JP2955642B2 - A new antagonistic bacterium SB-K88 belonging to the genus Xanthomonas and a method for growing healthy sugar beet seedlings using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a new antagonistic bacterium SB-K88 belonging to the genus Xanthomonas, which is effective for cultivating sugar beet, and a method for rearing healthy sugar beet using the new antagonistic bacterium. It relates to the control of blight and the use for promoting germination.

[0002]

2. Description of the Related Art Seedling blight is one of the important diseases of sugar beet and Rhizoctonia solani (Rhizoctonia solani) and Pishum ultimum (P
ythiumultimum), Aphanomyces cochlioides (A
phanomyces cochlioides) is known to be the main pathogen.

[0003] The occurrence of seedling wilt of sugar beet is caused by the use of chemicals such as tachigaren, lysolex, and validasin (all of which are registered trademarks, hereinafter the same) when raising seedlings using a paper pot (registered trademark, hereinafter the same). In the case where sugar beet seeds are directly sown in a field, they are controlled by mixing the seeds with cultivated soil or by irrigating the cultivated soil, or by dressing the seeds with the above-mentioned agent. On the other hand, various biological control methods have been proposed as alternatives to drugs. For example, Seikyo et al. (1988), Genetics, Vol. 42, No. 6, pp. 25-29, by antagonistic bacteria of sugar beet seeds. The results of a study on bacterization (a method of spraying microorganisms that are antagonistic to seeds or seedlings). As an effective antagonistic bacterium against the damping-off disease of seedling, Pseudomonas fluorescens.
(Pseudomonas fluorescens), Pseudomonas sputida (Pseu
domonas putida), Pseudomonas cepacia (Pseudomona
cepacia) and Bacillus sp., and among these antagonistic bacteria, those having a germination promoting effect are described. Tsukio (1993), Plant Protection, Vol. 47, No. 3, On pages 30 to 33, the addition of sugar beet seedling antagonizing bacteria to sugar beet pellet seeds (irregularly shaped sugar beet seeds coated spherically with clay minerals, etc.) was tested. It is described that Pseudomonas cepacia and Pseudomonas fluorescens have an inhibitory effect on sugar beet seedling damping-off.
RATH et al. (1992), Bulletin OILB / SROP, 15th
Vol. 1, No. 1, pp. 113-114, for biological control of seed rot and seedling blight by sugar beet antagonistic microorganisms, Pseudomonas fluorescens and the like are described as effective antagonistic microorganisms, JP-A-5-51305 discloses Bacillus subtilis (Baci
A method for controlling plant diseases by coating a plant seed with (llussubtilis) SC-3 or a mutant thereof is described.

[0004]

The above-mentioned method using a drug in the control means of the control of sugar beet seedling blight has been established and has been put into practical use with an established prescription, and provides an effective control method. However, since it is a chemical substance, it cannot be said that it is thorough in terms of drug resistance and safety or persistence to humans. In addition, since sugar beet seedling wilt tends to occur constantly, a large weight is imposed on farming costs. Inexpensive means are desired to occupy.

[0005] On the other hand, as mentioned above, various biological means have been proposed as a means of controlling with an antagonistic microorganism, but all of them remain at the research stage and still have problems that must be solved for practical use. . For example, Pseudomonas fluorescens (Pseudomonas fluorescens), which is mentioned as an antagonistic bacterium of sugar beet seedling blight in the above various studies
When Pseudomonas putida, Pseudomonas cepacia, and Bacillus subtilis SC-3 are to be used for controlling the damping-off of sugar beet, the activity of these bacteria is stable. Is an important requirement,
In view of the persistence of the activity of pyro-lunitrin, which is one of the substances produced by the bacteria of the genus Pseudomonas, there is still a problem in the stability of the activity. In order to save labor and reduce costs due to aging, in the future, direct sowing,
It is anticipated that the proportion of non-thinning cultivation will increase more and more. Therefore, it is desired to provide more effective biological control means using antagonistic microorganisms to improve germination and increase the seedling rate.

[0006]

Means for Solving the Problems In view of the above circumstances, the present inventors have conducted intensive studies on antagonistic microorganisms that are effective in controlling sugar beet seedling blight, and as a result, among the microorganisms isolated from the fine roots of sugar beet, the above-mentioned requirements were determined. The present inventors have found an antagonistic microorganism having a germination promoting effect particularly at a low temperature, and have identified it as a novel antagonistic bacterium which has not been known by identification. Based on this finding, the present inventors have conceived the present invention. Of the present invention relates to a new antagonistic bacterium S belonging to the genus Xanthomonas which does not show pathogenicity to sugar beet
The second invention relates to the use of the novel antagonistic bacterium SB-K88 of the first invention, and relates to a new antagonistic bacterium SB-K88 belonging to the genus Xanthomonas which does not show pathogenicity to sugar beet. The present invention is characterized in that seedling blight is controlled by coating K88 on sugar beet seeds. The third invention is directed to a genus Xanthomonas which also does not show pathogenicity to sugar beet. The present invention is characterized in that germination at a low temperature is promoted by coating the sugar beet seeds with a new antagonistic bacterium, SB-K88, to which the present invention belongs. A new antagonistic bacterium belonging to the genus Xanthomonas (Xanthomonas), which does not show any phenotype, is coated on sugar beet seeds and pelletized to promote germination at low temperatures. , And the As a result,
It is possible to stably control the control of sugar beet seedling blight by antagonistic microorganisms, improve germination at low temperatures, and secure seedlings with uniform growth. The present invention will be described in detail below including the operation.

The novel antagonistic bacteria effective for the pathogen of the sugar beet seedling blight of the present invention are microorganisms isolated from sugar beet fine roots cultivated in a sugar beet continuous cropping field in Sugigawa-cho, Obihiro-city, Hokkaido, and isolated by the following procedure. Things. The collected fine roots were washed with distilled water and then ground, and the ground liquid was sterilized with 10 ⁶
1 ml of the diluted solution, the medium composition was sucrose 10 g / l, casein hydrolyzate 8 g / l, yeast extract 4 g / l
l, suspended in a medium just before solidification in an agar medium (referred to as medium 1) containing 15 g / l of agar and 1000 ml of distilled water.
From the colony produced by culturing for 7 days, the medium composition was 200 g / l of potato decoction, 5 g / l of peptone, 3 g / l of glucose,
It was separated into a test tube slant medium (referred to as medium 2) containing 2 g / l of sucrose, 15 g / l of agar, and 1000 ml of distilled water.

[0008] The preservation of the isolate was carried out using sodium glutamate 1
g / l, skim milk 10 g / l, suspended in a dispersing medium of 1000 ml of distilled water, frozen and stored at −80 ° C., and in use, inoculate the preserved strain in the medium 1 and the medium 2 and cultivate statically at 25 ° C. .

The mycological properties are as follows. The mycological properties are reviewed in Bergey's Manual of Systematic Bacteriology.
Systematic Bacteriology (1984), Vol. 1, and by Takeharu Hasegawa, Classification and Identification of Microorganisms (1985), by Jikken Shuppan Center. (A) Morphology (1) Cell shape and size: 0.5 × 2.0 μm alone or in two or three straight rods (2) Flagella: no flagella (3) Sporulation: none (4) Gram staining: negative (b) Growth state The growth state when cultured on a 1.5% agar medium having the composition of the above-mentioned medium 1 at 25 ° C. for 7 days is as follows. (1) Shape: circular (2) Periphery: smooth (3) Ridge: swells. However, it is not nipple-like. (4) Surface: smooth (5) Color: yellow (c) Physiological properties (1) OF test (by Hugh Leifson method): negative (2) Growth range: optimal pH: 6.5 growth pH: 4 .
0-7.0 Optimum temperature: 28 ° C Growth temperature: 25-40 ° C (3) 5% sodium chloride: negative (4) Triphenyltetrazolium chloride 0.1
%: Negative (5) Methyl green 0.02%: d (6) Ubiquinone: Q8 (7) Lebanone productivity from sucrose: negative (8) Arginine hydrolyzability: negative (9) Oxidase reaction : Negative (10) Denitrification reaction: negative (11) Major bacterial fatty acid: 13-methyltetradecanoic acid (ic 15: 0) (12) GC (guanine, cytosine) content: 66.8% (d ) Utilization of carbon source Medium composition is ammonium sulfate 8g / l, potassium monohydrogen phosphate 0.3g / l, potassium dihydrogen phosphate 0.2g / l, magnesium sulfate heptahydrate 0.5g / l, chloride Potassium 10.2g / l,
Methionine 50 mg / l, agar 15 g / l, succinate 1 g / l, D
-A medium in which 2 g / l of galactose, 2 g / l of glucose, and 1 g / l of propionate (referred to as culture medium 3) was inoculated. (1) Utilized: glucose, trehalose, succinate (2) Unutilized: D-galactose, propionate, 2-ketogluconate, meso-inositol, L
-Valine, β-alanine, DL-arginine, hippurate

(E) Comparison with conventional analogous species Due to the above mycological properties and pathogenicity to the following plants, microorganisms that effectively antagonize the pathogens of sugar beet seedling blight of the present invention are aerobic. Is a gram-negative bacillus, has ubiquinone Q8, and the main bacterial fatty acid is a branched acid
Judging from having 13-methyltetradecanoic acid and having a GC (guanine, cytosine) content of 66.8%, it is clear that the bacterium belongs to the genus Xanthomonas, and its pathogenicity to plants and D -Xanthomonas campestris (Xanthomonas campestris) was negative because both galactose and propionate were negatively assimilated.
homonas campestris), and that the colony is not umbonate in shape, sodium chloride 4
% And triphenyltetrazolium chloride 0.
It is not a Xanthomonas maltophilia because it cannot grow at 1%. Therefore, it is judged to be a new strain belonging to the genus Xanthomonas, and Xanthomonas espi (Xanthomonas sp.)
s sp.) SB-K88. This new antagonistic bacterium has been deposited with the National Institute of Advanced Industrial Science and Technology as a FERM P-13821 on August 23, 1993.

(F) Culture method Xanthomonas esp.
s sp.) The culture of SB-K88 is basically the same as that of general bacteria, except that the above-mentioned medium 1, medium 2 and medium 3
It grows well in any of the above.

(G) Pathogenicity to Plants Proteose Peptone # 3 (Difco) 20.0 g / l, potassium monohydrogen phosphate 1.5 g / l, magnesium sulfate heptahydrate 1.5 g / l, glycero- The tip of a fingernail was pierced into a colony cultured at 25 ° C. for 3 days in a medium containing 15 ml of agar, 15.0 g / l of agar, and 1000 ml of distilled water to adhere bacteria, and the tip was radish and sugar beet seedlings. Radish, Chinese cabbage, cabbage, Sirona, Taina, Taasai, Chingensai and sugar beet are pierced and inoculated into the front and back leaf veins of each leaf, placed in a moist chamber and allowed to stand in a 23 ° C sunlight incubator, Xanthomonas campestris with known pathogens with known pathogens
homonas campestris pv.campestris), the Xanthomonas sp.
(Xanthomonas sp.) No pathogenicity was observed in SB-K88.

Next, the screening of the new antagonistic bacterium Xanthomonas sp. SB-K88 (hereinafter simply referred to as "SB-K88"), the suppression of sugar beet seedling blight and the promotion of germination are described above. Will be described with reference to test examples.

Test Example-1 (Screening of SB-K88) 374 strains of 374 strains isolated from sugar beet fine roots cultivated in a sugar beet continuous cropping field in Sugigawa-cho, Obihiro-city, Hokkaido
Sugar beet single embryo seed monoace S (variety, the same applies hereinafter) obtained by coating each of the cells cultured under the same conditions for one day is placed in a clay pot (diameter 12 cm, height 12 cm) filled with naturally contaminated soil. A seedling test was conducted in which 13 seeds per pot were sowed and seedlings were raised in a greenhouse for 2 weeks. As a result, 41 strains showing suppression of seedling blight were selected. From these 41 strains, the same seedling raising test as described above was repeated, and 11 strains showing high reproducibility in suppressing the establishment of seedling blight were selected.

Each of the 11 strains selected as described above was coated in the same manner as above with the sugar beet single-embryo seeds, and the naturally contaminated soil alone and Rhizoctonia solani (Rhizoctonia solani), a major pathogen of sugar beet seedling blight, were used. ) A
G-4, Pythium ultimum,
Aphanomyces coch
lioides) (light black and black soil)
Paper pot (diagonal diameter 0.9cm, height 13)
(a hexagonal column of 1 cm) was seeded at a rate of 1 grain / pot, and a seedling raising test was conducted in a vinyl house for 35 days. The test results were obtained by sowing the above seeds without the coating of bacterial cells on each of the above soils (no treatment), and adding the same seeds to each of the above soils by adding tatigalen (powder) and PCNB (powder) to the respective active ingredients. 50
Table 1 shows, as a control, the case where the seeds were sown on soil mixed with ppm (drug treatment).

[0016]

[Table 1]

In Table 1, each numerical value indicates the degree of seedling wilt, the degree of illness was investigated for each individual by using a 4-stage sickness index of 0 to 2, and the sickness was determined by the following equation. . The results were evaluated by Duncan's multiple test.

Disease index Index Criteria 2 Pre-emergence mortality 1 Establish post-emergence mortality 0.5 Mild (not mortal but affected) 0 Healthy illness Degree = ｛[sum of (individual body × index)] / (incidence index 2
X total population) x 100

According to the results, it was found that SB-K88 showed as good a control as the control of the chemical application in all of the soil naturally contaminated and the soil inoculated with the pathogenic bacteria. As for the remaining strains, some suppression was observed in some strains depending on the inhabited or inoculated pathogens, but it was far below that of SB-K88, and SB-K88 was effective as a strain effective for controlling sugar beet seedling wilt. That is, the novel antagonistic bacterium SB-K88 of the present invention.
Was selected as the most effective strain.

Test Example 2 (Concentration and Inhibition of SB-K88) The effective concentration of SB-K88 selected in Test Example 1 above was determined by using a naturally contaminated soil in the manner of the above-mentioned seedling raising test using a paper pot. Done. The concentration of bacterial cells coated on the seeds was 10 ⁶ , 10 ⁷ , 10 ⁸ cfu / grain seed (cfu: colony for
ming unit) and set 30 individuals per test group (1 grain /
(Pot) four times. The results are shown in Table 2 in the same manner as in Test Example 1 except that no treatment and drug treatment were used as controls.

[0021]

[Table 2]

From Table 2, it can be seen that the suppression tends to be enhanced as the bacterial cell concentration is increased, and almost no suppression can be expected at a concentration of 10 ⁶ cfu / one grain seed. 10 ⁷ cfu / 1 grain seed or more, preferably 10 ⁸ cfu
It was found that more than one seed was at a practical concentration.

In the above, the novel antagonistic bacterium SB-K8 of the present invention is described.
No. 8 was found to be an effective microorganism for controlling sugar beet seedling blight, but such an inhibitory effect was achieved by coating the seeds of sugar beet with SB-K88, and the treatment was carried out as described above. It may be simply adhered to the seed as in the test example, or may be processed into pellets coated with an appropriate coating agent after being adhered. What is necessary is just to select suitably according to conditions.

[0024] It is not clear what mechanism the favorable control of the establishment of seedling blight by the new antagonistic bacterium SB-K88 of the present invention depends on the mechanism of action, but the bacterial cells coated on the seeds are found in the rhizosphere of sugar beet seedlings. Future research awaits whether it will proliferate enough to overwhelm the pathogen and protect it from infection or activity, or whether it will produce some intense antimicrobial substances that will kill or inactivate the pathogen itself. However, it is significant that the provision of the novel antagonistic bacterium SB-K88 of the present invention greatly improves cultivation of sugar beet. In addition, it is known that the new antagonistic bacterium SB-K88 of the present invention is not only effective in controlling the above-mentioned blight of the sugar beet, but also has an effect of promoting germination especially at a low temperature (around 15 ° C.). Was. In this case, it was found that when the seeds were subjected to a coating treatment and further processed into pellets with a coating agent, the seeds tended to be better.
This will be described in Test Example 3.

Test Example-3 (Sprouting by Sugar Beet Seed Treatment) In the breeding of sugar beet, the initial germination 3 to 5 days after the start of the seedling is an important requirement for the completion of the seedling after harvesting. Establish conditions where initial germination is as high as possible. Therefore, it was examined how SB-K88 is involved in early germination.

After SB-K88 isolated from the cryopreserved strain was cultured in the above-mentioned medium 3 for 3 days, distilled water was added to the cells washed by centrifugation so that the concentration of the cells became 10 ⁷ cfu / one seed. The suspension was prepared by adding 40 g (approximately 4000 seeds) of sugar beet single germ seed monoace S (original seed) to the suspension, after which SB-K88 was adhered to the suspension and air-dried.
-K88 coated seeds were prepared. This SB-K88
The coated seeds were divided into two parts, one of which was SB-K88.
In the pot type granulator (specification: 44 cm in diameter, 14 cm in depth, flat bottom, spherical rotary pot with opening of 14 cm on top, 30 rpm, rotation angle 30-40 degrees)
The mixture is supplied in an amount of 0 g, and while being rotated, a mixed powder of water, a coating agent mainly composed of silicates, and a binder mainly composed of PVA (water-soluble glue) is sprayed, so that the particle diameter becomes approximately 4.5 mm. This was taken out and air-dried to prepare a seed treated with SB-K88 coat + pellet.

Separately, pellet-treated seeds are prepared by pelletizing the above-mentioned original seeds by the above-mentioned method, and the three forms of seeds and the original seeds prepared here are subjected to a filter paper method (official method).
The germination rate on the third day after the start of the germination test was measured under the conditions of a set temperature of 15 ° C. and 25 ° C., 100 grains per group, and 4 repetitions. Table 3 shows the results.

[0028]

[Table 3]

From Table 3, the germination rate by treatment is considered.
When the germination set temperature is 15 ° C., the germination rate of the pellet-treated seed is 24.3 as a real number based on the germination rate of the original seed.
% Of the germinability increase, as well as an 8.0% improvement in the germination increase of the SB-K88 coat-treated seeds.
In the case of the seed treated with -K88 coat and the pellet, an improvement in the germinability by 46.3% was observed. The 46.3% improvement in germinability of the SB-K88 coat + pellet-treated seeds included an improvement in the former two, but combined with an improvement in the former two. The value is 32.3%, which greatly exceeds this. From this, SB-K88
The improvement in the germinability of the seeds treated with the coating and pellets can be recognized as a result of the synergistic effect of the two treatments, SB-K88 coating and pelletizing.

On the other hand, when the set germination temperature is 25 ° C.,
The temperature factor has a large effect, and it is almost independent of the treatment type.
The germination rate was on the order of 0%, and almost no improvement in germination by SB-K88 coating treatment and pelletization was observed. For this reason, the SB-K88 coating and pelletizing treatment of sugar beet seeds provides an effective means when raising seedlings in a low-temperature environment such as 15 ° C., for example, in winter or early spring.

As described in detail above, the novel antagonistic bacterium SB belonging to the genus Xanthomonas of the present invention
-K88 is used for cultivation of sugar beet to control seedling blight, which is an important disease, and to promote early germination of seedlings in a low temperature environment to bring about uniform germination. It is a very useful microorganism.

[0032]

【Example】

In the manner of Example 1 in Test Example 3, the concentration 10 ⁷ cfu / l grain seeds SB-
K88 was coated on sugar beet single embryo seed monoace S, air-dried and pelletized to form SB-K88 coat with a particle size of 4.3 to 4.6 mm + pellet seed 20 g (about 20 g).
00), and as a control, a particle size of 4.3 to 4.6 obtained by simply pelletizing the sugar beet single embryo seed monoace S.
Approximately 2,000 pellets in the mm range were prepared.

A group of 50 hexagonal pillar-shaped paper pots having a diagonal diameter of 19 mm and a height of 130 mm is defined as A, B,
C. Three repetitions of the three sections were prepared, and the soil of the sugar beet cultivation field in which the pathogen of the sugar beet seedling blight was found was filled into each of the paper pots of the three sections by a conventional method. To form an approximately 15 mm cover soil space at the top,
In the plot, the above-prepared SB-K88 coat + pellet seeds were sown, and in the plots B and C, the pellet seeds obtained by simply pelleting were sown at a rate of 1 grain / pot on the compacted soil.
The soil of the sugar beet cultivation field was covered in the soil cover space of the ward and the ward C, and the drug-treated soil obtained by mixing 1 g of the soil of the sugar beet cultivation field with 18 g of the drug Resolex H (dust) was used in the soil cover of the field B. Seeding was completed, and the seedlings were grown under the same conditions in a vinyl house under the usual irrigation control.

Germination was observed from the 5th day after the start of raising seedlings.
The day was almost complete. Thereafter, seedlings were continued until the 40th day, and the seedlings were measured. From the 8th day to the completion of the seedling raising, the occurrence of seedling damping was observed. Early bud rate on day 8, 1
Table 4 shows the survey results of the total germination rate on the fourth day and the seedling yield on the 40th day (% of healthy seedlings based on the number of seeds).

[0035]

[Table 4]

The section A in which the SB-K88 coat + pellet seeds were sown had better initial emergence and better total emergence than the other two sections, and the seedling yield was as high as 90%. recognized.
The section B in which the soil covering was treated with chemicals slightly outperformed the section C in which no covering soil was treated, but the result was that the seedling yield was about 30% lower than that of the section A by a real number.

[0037]

According to the present invention, the new antagonistic bacterium SB-K88 belonging to the genus Xanthomonas can be cryopreserved, and a part thereof can be taken out and easily grown for use, so that it is easy to handle. It can be used simply by coating the grown new antagonistic bacteria SB-K88 on sugar beet seeds or by a simple treatment of pelletizing after coating, and is easy to handle. By sowing the seeds coated with SB-K88, healthy seedlings that are constantly germinated are obtained, and this results in the harvest of sugar beet of good quality. This is extremely useful for growing and cultivating healthy seedlings of sugar beet. is there.

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Mitsuru Sayama 1st farm shelter, Hitsugaoka, Toyohira-ku, Sapporo, Hokkaido (58) Field surveyed (Int.Cl. ⁶ , DB name) C12N 1/20 A01G 7/00 A01C 1/06 BIOSIS (DIALOG)

Claims (4)

(57) [Claims] 1. A new antagonistic bacterium SB-K88 belonging to the genus Xanthomonas or a culture thereof, which does not exhibit pathogenicity against sugar beet but has an antagonistic action against the pathogen of seedling blight of sugar beet. 2. A new antagonistic bacterium SB-K88 belonging to the genus Xanthomonas, which does not show pathogenicity against sugar beet but has an antagonistic action against the pathogen of seedling blight of sugar beet, and / or a culture thereof. A sugar beet seed that has been coated, or a pellet-treated sugar beet seed obtained by further processing the sugar beet. 3. A new antagonistic bacterium SB-K88 belonging to the genus Xanthomonas, which does not show pathogenicity to sugar beet but has an antagonistic action against the pathogen of seedling blight of sugar beet, and / or a culture thereof. A method for cultivating a healthy sugar beet seedling, comprising coating a sugar beet seed or, further, pelletizing the sugar beet seed to control the sugar beet seedling damping-off. 4. A new antagonistic bacterium SB-K88 belonging to the genus Xanthomonas and / or a culture thereof, which does not show pathogenicity to sugar beet but has an antagonism to the pathogen of sugar beet seedling blight. A method for cultivating healthy sugar beet seedlings, comprising germination is promoted by coating sugar beet seeds or by further pelletizing the sugar beet seeds.