JPH0775562A - Novel antagonistic bacterium sb-k88 belonging to genus xanthomonas and method for growing healthy beet seedling using the bacterium - Google Patents

Abstract

PURPOSE:To provide a novel microorganism useful for the growth of healthy seedling of beet and a process for the use of the microorganism. CONSTITUTION:This novel antagonistic bacterial strain SB-K88 is useful for the growth of healthy beet seedling, free from pathogenicity to beet and belongs to the genus Xanthomonas. This invention also relates to its cultured product and its treated material. The damping-off of seedling can be controlled and the initial germination is improved to enable the growth of a healthy seedling by treating seed of beet with the novel antagonistic bacterium SB-K88, its cultured product or its treated material.

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 effective for the cultivation of sugar beet and a method for growing beet seedlings using the new antagonistic bacterium, and particularly to the establishment of seedlings of sugar beet. Use for controlling blight and promoting germination.

[0002]

2. Description of the Related Art Rhizoctonia solani, which is a soil fungus that lives in the soil, and Pishum ultimum (P
ythiumultimum), Aphanomyces cochlioides (A
It is known that phanomyces cochlioides) is the main pathogen.

The occurrence of seedling blight of sugar beet is caused by the use of drugs such as Tachigaren, Risolex, and Validacin (all are registered trademarks, the same applies hereinafter) in the case of raising seedlings using a paper pot (registered trademark, the same applies below). Is mixed with the soil or irrigated with the soil, and in the case where the sugar beet seeds are directly sown in the field, the seeds are coated with the above-mentioned chemical agent for controlling. On the other hand, various biological control methods as alternatives to drugs have been proposed, for example, in Sakoshi et al. (1988), Genetics, Vol. 42, No. 6, pp. 25-29, using sugar beet seed antagonist bacteria. Based on the results of research on bacteriosis (a method of sprinkling seeds or seedlings with an antagonistic microorganism), C. fluorescens as an effective antagonistic bacterium against the pathogen of seedling wilt disease.
(Pseudomonas fluorescens), Pseudomonas putida (Pseu
domonas putida), Pseudomonas (Pseudomona)
s cepacia), Bacillus sp. (Bacillus sp.), and it is described that some of these antagonistic bacteria have a germination promoting effect, Tsukio (1993), Plant Protection, Volume 47, No. 3, On pages 30 to 33, addition of sugar beet pellet seeds (irregularly shaped sugar beet seeds spherically coated with clay minerals, etc.) to the addition of antagonistic bacteria for seedling wilting was tested. Pseudomonas cepacia and Pseudomonas fluorescens have been described to have an effect of suppressing sugar beet seedling blight, and MONIKA
RATH et al. (1992), Bulletin OILB / SROP, 15th
Volume, No. 1, pp. 113 to 114, for biological control of seed rot and seedling wilting by antagonistic microorganisms of sugar beet, Pseudomonas fluorescens, etc. are described as effective antagonistic microorganisms, [Patent Document 1] JP-A-5-51305
llus subtilis) SC-3 and its mutants are described as a method for controlling plant diseases by coating plant seeds with a coat.

[0004]

The above-mentioned method using a drug in the means for controlling sugar beet seedling wilt has been put into practical use with established application prescriptions and the like, but it provides an effective control method. However, since it is a chemical substance, it cannot be said to be perfect in terms of drug resistance and safety or persistence to humans and animals. Moreover, since sugar beet seedling wilting tends to occur constantly, a large weight is given to farming costs. Inexpensive means to occupy are desired.

On the other hand, various biological means have been proposed as means for controlling antagonistic microorganisms as described above, but all of them remain at the research stage and still have problems to be solved for practical use. . For example, Pseudomonas fluorescens, which has been cited as an antagonistic bacterium of sugar beet seedling wilt in the above-mentioned various studies.
, Pseudomonas putida, Pseudomonas cepacia, and Bacillus subtilis SC-3 are used for controlling sugar beet seedling blight, the activity of these bacteria is stable. Is an important requirement,
Pyrrolnitrin, which is one of the substances produced by the bacterium of the genus Pseudomonas, is used as a mercumal, and there is a problem in the stability of the activity even if it is seen from the sustainability of the activity. Direct seeding in the future for labor saving and cost reduction due to aging
It is predicted that the proportion of non-thinning cultivation will increase more and more. Therefore, it is desired to provide a more effective biological control means by antagonistic microorganisms that improves germination and increases seedling yield.

[0006]

[Means for Solving the Problems] In view of the above circumstances, the inventors of the present invention have earnestly studied an antagonistic microorganism that is effective in controlling seedling wilting of sugar beet, and as a result, have found that the above-mentioned requirement in the microorganism isolated from the fine root of sugar beet. In particular, the inventors have found an antagonistic microorganism that also has a germination-promoting effect also at low temperature, and identified it as a novel antagonistic bacteria that has not been known so far, and arrived at the present invention based on this finding. Of the invention is a new antagonistic bacterium S belonging to the genus Xanthomonas which is not pathogenic for sugar beet.
B-K88 itself, the second invention relates to the use of the new antagonistic bacterium SB-K88 of the first invention, which is a new antagonistic bacterium SB- belonging to the genus Xanthomonas that does not show pathogenicity to sugar beet. The present invention is characterized by controlling seedling blight by coating K88 on sugar beet seeds. The third invention also relates to the genus Xanthomonas which is not pathogenic to sugar beet. The present invention is characterized in that sugar beet seeds are coated with a new antagonistic bacterium belonging to the group, SB-K88, to promote germination at low temperature. The fourth invention is pathogenicity to the sugar beet of the third invention. A new antagonistic bacterium SB-K88 belonging to the genus Xanthomonas that does not show the characteristics of sugar beet seeds is coated and further pelletized to promote germination at low temperature. , And the As a result,
It is possible to stably control the seedling wilt of sugar beet by the antagonistic microorganisms and improve germination at low temperature to secure seedlings with uniform growth. The present invention will be described in detail below, including its operation.

A novel antagonistic bacterium effective for the pathogen of sugar beet seedling wilt of the present invention is a microorganism isolated from sugar beet rootlets cultivated in a sugar beet continuous cropping field in Suenokawa-cho, Obihiro City, Hokkaido, and isolated by the following procedure. It is a thing. The collected fine roots were washed after triturated with distilled water, trituration liquid in sterile water 10 ⁶
Dilute to about 1 ml and dilute 1 ml with medium composition of sucrose 10 g / l, casein hydrolyzate 8 g / l, yeast extract 4 g / l.
l, agar 15g / l and distilled water 1000ml agar medium (medium 1) suspended in a medium just before solidification, after solidification 25 ℃
From the colonies 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,
Sucrose (2 g / l), agar (15 g / l), and distilled water (1000 ml) were separated into a test tube slope medium (referred to as medium 2).

Preservation of isolates is sodium glutamate 1
After suspension in a dispersion medium of g / l, skim milk 10 g / l, and distilled water 1000 ml, it is frozen and stored at −80 ° C. In use, the preserved strain is planted in the medium 1 and the medium 2 and statically cultured at 25 ° C. .

The mycological properties are as follows. In addition, the examination of mycological properties was carried out by Bergey's Manual of Systematic Bacteriology.
Systematic Bacteriology (1984), Vol. 1, and Takeharu Hasegawa, Classification and Identification of Microorganisms (1985), Academic Society Publishing Center. (A) Morphology (1) Cell shape and size: 0.5 × 2.0 μm alone or in 2 to 3 continuous rods (2) Flagella: No polar flagella (3) Sporulation: None (4) Gram stain: Negative (b) Growth state The growth state when cultured in a 1.5% agar medium having the composition of the medium 1 at 25 ° C. for 7 days is as follows. (1) Shape: Circular (2) Edge: Smooth (3) Uplift: Raised. However, it is not a nipple (umbonate). (4) Surface: Smooth (5) Color tone: Yellow (c) Physiological properties (1) OF test (by Hugh Leifson method): Negative (2) Growth range: Optimum pH: 6.5 Growth pH: 4 ．
0-7.0 Optimum temperature: 28 ° C Growth temperature: 25-40 ° C (3) Sodium chloride 5%: Negative (4) Triphenyl tetrazolium chloride 0.1
%: Negative (5) Methyl green 0.02%: d (6) Ubiquinone: Q8 (7) Lebanone productivity from sucrose: Negative (8) Arginine hydrolysis: Negative (9) Oxidase reaction : Negative (10) Denitrification reaction: Negative (11) Major cell fatty acid: 13-methyl tetradecanoic acid (i-c15: 0) (12) GC (guanine, cytosine) content: 66.8% (d ) Assimilation 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 10.2 g / l potassium,
Methionine 50mg / l, Agar 15g / l, Succinate 1g / l, D
-Galactose 2 g / l, glucose 2 g / l, propionate 1 g / l medium (referred to as medium 3) were inoculated, and those showing growth after culturing at 30 ° C were regarded as positive. (1) Assimilated: Glucose, trehaloose, succinate (2) Not assimilated: D-galactose, propionate, 2-ketogluconate, mesoinositol, L
-Valine, β-alanine, DL-arginine, hippurate

(E) Comparison with Native Similar Species Due to the above-mentioned mycological properties and pathogenicity to the following plants, the microorganisms of the present invention which have an effective antagonistic effect on the pathogen of sugar beet seedling wilt are aerobic. Is a Gram-negative bacillus, has ubiquinone Q8, and the main microbial fatty acid is branched acid
Judging from the fact that it has 13-methyl tetradecanoic acid and the content of GC (guanine, cytosine) is 66.8%, it is clear that it is a bacterium belonging to the genus Xanthomonas. -Galactose and propionate are both negative in assimilation, so that Xanthomonas campestris (Xant
Unlike homonas campestris), the shape of the colony is not papillary (umbonate), sodium chloride 4
% And triphenyl tetrazolium chloride 0.
Since it is not Xanthomonas maltophilia from the point that it cannot grow at 1%, it was judged to be a new strain belonging to the genus Xanthomonas, and Xanthomonas espi
s sp.) SB-K88. This new antagonistic bacterium was deposited as FERM P-13821 on August 23, 1993 at the Institute of Biotechnology, Institute of Biotechnology, AIST.

(F) Culturing method Xanthomona named above
The culture of S. sp.) SB-K88 is basically the same as that for general bacteria, except that the above-mentioned medium 1, medium 2 and medium 3 are used.
It grows well in both cases.

(G) Pathogenicity against plants Proteose-peptone # 3 (Difco) 20.0 g / l, potassium monohydrogen phosphate 1.5 g / l, magnesium sulfate heptahydrate 1.5 g / l, glycero- 15 ml of agar, 15.0 g / l of agar, and 1000 ml of distilled water at 25 ° C for 3 days, a colony was cultivated for 3 days at the tip of the picking tip of the nail to attach the fungus, and the tip was radish and sugar beet seedlings. Inoculate by piercing the front and back leaf veins of the leaves of radish, Chinese cabbage, cabbage, sylona, taina, taa sai, bok choy and sugar beet leaves, put them in a humid chamber and leave them in a 23 ° C sunlight incubator, Xanthomonas campestris of known pathogenicity Xanthomonas campestris (Xant
homonas campestris pv. campestris), the result was that the Xanthomonas spirit
No pathogenicity was observed in (Xanthomonas sp.) SB-K88.

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

Test Example-1 (Screening of SB-K88) 3 strains of 374 strains isolated from sugar beet rootlets cultivated in a sugar beet continuous cropping field in Sugawara-cho, Obihiro City, Hokkaido, were used in Medium 3 described above.
Biscuit pots (diameter 12 cm, height 12 cm) filled with naturally contaminated soil of sugar beet single embryo seed monoace S (cultivar, same hereafter) obtained by coating each of the cells cultured under the same conditions for 1 day. As a result of a seedling raising test in which 13 seeds per pot were sown and seedlings were raised in a greenhouse for 2 weeks, 41 strains showing suppression of seedling wilt disease were selected. From these 41 strains, the same seedling raising test as described above was further repeated, and 11 strains showing high reproducibility in suppressing the onset of bacterial wilt disease were selected.

Each of the 11 strains selected as described above was coated with sugar beet single embryo seeds in the same manner as described above to isolate naturally contaminated soil alone and Rhizoctonia solani (Rhizoctonia solani), which is a major pathogen of sugar beet seedling wilt disease. ) A
G-4, Pythium ultimum,
Aphanomyces coch
lioides) inoculated in each of the sandy loam soils (light black my soil)
Filled with paper pot (diagonal diameter 0.9 cm, height 13
1 seed / pot was sown in a hexagonal column (cm) and a seedling raising test was carried out in a vinyl house for 35 days. When the above-mentioned seeds without the coat treatment of the bacterial cells were sown in each of the above-mentioned soils (untreated), and the seeds were added to each of the above-mentioned soils as tachygalene (powder) and PCNB (powder) as active ingredients. Fifty
Table 1 shows the case of sowing in soil mixed with ppm (chemical treatment) as a control.

[0016]

[Table 1]

In Table 1, each numerical value shows the morbidity of seedling wilt disease, and the degree of morbidity was investigated for each individual with a four-stage disease index of 0 to 2, and the morbidity was determined by the following formula. . The results were judged by Duncan's multiple test.

Onset index Judgment criteria 2 Pre-emergence withering dying 1 Post-emergence withering dying 0.5 Mild (no dying but ill) 0 Healthy Degree = {[(individual body x index) total] / (Sickness index 2
X total number of individuals)} x 100

According to the results, SB-K88 was found to have the same good suppression of drug application in all naturally contaminated soils and soils inoculated with pathogens. Regarding the remaining strains, some strains were observed to be suppressed to some strains depending on the inhabited or inoculated pathogens, but they were far below those of SB-K88, and SB-K88 was effective as a strain effective for controlling sugar beet seedling blight. That is, the new antagonistic bacterium SB-K88 of the present invention
Was selected as the most effective strain.

Test Example-2 (Concentration and Suppression of SB-K88) Regarding the effective concentration of SB-K88 selected in Test Example-1 above, naturally contaminated soil was used in the same manner as the seedling raising test in the above paper pot. I did. The concentration of bacterial cells coated on the seed was 10 ⁶ , 10 ⁷ , 10 ⁸ cfu / 1 grain seed (cfu is colony for
ming unit), and 30 animals (1 capsule /
4 times). The results are shown in Table 2 with no treatment and drug treatment as controls, as in Test Example 1 above.

[0021]

[Table 2]

It can be seen from Table 2 that the inhibition tends to be strengthened as the cell concentration is increased. Almost no inhibition can be expected with a concentration of 10 ⁶ cfu / 1 seed, and the same inhibition can be expected with the drug treatment. 10 ⁷ cfu / 1 seed or more, preferably 10 ⁸ cfu
It was known that the practical concentration was 1 seed or more.

In the above, the new antagonistic bacterium SB-K8 of the present invention is used.
It was revealed that No. 8 is an effective microorganism for controlling the seedling wilt of sugar beet, and such inhibitory effect was achieved by coating SB-K88 on sugar beet seeds, and the treatment was carried out as described above. It may be simply attached to the seed as in the test example, or may be processed into pellets coated with a further suitable coating agent after being attached, and the suppression is not affected by the mode of treatment, and cultivation It may be appropriately selected according to the conditions.

Although it is not clear what mechanism of action the successful suppression of seedling wilt pathogenesis by the novel antagonistic bacterium SB-K88 of the present invention is, the bacterial cells coated on the seeds are in the rhizosphere of sugar beet seedlings. Whether further growth is sufficient to outweigh the pathogens to protect them from infection and activity, or to produce some kind of intense antibacterial substance to kill or inactivate the pathogens themselves, is left for future research. However, it is significant that the provision of the new antagonistic bacterium SB-K88 of the present invention greatly improves the cultivation of sugar beet. It is known that the new antagonistic bacterium SB-K88 of the present invention is not only effective for controlling seedling wilting of sugar beet, but also has an action of promoting germination especially at low temperature (around 15 ° C). It was Then, in this case, it was observed that the seeds tended to be better when they were coated and further processed into pellets with a coating agent.
This will be described in Test Example 3.

Test Example-3 (germination of sugar beet seeds by treatment) In the seedling raising of sugar beet, the initial germination on the 3rd to 5th day after the start of seedling raising is an important requirement for the subsequent completion of seedlings and the harvest of sugar beet. , Set the condition that the initial germination is as high as possible. Therefore, it was examined how SB-K88 participates in early germination.

SB-K88 separated from the cryopreserved strain was cultured in the above-mentioned medium 3 for 3 days, and then distilled water was added to the washed bacterial cells so that the bacterial cell concentration would be 10 ⁷ cfu / 1 seed. A suspension of cells was prepared by adding 40 g (about 4000 seeds) of sugar beet single embryo seed monoace S (original seed) to SB-K88, which was taken out and air-dried to give SB.
-K88 coat treated seeds were prepared. This SB-K88
Coat treated seeds were divided into two and one of them was SB-K88
2) in a pot-type granulator (specification: diameter 44 cm, depth 14 cm, flat bottom, spherical rotary pot with 14 cm opening at the top, 30 rpm, rotation angle 30-40 degrees)
It is supplied in an amount of 0 g, and while being rotated, sprinkles water, a mixed powder of a coating agent mainly composed of silicates and a binder mainly composed of PVA (water-soluble glue), and the particle size becomes about 4.5 mm. Granules were taken up, taken out, and air-dried to prepare SB-K88 coat + pellet-treated seeds.

Separately, pellet-treated seeds obtained by pelletizing the above-mentioned original seeds by the above-mentioned method were prepared, and the three types of seeds and original seeds prepared here were prepared by the paper method (official method).
The germination rate was measured on the third day after the start of the germination test by setting the conditions of set temperature of 15 ° C., 25 ° C., 100 grains in 1 section, and 4 repetitions. The results are shown in Table 3.

[0028]

[Table 3]

From Table 3, considering the germination rate by treatment,
When the germination setting temperature is 15 ° C., the germination rate of the pellet-treated seeds is 24.3 in terms of the actual germination rate based on the germination rate of the original seed.
% Improvement in germination as well as 8.0% improvement in germination in SB-K88 coated seeds.
-K88 coat + pellet-treated seeds showed an improvement of 46.3% increase in germination. The improvement of 46.3% increase in germination resulting from SB-K88 coat + pellet-treated seeds included the improvement of germination increase of the former two, but the improvement of germination increase of the former two was added. The value is 32.3%, which greatly exceeds this value. From this, SB-K88
The improvement in germination increase caused by the coat + pellet-treated seeds can be recognized as a result of the synergistic effect of the two treatments of SB-K88 coat and pelletization.

On the other hand, when the germination set temperature is 25 ° C.,
The temperature factor has a large effect, and it is almost irrelevant to each treatment.
The germination rate was in the 0% range, and almost no improvement in germination due to SB-K88 coat treatment or pelletization was observed. From this, the SB-K88 coat and pelletizing treatment of sugar beet seeds provide an effective means for raising seedlings in a low temperature environment such as 15 ° C., for example, in winter or early spring.

As described in detail above, the new antagonistic bacterium SB belonging to the genus Xanthomonas of the present invention
-K88 is used for the cultivation of sugar beet to control seedling wilt, which is an important disease, and also promotes early germination in seedlings in a low temperature environment to bring about germination evenness. Above all, it is a very useful microorganism.

[0032]

【Example】

Example 1 SB-at a concentration of 10 ⁷ cfu / l grain seed was prepared in the same manner as in Test Example 3 above.
K88 was coated with sugar beet single embryo seed monoacet S, air-dried and pelletized, and SB-K88 coat + pellet seed 20 g (about 20 g) having a particle size range of 4.3 to 4.6 mm was prepared.
Particle size of 4.3 to 4.6 prepared by simply pelletizing sugar beet single embryo seed monoace S as a control.
About 2000 pellet seeds in the mm range were prepared.

50 hexagonal column-shaped paper pots having a diagonal diameter of 19 mm and a height of 130 mm are defined as one section, which are designated as A, B, and
Three replicates of 3 wards of C were prepared, and the soil of the sugar beet cultivation field in which the pathogen of the sugar beet seedling wilting disease was found was filled in each of the 3 wards' paper pots by a conventional method, and compression was performed. To form an approximately 15 mm soil cover space at the top,
SB-K88 coat + pellet seeds prepared as described above were sown in the plots, and pellet pellets that were simply pelletized were sown at 1 grain / pot in the B and C plots, respectively.
The soil of the sugar beet cultivation field is covered in the soil covering space of the ward and the C ward, and the chemical treatment soil in which 1 g of the soil of the sugar beet cultivating field is mixed with 18 g of the drug Resolex H (powder) is covered in the soil covering space of the ward B. Then, sowing was completed, and seedlings were grown under the same irrigation control in the vinyl house under the conventional irrigation control.

Germination was observed from the 5th day after the start of seedling raising,
On the day of the day, they were almost all present. After that, the seedlings were continuously grown until the 40th day, and the adult seedlings were measured. Occurrence of seedling wilt was observed between the 8th day and the completion of seedling raising. Initial germination rate on day 8, 1
Table 4 shows the examination results of the total emergence rate on the 4th day and the seedling yield rate on the 40th day (% of healthy seedlings to the number of seeds).

[0035]

[Table 4]

Group A, in which SB-K88 coat + pellet seeds were sown, had a better initial germination and a better total germination than the other two groups, and the seedling yield was 90%. Admitted.
The B-district where the soil covering was treated with a chemical was slightly superior to the C-district where the soil covering was not treated, but the seedling yield was 30% lower than that of the A-district by a real number.

[0037]

Industrial Applicability According to the present invention, the new antagonistic bacterium SB-K88 belonging to the genus Xanthomonas can be cryopreserved, and a part of it can be taken out and easily propagated before use, so that it is easy to handle. The above-mentioned new antagonistic bacterium SB-K88 may be simply treated by coating the seeds of sugar beet with the proliferated new antagonistic bacterium, or by a simple treatment of pelletizing after the coating, which is easy to handle. Sowing seeds coated with SB-K88 results in healthy seedlings that are always germinated, which results in good quality sugar beet harvest, which is extremely useful for growing and growing healthy seedlings of sugar beet. is there.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor, Katsukazu Kanzawa, 19 West 9th Line, Minami Line 9, Inada-cho, Obihiro, Hokkaido (72) Inventor, Mitsuru Sayama, No. 1, Hitsujigaoka, Toyohira-ku, Sapporo, Hokkaido

Claims (4)

[Claims] 1. A new antagonistic bacterium SB- which belongs to the genus Xanthomonas and which is not pathogenic for sugar beet.
K88, its culture or its processed product. 2. Sugar beet seedling blight is controlled by coating a new antagonistic bacterium SB-K88 belonging to the genus Xanthomonas, a culture thereof or a treated product thereof to a sugar beet seed. Healthy seedling raising method. 3. A new antagonistic bacterium SB-K88 belonging to the genus Xanthomonas, a culture thereof or a treated product thereof is coated on sugar beet seeds to promote germination, thereby promoting germination of healthy sugar beet seedlings. Method. 4. A new antagonistic bacterium SB-K88 belonging to the genus Xanthomonas, a culture thereof or a treated product of sugar beet seeds coated with sugar beet seeds is pelletized to promote early germination at low temperature. A method for raising healthy seedlings of sugar beet, which comprises: