JPH04179475A - Controlling agent for soft rot and control of soft rot - Google Patents

Abstract

NEW MATERIAL:Erwinia carotovora subsp. carotovora CGE 234 M 403 strain (FERM P-11792) having no soft rot pathogenicity by deleting with a mutagenic treatment. USE:A controlling agent for plant soft rot. Free from chemical injury and with safety. PREPARATION:Erwinia carotovora CGE 234 strain is put to a mutagenic treatment using e.g. ultraviolet rays, a mutagenic reagent such as nitrosoguanidine, and pathogenicity-deleted strains are screened by determining the presence or absence of the decline in pectinase secretion activity. Furthermore, strains with high lesion-inhibitory ability are selected from said pathogenicity-deleted strains.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to the scientific name Erwinia carotovora subsubi.
ubsp, carotovora) CG E234
The present invention relates to a method for controlling soft rot by spraying the M403 strain and bacteria that infect plants with the live bacteria.

Many plants are targeted for disease control due to soft rot, including Chinese cabbage, cabbage, celery, lettuce, carrot, radish, wasabi, potato, tobacco, tomato, and cyclamen. (Soft rot dise
Ase) is the target disease.

[Conventional technology]

caused by the Erwinia carotovora bacterium,
As a method of controlling so-called soft rot, which softens and rots plant tissue, spraying of antibiotic preparations such as streptomycin and copper agents such as Bordeaux liquid is generally performed.

[Problem that the invention seeks to solve]

However, when these pesticides are used, not only are their control effects unsatisfactory, they also kill beneficial bacteria other than pathogenic bacteria, and there are problems with environmental pollution and drug damage. . Furthermore, the emergence of bacteria resistant to antibiotics has become a problem.

The Erwinia rotophora bacterium causes soft rot in the storage tissues of many plants, and is thought to be responsible for its ability to produce pectin-degrading enzymes that degrade pectin substances that act as intercellular bonding substances in plant tissues. It has been reported that it exists uniformly in soil. Soft rot outbreaks may be observed even in fields where crops that host this fungus have not been grown for five years or more. The general ecology of this bacterium is, for example, in the case of Chinese cabbage, this bacterium proliferates around the roots from about 40 days after sowing, and its presence is recognized in almost every place, including the rhizosphere soil and leaves. . When the Chinese cabbage is damaged by typhoons, insects, or daily work,
Bacteria invade from there, and if the climatic conditions are right, the concentration of pathogens increases overnight and lesions become visible. Therefore, the Erwinia carotovora bacterium, which lacks pathogenicity and has antibacterial properties against the pathogenic strain, can be grown in the rhizosphere soil and leaves to the same extent as the pathogenic strain. If possible, we can expect to control these soft rot diseases.

[Means for solving the problem; The present invention is directed to the use of mutant-treated strains of Erwinia virulotovora bacteria that grow well in competition with pathogenic strains of the same bacterium and that also have no pathogenicity. We have found that soft rot disease can be effectively controlled by selecting strains that do not have the same pathogenicity and inoculating the roots or leaves of the target plants with live Erwinia carotovora bacteria lacking these pathogenic qualities.

That is, the present invention is directed to the Erhinia subrotovora CG E234M403 strain in which the pathogenicity of soft rot disease has been deleted through mutation treatment. Or Erwinia carotovora in which the pathogenicity of soft rot disease has been deleted by mutation or mutation treatment - Lasabsubicarotovora CG
A soft rot control agent containing E234M403 bacteria as an active ingredient. A further object of the present invention is to provide a method for controlling soft rot, which comprises applying the Erwinia carotopora subsp. rotovora CG E234M403 bacteria onto soil or plants.

In order for non-pathogenic Erwinia bacteria to grow competitively with pathogenic bacteria, it is advantageous to produce some kind of antibacterial substance, and examples of such antibacterial substances include bacteriocins and phages. Regarding the bacteriocin produced by the Erwinia rotovora bacterium, Tsuyama et al.
5 years) and Takahashi et al.
nd H, Takahashi.

J, Gen, Appl, Microbiol, , 24
．． 27-39 (1978)), some of them were purified and their properties were investigated. When used as pesticides, the action of these antibacterial substances is preferably effective against a wide range of pathogenic strains of Erwinia carotovora; Since it is limited to related species, it is desirable to kill only soft rot fungi and not kill other bacteria that are useful to plants.

The configuration of the present invention will be described in detail below.

The present inventors collected a large number of Erhinia bacteria from vegetables with soft rot spots or healthy vegetables and examined the antibacterial activity of these bacteria. We obtained several bacterial strains with antibacterial activity against. Among them, the CGE 234 strain is a preferred bacterial strain that has a broad antibacterial spectrum and exhibits low susceptibility to bacteriocins produced by other similar bacteria.

The bacteriological properties of CGE234 strain are shown below.

Next, Erwinia rotovora CGE234 strain was mutated to create a pathogenicity-deficient strain. As a mutation processing method,
- Methods using mutation agents used in boat fishing, such as ethylmethanesulfonyl, nitrosoguanidine, or ultraviolet light, are known [Microbial Experimental Methods, pp. 288-306, published by Kodansha (1982) 3. Bye.

Screening for pathogenicity deficiency was performed by selecting strains with reduced pectinase secretion ability and conducting a pathogenicity test using Chinese cabbage sections. In the pathogenicity test, a highly concentrated test bacteria solution was applied to a wound on a Chinese cabbage leaf section, and the lesion length was measured after leaving it in a constant temperature bath at 28°C in the presence of water for 24 hours.
Among the deletion types, there were strains with decreased or no pectinase production ability, and strains with production but decreased secretion ability. The pathogenicity of Erwinia carotovora bacteria is determined by the presence or absence of pectinase, and in particular, pectic acid lyase is said to be the cause of soft rot (Masao Goto, New Plant Bacterial Pathology, p. 166, Soft Science Publishing, 1981).
.

In the present invention, the pathogenicity-deficient Erwinia carotovora bacterium obtained in this manner is mixed with a pathogenic strain and inoculated onto wounded Chinese cabbage sections to produce C G E 2 in which the pathogenicity has been deleted.
Among the 34 mutant strains, we obtained strains that suppressed the growth of pathogenic strains and did not produce lesions or significantly reduced the rate of lesion formation. In particular, it was found that an effective lesion inhibiting effect was observed against pathogenic bacteria at low concentrations.

Among these pathogenic deletion strains, strains with high ability to inhibit lesions have been deposited at the Microtech Institute and have been assigned the following deposit numbers.

Erhinia, Carotonera Subspi Power ■Tonera
CGE234M403 Microtechnology Research Institute No. 11792 (FE
RM P-11792) These pathogenic deletion patterns were unchanged from the parent strain except for the mutation site S and the potato tuber test (-).

Examples will be shown next, but the present invention is not limited to the following examples.

The composition of the medium used in the examples is shown below.

802 medium: polypeptone 10g, yeast extract 2g, M
g5Oa・7HzOIg, water 1 (1, pi(7,0(
For plates, include 15 g of agar) YCP medium:
(NH4) 25042g, Mg5Oa・IHzOO,
2g, Kazaamino acid 3g, yeast extract 2g1, pectic acid 7g, agar 15g, water 1l, pH 8.0 Tricarski improved medium: meat extract 4g, lactose 10g,
Peptone Log, Bromthymol Blue 0.04g, Agar 16g, Water 11, pH 7
,4 Minimal medium: NazHPOa-78208,2g-,
, KHzPOa 2.7g, (NH4)2SO41,
Og, FeSO4-7H200, 25g, MgSO
4.7HzO0,1g-Ca (NO3) z 5mg
, water LA, pH 7, 2PG medium: 5 g of vectic acid,
31g of NaNO.

K2HPO44g, Mg5O4・7Hz00.2g-
9 g of agar, 1N of water, pH 7.0 Example 1 (Method for creating mutants) Erwinia rotovora CGE234 strain was cultured in 802 medium at 30"C until the mid-log phase. 2- Add 2 d of minimal medium to the culture solution. In addition, 2% ethylmethanesulfonyl was added and incubated for 80 minutes.The bacterial cells were centrifuged,
After washing once with 802 medium, add 5 ml of fresh 802
A medium was added and the shaking culture was continued overnight. 0.1 ml of culture solution was added to 5 ml of PG medium and cultured with penicillin GK salt (final concentration 2F30 u/me) at 30° C. for 6 hr. After dilution, it was spread on an 802 medium plate and cultured overnight.

Next, the cells were inoculated onto a YCP plate and cultured overnight. A 10% calcium chloride solution was added to the plate to select colonies with small or almost no pectinase halo, and a CGE234M4 mutant strain was obtained from the CGE234 strain. Next, the CGE234M4 strain was further subjected to the same mutation treatment with ethylmethanesulfonyl, and CG
E234M403 strain (Feikoken Bibori No. 11792) was obtained.

802 medium and Y of the obtained CGE234A403 strain
Pectic acid lyase, pectin lyase, and polygalacturonase activities in the medium after overnight culture in CP medium were examined and all were 0.010100.
−1 or less.

Example 2 (Settlement) A 1:2000 Wagner Bosoto consisting of three groups A, B, and C was filled with Akadama soil and humus at a ratio of 2:1, and used as fertilizer per pot (N: P: K-8: s :
8), 25 g of each were mixed. After sowing Chinese cabbage (inspection product No. 2), cultivation was carried out, and about 30 days after that, 100 - of CGE234M403 strain bacterial body fluid (1 x 108/mf) was sprayed on the roots and leaves. Then, 63rd and 71st day gold leaves lcn
Then, 1 g of the stem was collected, and the diluted solution was applied to Trigalski's improved medium to determine the bacterial cell concentration.

Table 1 shows the bacterial cell concentrations on leaves and in stems.

From Table 1, it can be seen that the dispersed mutant fungi were stably colonized on the leaves and in the stems.

Example 3 CGE about 30 days after sowing for box-grown bok choy
234M403 strain bacterial body fluid (l x 10'/me) 30
One week after spraying 0-, pathogenic bacteria (l
/mN) was sprayed at 300+nf-. 5 after sowing in Table 2
The results of the disease control effect after 6 days are shown.

Table 2 Example 4 For Chinese cabbage grown in open field, the bacterial body fluid of CGE234M403 strain prepared to a bacterial concentration of 1X10''/- was added at 1 time per 10 ares.
Sprayed at a ratio of 1:l 20OL. Spraying was performed three times every week from about 30 days after sowing. Table 3 shows the results of disease control effects. In addition, as a control drug, a plan using Shikibetsu Kagaku (500 times diluted) is also listed.
゛Table 3 index O: No disease onset 1: Disease onset only on a part of outer leaves (can be shipped) [Effects of the invention] The present invention has been able to cure one of the major bacterial plant diseases that have been considered difficult to control in the past. It has become possible to effectively control soft rot. The present invention is a method of using live bacteria as a so-called biological control measure, and provides a safe agent and method for controlling soft rot that is free from phytotoxicity.

Claims (3)

[Claims] (1) Erwinia carotovora subspicalotovora CGE23 in which the pathogenicity of soft rot disease has been deleted by mutation treatment
4M403 strain. (2) A control agent for soft rot disease, which contains the bacterial strain according to claim 1 as an active ingredient. (3) A method for controlling soft rot disease, which comprises applying the bacterial strain according to claim 1 onto soil or plants.