JP2801164B2 - Root fungi having ability to control Chinese cabbage soil disease, method for producing the same, and method for suppressing Chinese cabbage soil disease - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a root fungus having an ability to suppress soil disease of Chinese cabbage, a method for producing the same, and a method for controlling soil disease of Chinese cabbage using the same.

[0002]

2. Description of the Related Art Continuous cropping of many crops causes continuous cropping failures such as poor growth, reduced yield, and poor quality. If the continuous cropping failures become severe, they may die. Such continuous cropping disorders are mainly due to soil diseases. For example, a typical root disease of Chinese cabbage is Chinese cabbage root disease. This root-knot disease of Chinese cabbage (Plasmodiophora) lurks in the soil.
brassicae), the spores of the clubroot fungus invade the roots of Chinese cabbage cultivated in soil, multiply and cause the clubroot. The roots of Chinese cabbage that caused this clubroot are formed with bumps, as the name suggests, and the absorption of moisture and nutrients from the soil is hindered. As a result, the growth of Chinese cabbage deteriorates, resulting in a decrease in the yield and quality of Chinese cabbage.

Conventionally, as a countermeasure against soil diseases, soil disinfection using chemical pesticides such as PCNB agents has been widely and generally performed. However, continuous use of soil disinfectants due to continuous cropping
It causes the emergence of new bacteria that are more resistant to soil disinfectants in the soil, resulting in a reduced control effect.
In addition, the use of chemically synthesized soil disinfectants is feared to have an adverse effect on the environment including consumers, producers and natural ecosystems, and their use is decreasing.

On the other hand, in recent years, biological control of soil diseases has been tested in various countries around the world, but there have been few reports of success.
The number of microorganisms having the ability to suppress soil disease bacteria is also limited to several types, and most of them are bacteria and actinomycetes. These microorganisms mainly produce antibiotics outside the body and suppress the onset of soil-borne bacteria by a mechanism that suppresses the propagation of pathogenic bacteria.

[0005] On the other hand, there have been few reports on the success of biological control using fungi, and Trichoderma (Tri
biocontrol using the genus choderma). The Trichoderma spp. Is parasitic on other fungi and can kill the fungi. In Japan, the control of tobacco white silk disease by Oshima in 1966
Is the first report, and has been registered as a pesticide as a biocontrol agent. However, there have been no reports of practical use of biological control using fungi.

[0006]

SUMMARY OF THE INVENTION As described above,
Soil disinfection with a chemical pesticide such as a PCNB agent cannot effectively suppress the soil damage of crops, and has unfavorable effects on the environment including humans and natural ecosystems. Against this background, there is a demand for a new crop soil disease control technology that does not affect people or the environment.

[0007] Various microorganisms are present in the soil and the plant body while influencing each other. Most of these microorganisms are non-pathogenic without causing disease in plants. In particular, in the root of the plant,
It is already known that there are symbiotic fungi in roots (endophytes) that coexist with plants without causing disease in the plants.
The present invention pays attention to such root symbiotic fungi, and selects and selects those having a function of suppressing soil pathogenic fungi from among such root symbiotic fungi, and selects crop soil non-pesticidal fungi that do not depend on pesticides. It is an object of the present invention to enable the suppression of

[0008]

Means for Solving the Problems From the above viewpoints, the present inventors have selected symbiotic fungi in Chinese cabbage roots for the purpose of controlling Chinese cabbage root-knot disease, which is difficult to control among soil diseases. , A root bacterium effective in controlling Chinese cabbage clubroot was identified. That is, a root fungus symbiotic in the root is cultivated from the roots of Chinese cabbage cultivated in the soil where wheat, soybean and the like are continuously cropped, and the cultured strain is separated and transplanted to a storage medium. At this time, the root fungus is identified, and the root fungus is classified according to the species. For bacteria that cannot be identified,
Classify by colony type. Chinese cabbage seeds are sown in a medium for isolation of these strains, germinated, and a root fungus having the ability to control Chinese cabbage soil disease is inoculated to the Chinese cabbage roots. Furthermore, while cultivating the Chinese cabbage, clubroot fungi are inoculated into the cultivation soil, and the Chinese cabbage is cultivated for a required period during which the clubroot occurs. Thereafter, the presence or absence of clubroot disease of Chinese cabbage is examined, and the root fungus inoculated on the roots of Chinese cabbage with a low incidence of clubroot is identified and selected.

It is noted that some of the rhizobacteria tested in this way have a remarkable function of suppressing the clubroot disease of Chinese cabbage by inoculating them on the roots of Chinese cabbage. Was done. That is, the incidence of clubroot disease of Chinese cabbage is extremely low, including those in which Chinese cabbage root disease is not developed at all. The characteristics of the root bacterium having an inhibitory function against the clubroot disease of Chinese cabbage include a bacterium in which the colony on the medium is black or brown and spore formation is not recognized on the medium (Black Steryl Meisseria:
Black stereo mycelia). Another type is a bacterium that forms a yellow-white spore, and is known as Westerdyk multispora.
ella multispora).

[0010]

Next, embodiments of the present invention will be described specifically and in detail. The seeds of Chinese cabbage are sown on the soil where wheat, soybean, rape, napier grass and the like are continuously grown, grown for about two months, and then the Chinese cabbage is collected from the soil. The roots of the Chinese cabbage are cut off, and the root pieces are washed and left still in a separation medium. After leaving the root piece in a separation medium, the root piece is transplanted to a storage medium of a colony that has grown on the medium, thereby obtaining an isolated strain. In the isolation, strains are identified and separated by classifying each strain, but strains that cannot be identified are classified and separated by colony type.

The strain thus isolated on the storage medium is cultured, and as shown in FIG. 1 (a), when the colony grows, seeds of Chinese cabbage are sown on it and the seedling is grown as shown in FIG. 1 (b). raise. This causes the fungi to enter the roots of Chinese cabbage seedlings. Next, as shown in FIG. 1 (c), the Chinese cabbage seedlings are transplanted in sterile soil and grown so that the fungi that have invaded the Chinese cabbage roots spread throughout the roots. Next, as shown in FIG. 1 (b), the soil in which the Chinese cabbage is growing is inoculated with the causal agent of the clubroot, and then the Chinese cabbage is cultivated for a period sufficient for the cabbage to develop. Then, the Chinese cabbage is dug out of the soil and its roots are observed to determine whether clubroot has developed.

As a result of the examination, a remarkable effect of inhibiting the clubroot of Chinese cabbage was found for the seven fungi. One of the seven fungi is Westerdykella mulspora
tispora), and the other six species do not show any sporulation on the culture medium, so that the bacterial species cannot be identified. The characteristics of these bacteria are shown in Table 1 below. In addition, the colony diameter shown in Table 1 is a value measured after culturing on a PDA culture medium for cultivation at room temperature for 3 weeks, and the number of established strains is as follows. The number of days until transplantation.

[0013]

[Table 1] 番号 Strain number Colony morphology and type Colony diameter (cm 1.) Number of days of strain establishment M5009 Black, felt, surface irregularities 74 16 M5016 Black-brown, felt, surface smooth 3.91 1 M5010 Brown, felt, surface smooth 4.09 16 M5019 Gray center brown, fluff, surface smooth 4.50 16 M4005 Gray-brown, felt, surface smooth 3 0.037 M4027 Westerdykella multispora 5.487 M4006 grey-brown, felt, surface smooth 5.009 11 ─────────────────────────── ────────

The accession number of the unidentified bacteria at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (M4005) is FERM P-1.
5126, M4006 is FERM P-15127, M
5009 is FERM P-15128, M5010 is F
ERM P-15129 and M5016 are FERM P-
15130 and M5019 are FERM P-15131.

[0015]

Next, embodiments of the present invention will be described specifically. 2 crops of wheat and soybean in Shimodate City, Ibaraki Prefecture, 10
Test soil was collected from the annually grown soil on February 22, 1993. Chinese cabbage (variety: Muso) whose surface was sterilized in a plastic pot with a volume of 500 ml containing this soil
Seeds, temperature 20 ℃, humidity 80% RH, light season 1
Chinese cabbage was grown for 2 months under the conditions of 6 hours and 8 hours of dark period. Thereafter, the Chinese cabbage was collected from the soil, the tip of the root was cut to a length of 1 cm, and the root pieces were washed by a modified Harley and Waid washing method (Harley and Waid, 1955). It was left still on the separation medium. After the root pieces were allowed to stand, the colonies that had grown on the separation medium were transplanted onto the storage medium to obtain isolated strains. The separation medium and the preservation medium used had the compositions shown in Table 2. All medium materials are manufactured by Difco.

[0016]

[Table 2] ───────────────────────────── Composition ratio (g / l) Medium material ───────培 地 Separation medium Preservation medium ｍｅ Corn meal agar 8.5 8.5 Bacto agar 7.5 7.5 Malt extract 10 Yeast extract 2

As a result, 186 strains were isolated from 450 root pieces and classified into 24 taxonomic groups. The formation of spores on the medium and the high frequency of isolation among the identified strains were Fusarium spp. And Codinaea spp. No sporulation was observed on the medium, and unidentified ones were isolated at a high frequency of about 50% in total. Table 3 shows the results.

[0018]

[Table 3] 類 Classification of root fungi Separation frequency (%) ────── Ｃｏ Ascomycetes Westerdykella multispora 0.5 Actinomycetes Isolates 2.7 Basidiomycetes White sterile mycelia 0.5 Deuteromelaces spp. 2.7 Codinaea spp. 6.5 Curvularia sp. 0.5 Cylindrocarpon sp. 1 4.3 Cylindrocarpon sp. 20.5 Cylindrocarpon sp. 30.5 Drechslera sp. 0.5 Fusarium sp. 1 10.2 Fusarium sp. 2 3.5 Fusarium spp. 7.5 Pdecillomyces sp. 1.1 Penicillium spp. 1.1 Phialophola sp. 1.1 Pithomycetes sp. 1.1 Rhizoctonia zeae 1.1 Trichoderma koningi 1.6 Trichoderma hamatum 1.6 Trichoderma hartianum 1.1 Trichodermie tyre tymey tere thyme tyre thyme tyre tyme tere tyme tere tyme ti Ｚ ｍ Ｗ Ｗ ２ ２ ２ ２ ２ ２ ２ ２ Ｒ Ｒ Ｒ Ｒ Ｒ ────────────────────

Next, the unidentified fungi were classified by colony type, and one strain of each of the 24 taxonomic groups was added thereto, and the 186 isolated strains were classified into a total of 51 strains. These were used as the root-knot fungus of Chinese cabbage (Plasmodiophora).
brassicae). That is, the root fungus was cultured on a PDA medium, and surface-sterilized Chinese cabbage seeds were sown on the colony and grown at room temperature for 2 to 3 days. Thereafter, the Chinese cabbage seedlings growing on the colony were transplanted together with the colony into a plastic pot containing 200 ml of sterilized soil, and grown in a greenhouse at 20 to 25 ° C. One month after transplantation, dormant spores of the clubroot fungus of Chinese cabbage were inoculated to a concentration of 4 × 10 ⁶ / g (dry soil), and then growth of Chinese cabbage was continued under the same conditions for two months.

Thereafter, the Chinese cabbage was collected from the plastic pot, and the roots thereof were observed to determine whether or not the clubroot was developed. This test was repeated three times. Evaluation of the presence / absence of the disease is described in Yoshikawa et al. (1977
Year) according to the evaluation method, the incidence is classified into four stages of 0 to 3,
1 stage onset, 2 stages onset, 1 stage for 3 stages onset
An index from 0 to 100 was calculated by weighting each of 0, 60, and 100 points.

As a result, there were 7 outbreak indexes of 20 or less. One of them is yellow-white spore-forming spores, and from the spores Westerdykella mul
tispora. The other six are black or brown mycelia and are unidentified bacteria (Black sterile myceli) in which sporulation is not observed on the medium.
a). Colony morphology of the latter group bacteria are felted or fluffy, and 5cm or less colony diameter 3 weeks of culture on PDA medium at room temperature, many of slow growth on medium until strain established Had the feature of requiring days. The characteristics of each strain are as shown in Table 1 above. Most of these strains were re-isolated at high rates. That is, it was confirmed that the Chinese cabbage had a fixing property on the root surface. Table 4 shows the disease incidence index and the re-isolation rate of those strains. Here, the disease incidence index and the re-separation rate are as follows. Disease index = Σ (number of individuals for each index x weighted points) / total number of individuals Reseparation rate = number of re-isolated root pieces / number of root pieces left in separation medium x 100

[0022]

[Table 4] 菌 Strain number Disease index Reseparation rate (%) ────────────── ──────── H4027 050 M5016 067 M4005 3.3 83 M5009 6.7 33 M5019 6.7 67 M4006 100 M5010 20 33 Control 100 − ─────────── ───────────

Further, the roots of Chinese cabbage, which did not show any disease, were stained with 0.005% cotton blue and observed with an optical microscope. As a result, the growth of the fungus was observed in the roots. In this manner, the root bacterium identified as having a function of suppressing clubroot disease of Chinese cabbage was selected from P.
It can be obtained in a large amount by culturing a DA medium or the like. Then, by inoculating it into Chinese cabbage cultivated in a field or the like, it can be used as an effective means of controlling clubroot. How to inoculation, as described above, were seeded Chinese cabbage seeds were washed on the culture medium of the root surface fungi, germinated, methods der of obtaining seedlings for planting in the soil
You . The root fungus can be stored in a dormant state by drying or the like.

[0024]

As described above, according to the present invention, a root fungus symbiotic in the roots of Chinese cabbage, which has an inhibitory function against the clubroot of Chinese cabbage, is used. Can be controlled and controlled. In other words, it is possible to control and control root-knot disease of Chinese cabbage using the action of nature, so that it is possible to control soil-borne fungi on crops without using pesticides, and it is possible to control soil-borne disease with little effect on humans and the environment. Can be realized.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the steps of collecting and cultivating Chinese cabbage root bacilli according to the present invention and a control test thereof.

──────────────────────────────────────────────────続 き Continued on the front page Microorganism accession number FERM P-15131 (58) Field surveyed (Int. Cl. ⁶ , DB name) C12N 1/14 BIOSIS (DIALOG) JICST file (JOIS) WPI (DIALOG)

Claims (8)

(57) [Claims] 1. A symbiotic in roots Chinese cabbage in the soil, it has a suppression factor of clubroot of Chinese cabbage, colonies on medium black
It grows slowly with color or brown, and sporulation on the medium
Unacceptable Black Steril Meisseria and C
A root bacterium having an ability to inhibit a soil disease selected from the genus Estherdichella multispora. 2. The root bacterium according to claim 1, wherein the root bacterium is Black Steryl Myceria. 3. The root bacterium according to claim 1, wherein the root bacterium is Westerdichella multispora. 4. The root fungus symbiotic in the roots is isolated, cultivated and categorized from the roots of Chinese cabbage cultivated in soil, and the separated bacterial strain is inoculated into Chinese cabbage roots. Inoculate the clubroot disease in the cultivation soil,
After cultivating Chinese cabbage for the required period of root-knot disease, check for the occurrence of club-root disease in Chinese cabbage, confirm the rhizobacteria inoculated on the roots of Chinese cabbage with a low incidence of clubroot, and select Chinese cabbage. A method for producing a root fungus having an ability to inhibit soil disease. 5. The root bacterium according to claim 4, wherein the soil for cultivating the Chinese cabbage is a continuous cropping crop to isolate the root fungus symbiotic in the root from the Chinese cabbage root. Manufacturing method. 6. The method for producing a rhizobia having an ability to inhibit Chinese cabbage soil disease according to claim 4 or 5, wherein the cultured rhizobacteria, which have not been identified, are classified according to the colony type of the strain. 7. A method for controlling Chinese cabbage soil disease, wherein roots of the Chinese cabbage roots having the ability to inhibit the root-knot disease of Chinese cabbage coexist in the roots of the Chinese cabbage and have the ability to inhibit the clubroot disease of Chinese cabbage. 8. A method for controlling Chinese cabbage soil disease in which Chinese cabbage seeds are sown in a medium for cultivating Chinese cabbage root fungi, germinated, and inoculated into Chinese cabbage roots with Chinese cabbage root disease-inhibiting fungi.