inoculation source. Pisium spp. Form spore or spore spore in the soil and survive the endurance, but it is suggested that they start infectious behavior in response to specific substances in exudates from seeds and roots . However, it has been reported that spores formed in a synthetic medium react not only with specific substances but also with substances such as sugars and amino acids. Thus, what was formed in the synthetic culture medium differs in the property with respect to infection (Nelson and Craft. Phytopathology, 79, 1009-1013), and is not preferable.
The contaminated soil is prepared so that the number of Pisium spp. In the Psium spoiled soil becomes the minimum number of bacteria necessary for causing all of the plants sowed and cultured in the contaminated soil. Here, the number of bacteria refers to the total number of Psium bacteria contained in the contaminated soil, the total number of oospores and spores, and the minimum number of bacteria required to cause disease refers to the plant seeds in the contaminated soil. Necessary to cause all plant solids to die and all to die when seeds are sown and cultured at a distance that does not affect each other's individual plants in plant disease when seeded and grown. Means the smallest number of bacteria. Usually, the number of Psium species contained in Psium-contaminated soil is 10 to 1,000 cfu, preferably 50 to 500 cfu, and more preferably 100 to 200 cfu per 1 g of soil. The number of bacteria is measured by a dilution plate method or the like using a selective medium of Psium genus such as VP ₃ medium (Ali-Shitaehe et al. Trans. Br. Mycol. Soc. 86: 39-47, 1986). For example, Pythium aphanidermatum FERM P-19018 and Pythium ultimum FERM P-19017 can be used as the target Psium genus. Can be used similarly.

The contaminated soil prepared as described above is filled in a pot, preferably a minipot. In order to prevent the outflow of assay bacteria, it is preferable to use a pot without a drain hole. It is preferable to provide a soil conditioner for retaining soil moisture below the contaminated soil. Examples of such a soil conditioner include pearlite, vermiculite, zeolite, charcoal and the like, and pearlite and vermiculite are particularly preferable. Specifically, for example, a plastic container for silkworm radish of 85 mm (length) x 85 mm (width) x 140 mm (depth) is used, and 100 ml of contaminated soil is packed on 75 ml of pearlite.
It is preferable to irrigate the pot to such an extent that the liquid level exists in the soil improvement material layer such as pearlite in the lower part. The presence of a soil amendment layer such as pearlite keeps the soil moisture during the assay moderate. In the case of the above optimum form, 50 ml of irrigation is an appropriate amount.

  In general, filamentous fungi or bacteria are preferably used as the test bacteria to be tested because of their easy collection and high growth ability. After culturing the test bacteria in a medium for a certain period and growing them, the cells are mixed with contaminated soil. For example, after inoculating bacteria in a liquid medium after shaking culture, the medium components are removed by centrifugation and suspended in sterile distilled water. Filamentous fungi are cultured in soil bran medium or the like as an inoculum. Since saccharides and the like in the culture medium for cultivating the test bacteria activate the genus Psium, it is preferable to remove the medium components at the time of inoculation.

  About 3 to 10 plant seeds are usually sown in a pot in which contaminated soil and test bacteria are mixed. The sowing is preferably performed after standing overnight at 25 ° C. to 30 ° C. in consideration of colonization of the test bacteria on the soil. The plant seeds may be any as long as they are within the host range of the genus Psium to be used. For example, since Pisium aphanidermartam is a thermophilic (27 ° C. to 31 ° C.) seed, it is preferable to use a seed having a temperature suitable for germination in this temperature range. In addition, it is preferable to use seeds having a high germination rate because they can be easily distinguished from seed rot due to disease when they have not germinated. The seeds of Cucurbitaceae plants are moderately sized for observing the state of the seeds, have a relatively high germination rate, and are suitable for assaying suitable germination temperatures. In particular, cucumber seeds have a high germination rate of almost 100% and are optimal as test plants. Further, it is preferable to investigate the germination rate of the test plant seed in the non-contaminated soil under the same conditions in advance and use it to determine whether the ungerminated seed at the time of the assay is due to germination.

After sowing, coat with vinyl or the like to prevent drying and grow in a growth chamber or greenhouse. At this time, care should be taken to maintain an appropriate temperature for infection with Psium. After the seeds germinate and are cultivated for a sufficient period of time for the cotyledons to fully develop, the state of the plant is observed. Compared with the non-inoculated test pot, the pot in which the number of healthy seedlings in which seed rot and seedling wilt did not appear was significantly large is considered that the inoculated test bacteria have a disease control effect. The same test is repeated 2 to 5 times, and the test bacteria that have been continuously effective are selected as antagonistic microorganisms that suppress diseases caused by Pythium spp.
According to the present invention, a screening system for antagonistic bacteria against Psium disease can be established by establishing an optimal number of Psium species, Psium species, plant species, and environmental conditions. In other words, after mixing the test bacteria in the contaminated soil of the genus Psium, plant seeds are sown and the disease control effect of the test bacterium is evaluated, so that antagonistic microorganisms that may be useful for biological control of the disease caused by the genus Psium You can get it. The present invention is an optimal method for detection and acquisition of antagonistic microorganisms against Pythium under conditions close to the field environment.

  According to the detection method of the present invention described above, the genus Arthrobacter sp., Paecilomyces, Malbranchea or Cunninghamella is described in detail in the examples described later. An antagonistic microorganism against the genus Psium was obtained. More specifically, Arthrobacter sp. Gb4a (FERM P-19020), Arthrobacter sp. Ib2a (FERM P-19022), Arthrobacter histodinovo Arthrobacter histodinovorans Rb5a (FERM P-19023), Arthrobacter histodinovorans Rb5b (FERM P-19024), Paecilomyces sp. Malbaranchea sp. Mf2 (FERM P-19019) and N'ninguhamera belonging to the genus (Cunninghamella sp.) Uf2 (FERM P-19020) was obtained.

These antagonistic microorganisms are effective as biological control agents for soil diseases caused by Psium genus because they exhibit an inhibitory effect on plant diseases caused by Psium species. In order to apply an antagonistic microorganism to a plant, a method is generally employed in which the antagonistic microorganism is cultured by tank culture, shaking culture, or the like, and the culture solution is sprayed as it is. Alternatively, the culture solution may be diluted appropriately and sprayed with the diluted solution. If necessary, commonly used agricultural additives and the like may be added to these culture solutions. Application of antagonistic microorganisms, as the culture liquid, usually 1~1,000ml / ^{m 2,} preferably in an amount of 10 to 100 ml / ^{m 2.} The antagonistic microorganism of the present invention can also be used as a control agent in combination with other suitable agricultural additives. As these additives, commonly used ones can be used as they are.

EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited to these Examples.
Example 1
In order to efficiently detect antagonistic microorganisms by the target plant assay, the aim is to conduct the assay with the smallest number of Psium bacteria as possible, and Psium aphanidermartam ( Pythium aphanidermatum OPU431: FERM P-19018) and Pythium ultramum UOP407: FERM P-19017) were used to investigate the relationship between the number of bacteria in the soil and the pathogenesis of the bacteria, and the lower limit of disease incidence in non-sterile soil The following experiment was conducted to determine the number of bacteria.

Method i) A water-purified cake sieved to 6 mm (using a water-purified cake having a fungal count of 2,500 cfu / g dry soil and a bacterial count of 5,000,000 cfu / g dry soil) was spread on a seedling box and cucumber ( Variety: Yotsuba Suzui) was sown and germinated. The cells of Psium aphanidermatum or Psium Ultimum previously cultured in a liquid medium were inoculated there to cause cucumber disease. When the plant body died, the plant body was removed and air-dried and crushed. This was used as an inoculation source for the psium-contaminated soil.
ii) Contaminated soil was prepared in 5 treatment zones by changing the mixing ratio of the inoculation source of Psium and non-contaminated soil. The number of Psium bacteria in the contaminated soil was measured by a dilution plate method using a VP ₃ medium that is a selective medium for Psium after producing the contaminated soil.
iii) A 85 mm (length) x 85 mm (width) x 140 mm (depth) plastic container for silkworm radish filled with 75 ml of pearlite for soil moisture adjustment, 100 ml of contaminated soil prepared in ii) above, and 50 ml of tap water Poured. Five cucumber seeds were sown per pot, and a plastic container was covered with vinyl and cultured in a growth chamber with illumination for 12 hours. The culture temperature was 30 ° C. for the Psium-Afanidelmartam contaminated soil and 26 ° C. for the Psium Ultimum contaminated soil.
iv) After culturing for 7 days, which is a sufficient period required for the development of cotyledons and the infection of the genus Psium, the state of the plant was observed, and the incidence of seed rot and seedling wilt caused by the genus Psium was investigated.

Results As a result of measuring the number of bacteria in each prepared contaminated soil, the amount of Psium afanidelmartam contaminated soil was 0, 5, 22, 80, and 133 cfu, respectively, per 1 g of soil. The amount of Psium Ultimum contaminated soil was 0, 25, 53, 77, and 105 cfu, respectively, per 1 g of soil. Tables 1 and 2 show the degree of occurrence of seedling blight of cucumber by plant assay. The incidence was shown as the average of three experiments.
In the case of Psium afanidelmartam contaminated soil, the disease was unstable at 80 cfu / g soil, but the disease incidence was 100% at 133 cfu / g soil. In the Psium Ultimum contaminated soil, the disease incidence was 100% at 77 cfu / g soil or higher. Therefore, it was clarified that the genus Psium is surely diseased when 100 to 200 cfu / g soil exists in the contaminated soil. This number of bacteria is considered to be the best condition used for selection of test bacteria.

Example 2
The following experiment was conducted in order to test the fungi and bacteria isolated from the target soil for the detection method of Example 1 and to obtain antagonistic microorganisms against the genus Pythium.

Method i) Microorganisms were separated from several arbitrary soils by a dilution plate method, and bacteria and filamentous fungi that appeared visually on the medium were isolated. The obtained 224 bacterial strains and 46 fungal strains were used.
ii) Psium aphanidermatum contaminated soil was prepared by the method shown in Example 1 and prepared so that the number of bacteria was 1 × 10 ² to 2 × 10 ² cfu per 1 g of soil. 100 ml of this contaminated soil was put in a plastic container [85 mm (length) × 85 mm (width) × 140 mm (depth)] containing 75 ml of pearlite in advance.
iii) The test bacteria were inoculated as follows. When the test bacteria were filamentous fungi, they were cultured for 7 days at 25 ° C. in a soil bran medium (soil: brass = 4: 1), mixed with 10 g of the contaminated soil, and 50 ml of distilled water was poured into the container. When the test bacterium is a bacterium, after shaking culture at 30 ° C. for 48 hours in a MYPG liquid medium, the medium component (supernatant) is removed by centrifugation, and the resulting microbial cell (precipitate) is diluted with 50 ml of sterile distilled water. And uniformly sprayed with the contaminated soil.
iv) After inoculation with test bacteria overnight at 27-30 ° C., seeded with 5 cucumber seeds dropped with running water from a commercially available cucumber seed drug-coated with drug, and plastic container with vinyl Covered. After culturing for 7 days in a growth chamber illuminated at 30 ° C. for 12 hours, the state of the plant was observed, and the disease inhibition effect of the test bacteria was evaluated. For the test strains that were confirmed to have a disease-suppressing effect, the same test was repeated three times, and antagonistic bacteria that were stably recognized were selected.

The results are shown in FIGS. In contrast to P. aphanidumartam, 4 bacterial strains, Gb4a, Ib2a, Rb5a, and Rb5b, and 3 fungal strains, If2, Mf2, and Uf2, were found to have disease-inhibiting effects.

Example 3
Objective Antimicrobial action mechanism of 7 antagonistic microorganisms detected by the present invention (4 bacteria: Gb4a, Ib2a, Rb5a, Rb5b, 3 fungi: If2, Mf2, Uf2) against Psium aphanidermartam In order to investigate, the following experiment was conducted.

Method i) Filamentous fungi in which antagonistic force was recognized in Example 2 were pre-cultured on a PDA plate, and a disc in which the flora was punched out with a cork borer having a diameter of 8 mm was produced. This was newly placed on one end of the PDA plate and cultured until the fungus layer expanded to 2-3 cm. On the other end of the flat plate, a Pichia aphanidermatum flora disk was placed in the same manner and cultured at 28 ° C.
ii) If the bacterium with antagonistic activity is a bacterium, streak at one end of the MYPG plate, and after culturing for 2 days, place the Pichium aphanidermatam flora disc on the other end of the petri dish at 30 ° C Cultured.
iii) After 2 to 3 days, the state of the Psium aphanidermatum flora was observed and the presence or absence of growth inhibition was observed.

Results None of the 3 filamentous fungi and 4 bacterial strains used in the experiments showed growth inhibition, suggesting a disease-inhibiting mechanism that does not produce antibacterial substances.

Example 4
Purpose The identification of 7 antagonistic microorganisms (4 bacteria, Gb4a, Ib2a, Rb5a, and Rb5b, and 3 fungi, If2, Mf2, and Uf2) detected by the present invention was carried out.

In the case of bacteria, the method strain was identified by analyzing a partial base sequence of about 16 bp of 16S rDNA and estimating the belonging taxon of the strain by homology search. Filamentous fungi were identified by macroscopic and microscopic morphological observation.

Results The partial base sequences of Gb4a strain, Ib2a strain, Rb5a, and Rb5b were as shown in SEQ ID NOs: 1, 2, 3, and 4, respectively.
From the partial base sequences of the Gb4a strain and the Ib2a strain, MicroSeq ^™ Microbiological Identification System software Ver1.4.1 and MicroSeq ^™ Bacterial 500 Library v. As a result of homology search by 0023, it has 96.42% homology and the highest homology to Arthrobacter oxydans. (A. pascens) and a cluster were formed. Blast (BLAST) homology search showed the highest homology to the Arthrobacter sp. SMCC ZAT262 strain with a homology rate of 99.0%. Therefore, these two strains can be estimated as belonging to the genus Arthrobacter and closely related to Arthrobacter passense (A. pascens).
From partial base sequences of Rb5a and Rb5b, MicroSeq ^™ Microbiological Identification System software Ver1.4.1 and MicroSeq ^™ Bacterial 500 Library v. When the homology search was performed using 0023, the homology was 100%, which was consistent with Arthrobacter histodinovorangans. Even in the molecular phylogenetic tree, it was located at the same place as A. histidinolovorans. Therefore, it was suggested that these two strains belong to Arthrobacter histodinovorans.

As a result of macroscopic observation of the If2 strain, the colonies showed low convexity and were dry. It was fluffy and white (A1), the basal mycelium showed moderate stagnation in the agar, and the growth was moderate, with the specimens cultured for 1 week at 25 ° C. growing about 26 mm-28 mm. Conidia were formed 3 days after the start of culture, and the surface color changed to pinkish-grayish rose (12A-B2-3). Even long-term culture specimens showed no production of soluble pigment or exudate. As for the microscopic observation results, formation of a fiaro-type penicillus-like conidia pattern was confirmed, and many conidia patterns were similar to the genus Penicillium but varied. The phialide is a needle with an elongated neck, and the handle, metre, and phialide are smooth to slightly rough, the conidia are single-celled, the surface is smooth, the size varies, and the shape is oval. It was spindle-shaped. Conidia were chained and some connections were observed. The formation of thick-wall spores and teleomorphs could not be confirmed from long-term culture specimens.
From the above observation results, the If2 strain was attributed to the genus Paecilomyces sp.

From the macroscopic observation of the Mf2 strain, the colonies were dry and showed velvety to fluffy. The color tone of the colony surface was white (A1) and the color tone of the back surface was almost the same. The mycelium hardly penetrated into the agar. Growth was fast, and the specimens cultured at 25 ° C. for 1 week had a diameter of 66 mm on the PDA plate, 59 mm on the MEA plate, and about 54 mm on the OA plate. In microscopic observation at high magnification, conidia linked to the curvature were formed. Even long-term culture specimens showed no production of soluble pigment or exudate. As for the microscopic observation results, only segmental conidia formation structures were observed. All segmented conidia were formed from aerial hyphae. The mycelium surface was slightly rough, the mycelium grew linearly, and there were relatively many branches. The septum was sparse and the hypha width was relatively narrow. The segmental conidia were formed with a curved structure from the tip of the aerial hyphae, and no clear boundary with the hyphae was observed. The conidia were unicellular and cylindrical, and the surface was slightly rough. The formation of thick-wall spores and teleomorphs could not be confirmed from long-term culture specimens.
Based on the above observation results, the Mf2 strain was assigned to the genus Malbaranchea sp.

As a result of macroscopic observation of the Uf2 strain, the mycelium was wooly and white, and there was no color change. The growth rate was extremely fast, and reached the entire surface of a petri dish having a diameter of 85 mm by the fifth day of culture in all plates cultured at 25 ° C. There was no odor and no soluble pigment, a spore stalk pattern was observed, and the colony was colored pale gray (B1). As a result of microscopic observation, a Cunninghamella-like spore sac was observed. The mycelium had very few partition walls, and fluff branches and temporary roots were formed. An apical sac was formed at the tip of the spore sac, and irregular or ring-like branches were observed below it. A small apical sac was also found at the tip of the branch. The apical sac was spherical to pear shaped. A monospore microspore sac was formed from the apical surface. The microspore sac was spherical to elliptical and brown, and the surface was short needle-like. No zygote was formed from the long-term culture specimen.
From the above, the Uf2 strain was assigned as Cunninghamella sp.

These acquired nine strains were deposited at the National Institute of Advanced Industrial Science and Technology patent biological deposit center on September 17, 2002, and the following deposit numbers were assigned respectively.
Paecilomyces sp. If2
FERM P-19016
Malbaranchea sp. Mf2
FERM P-19019
Cunninghamella sp. Uf2
FERM P-19020
Arthrobacter sp. Gb4a
FERM P-19021
Arthrobacter sp. Ib2a
FERM P-19022
Arthrobacter histodinovorans Rb5a
FERM P-19023
Arthrobacter histodinovorans Rb5b
FERM P-19024

Claims (7)

In a method of detecting an antagonistic microorganism that exhibits a biological control effect of a disease caused by Psium,
After preparing the inoculation source obtained by air-drying and pulverizing the soil containing the genus Psium , and using the soil generated from the water purification plant as the soil , inoculating and mixing the test bacteria in the contaminated soil, A method for detecting antagonistic microorganisms by seeding and cultivating plant seeds and evaluating antagonistic microorganisms by evaluating the inhibitory effect of the test bacteria against plant diseases caused by Psium spp. Use Psium-contaminated soil prepared so that the number is the minimum number of bacteria necessary to cause all the seeded plants to become diseased.
A method for detecting antagonistic microorganisms.   The detection method of Claim 1 using the Psium genus contaminated soil prepared so that the number of Psium bacteria may be set to 10-1,000 cfu / g soil.   The detection method according to claim 1 or 2, wherein the genus Psium is Pythium aphanidermatum or Pythium ultimum.   The detection method in any one of Claim 1 to 3 which uses the pot filled with the soil improvement material for hold | maintaining soil moisture in the lower layer of Psium bacteria contaminated soil.   The detection method according to claim 1, wherein a Cucurbitaceae plant is used as the plant. The detection method according to claim 5 , wherein the cucurbitaceae plant is cucumber. The soil for producing Psium spp. Contaminated soil is a soil having a filamentous fungus count of 100 to 10,000 cfu / g and a bacteria count of 100,000 to 10,000,000 cfu / g dry soil. 6. The detection method of any one of 6 .

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