JPH03101606A - Control method for soft rot - Google Patents

Abstract

PURPOSE:To accomplish safe, effective control, without causing any adverse reaction, of soft rot having been considered to be difficult to control, by using Erwinia carotovora with its pyrogenicity deleted by mutation treatment. CONSTITUTION:Soft rot can be effectively controlled by inoculating the root or leaves of an object plant with viable cells of Erwinia carotovora bacteria with the pyrogenicity of soft rot deleted by mutation or mutation treatment, well-growable in competition with the same kinds of bacteria of pyrogenic strain, also giving effective antagonistic action on pyrogenic strains. The pyrogenicity-deleted strains with high lesion-inhibitive ability are: CGE6M14 strain (FERM P-10998) and CGE6M16 strain (FERM P-10999) from CGE6 strain, CGE10M2 strain (FERM P-11000) from CGE10 strain, and CGE11M5 strain (FERM P-11001) from CGE11 strain.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to the scientific name Erwinia carotovora subsubi.
ubsp. This invention relates to a method for controlling soft rot disease by spraying live bacteria belonging to the genus Carotovora on plants.

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 causes plant tissues to soften and rot, generally, antibiotic preparations such as strebtomycin and spraying of copper agents such as Bordeaux liquid are used.

[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 carotovora bacterium causes soft rot in the storage tissues of many plants, and is said to be caused by its ability to produce pectin-degrading enzymes that degrade the vectin substances that act as intercellular junctions 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 Chinese cabbage is damaged by typhoons, insects, or everyday work, bacteria can invade the cabbage, and if the weather conditions are right, the concentration of pathogenic bacteria will increase overnight and lesions will appear. 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 problems]

The present invention aims to eliminate the pathogenicity of Erwinia carotovora bacteria by selecting strains that grow well in competition with pathogenic strains of the same bacteria and that are not pathogenic. The object of the present invention is to provide a method for effectively controlling soft rot by inoculating the deleted Erwinia carotovora bacteria into the roots or leaves of the target plant.

That is, the present invention is directed to a method for treating soft rot disease, which is characterized by using Erwinia carotovora bacterium, which has its pathogenicity deleted by mutation or a mutation treatment method, and which has an effective antagonistic effect against the pathogenic strain. It is a control method, and the Erwinia carotovora bacterium is Erwinia carotovora CG6.
CGE 6M14 obtained by mutating the strain
strain and/or strain CGE6M16, Erwinia carotovora bacteria are Erwinia carotovora CG1
CGE10M2 obtained by mutation treatment of 0 strain
The present invention is also characterized in that the bacteria and Erwinia carotovora bacteria are CGE11M5 strain obtained by mutationally treating the Erwinia carotovora CG11 strain.

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 batatteriocin and phages. Regarding the bacteriocin produced by Erwinia carotovora bacterium, Tsuyama et al.
1975) and Takahashi et al.
and H. Takahashi, J. Gen. 8pp
l. Microbio1. , 24.27-39 (197
8)), some of them have been purified and their properties have been 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 this 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 structure of the present invention will be described in detail below.

The present inventors collected a large number of Erwinia bacteria from vegetables with soft rot lesions or healthy vegetables and examined the antibacterial activity of these bacteria. We obtained several bacterial strains with For example, the bacteriocin of the CGE6 strain is a so-called protein aggregate with low diffusibility, and showed antibacterial activity against many of the Erwinia carotovora strains tested. In addition, the bacteriocin of the CGE10 strain has 2 MOs, one with high diffusibility and one with low diffusivity, and has antibacterial activity against many Erwinia carotovora bacteria. It was confirmed that the CGEll strain also produces bacteriocin with low diffusibility and batateriocin with high diffusibility.

Next, the Erwinia carotovora CGES strain was mutated to create a pathogenicity-deficient strain. As mutation treatment methods, methods using commonly used mutation reagents such as ethylmethanesulfonyl, latrosoguanidine, or ultraviolet light are known [Microbial Experimental Methods, pp. 288-306, published by Kodansha (1982)]. It can be processed according to the following.

Screening for pathogenicity-deficient strains was performed by selecting strains with reduced pectinase secretion ability and conducting a pathogenicity test using Chinese cabbage sections. In the pathogenicity test, a leaf section of Chinese cabbage was wounded, a highly concentrated test bacterial solution was applied, 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 strains, there were strains with reduced or no pectinase production ability, and strains that produced pectinase but with reduced secretion ability. The pathogenicity of Erwinia carotovora bacteria is judged by the presence or absence of pectinase, and in particular, pectate 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 strain of Erwinia carotovora bacterium obtained in this manner is mixed with the pathogenic strain and inoculated onto wounded Chinese cabbage sections, thereby producing a mutant of the CGE6 strain in which the pathogenicity has been deleted. We obtained a strain that suppressed the growth of the pathogenic strain 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.

Furthermore, when Chinese cabbage sections were inoculated with the pathogenic strain after inoculating the pathogenic strain, an even more effective lesion-preventing effect was observed, and similar operations were performed on strains other than the CGE 6 strain. , a pathogenic deletion strain was obtained.

Among these pathogenicity-deficient strains, strains with high lesion-inhibiting ability have been deposited at the Microtech Institute and have been assigned the following deposit numbers.

Engineering 1 Tobini 7 ・ Karotobora Subsububi Power Rotopora CGE6M14 Microtechnical Laboratory Bacteria No. 10998 (FE
RM P-10998) Erwinia carotonella sapuspi Wholesale Tohonichira CGE6M16 Microtechnical Research Institute No. 10999 (FBRM P-10999) Erwini 7
・Karototora Subspi Power Rototora CGE10
M2 Microtechnical Laboratory Bacteria No. 11000 (FERM P-110
00) 11L Bini7 - Carotovora subsubika Tonera CGE11M5 Microtechnical Laboratory Bacteria No. 11001 (FERM P-11001) These pathogenicity-deficient strains did not differ from the parent strain in terms of mutation sites other than vectinase.

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

The composition of the culture medium used in the Examples is shown below.

? 02 medium: voribeptone 10g5 yeast extract 2g, M
gSO4・7HzO Ig, water il, pH 7. (For plates, include 15 g of agar) YCP medium: (NH
JzSO42g
Bebuton 10g, promthymol blue 0.04g, agar 16g 1 water 16, pH 7
．． 4 Minimal medium: NatHPOa 711zO 8.2g
, KIl2PO4 2.7g- (NH4)tsOa
1. Og1FeSOn ・711200.25g,
MgSOa・71120 0.1g, Ca(NOz)z
5 mg, 1 water, pl+7.2 PC medium: 5 g of vectic acid, 4 g of NaNO, Ig, KJPOa. ．． M
gSOa・711■0 0.2g, agar 9g, water 11,
pH 7. Q Example 1 (Method for cultivating mutants) Erwinia rotovora CGE6 strain in 802 medium.
The cells were cultured at 30°C until the mid-log phase of growth. Minimal medium 2- was added to the 2d culture solution, 2% ethylmethanesulfonyl was added, and cultured for 80 minutes. Centrifuge the bacterial cells, 802
After washing once with the medium, fresh 5-802 medium was added and the shaking culture was continued overnight. 0.1-culture solution to 5-P
Penicillin GK salt (final concentration 280 u
/d) at 30°C for 6 hours. After dilution, 802
It was spread on a medium plate and cultured overnight. Next, inoculate the YCP plate and continue culturing overnight, and then inoculate the plate with 10
% calcium chloride solution was added and colonies with small or almost no pectinase halo were subjected to pathogenicity tests. From the CGE6 strain, CGE6M14 (FEI Deposit No. 10998) and CGE6M16 (FEI Deposit No. 10)
No. 999) mutant strain was obtained. In addition, by the same method, CGE10M2 strain (Feiberal Institute Deposit No. 1
1000), but CG811M5 strain (
A mutant strain of the microtechnical research institute Deposit No. 11001) was obtained. Obtained CGE6M14, CGE6M16, CGB10M2
and pectate lyase (PA) of each CGE11M5 strain.
L), pectin lyase (PL) and polygalaculonase (PC) activities are shown in Table 1 along with the pathogenic strains.

Table 1 Example 2 (In vitro lesion suppression test) The lower end of a Chinese cabbage section (1.5 cm x 3 cm) was scratched in 4 places with a syringe needle, and the filter paper, glass plate, and Chinese cabbage section were placed in that order in a glass petri dish. ．． Next, the mutant strain and the pathogenic strain were mixed at a concentration of I x 10"/1, and 20 μl of the mixture was dropped onto the wound part of the Chinese cabbage section. The filter paper in the glass Petri dish was sufficiently moistened with water, and the mixture was placed in a constant temperature bath at 28°C. After standing for 24 hours, the length of the soft rot lesion from the wound was measured.Table 2 shows each mutant strain and pathogenicityTable 3 shows the concentration of the test bacteria on the leaves.Table 3: Mixed inoculation of strains The lesion length of Chinese cabbage sections is shown.

Table 2 Suppression of soft rot lesions caused by pathogenicity-deficient bacteria Table 3 shows that the sprayed mutant fungi are stably colonized on the leaves. Example 4 Chinese cabbage grown in pots in the same manner as in Example 3 was treated with soft rot fungi [3 strains (CGE used in Example 2)] about 30 days after sowing.
14, CGE15, CGE16) mixed bacterial cell concentration Example 3
(Settlement) 1/2000th Wagnerbot with 2 pieces of Akaohsoil and mulch
The pots were packed at a ratio of 1 to 1, and 2 g of N%PSK was added per pot as fertilizer. After sowing Chinese cabbage (Matsushima No. 2), about 3
Mutant strain bacterial fluid (I x 10”/d) 100 m on day 0
was sprayed on the roots and leaves. Thereafter, 1 d of leaves were collected at the following number of days, and the diluted solution was applied to Drygalski's modified medium to determine the bacterial cell concentration.

After spraying 10 OIn1 (10'/d),
One week later, 100 times of the test strain (10"#tl!) was sprayed. Table 4 shows the results of the disease suppression effect.

Table 4 Example 5 Chinese cabbage grown in pots in the same manner as in Example 3 was sprayed with 100 mffi of test bacteria (10"/1) about 30 days after sowing, and 1 week later, soft rot fungal strains [3 strains ( 100 ml of mixed bacterial cells (same as in Example 4) (10 6 /me) each was sprayed.Table 5 shows the results of the disease suppression effect.

Table 5 [Effects of the Invention] According to the present invention, it has become possible to effectively control soft rot, which is one of the major bacterial plant diseases that have conventionally been considered difficult to control. The present invention uses live bacteria as a so-called biological control measure, and provides a safe method for controlling soft rot disease without chemical damage.

Claims (4)

[Claims] (1) A method for controlling soft rot disease, which is characterized by using Erwinia carotovora bacteria whose pathogenicity of soft rot disease has been deleted by mutation or a mutation treatment method and which has an effective antagonistic effect against the pathogenic strain. . (2) CG obtained by mutating Erwinia carotovora strain CG6 with Erwinia carotovora bacteria
The bacterium according to claim 1, which is E6M14 strain and/or CGE6M16 strain. (3) Erwinia carotovora bacteria obtained by mutating Erwinia carotovora strain CG10
The bacterium according to claim 1, which is the GE10M2 strain. (4) Erwinia carotovora bacteria obtained by mutating Erwinia carotovora strain CG11
The bacterium according to claim 1, which is the GE11M5 strain.