JPH0815428B2 - Soil disease control method for solanaceous plants - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to Pseudomonas solanacearum, which is a kind of phytopathogenic bacteria.
The present invention relates to a method for controlling soil diseases of Solanaceae plants caused by (parasitism) bacteria.

[Prior Art] Tobacco and other solanaceous plants, such as eggplant, tomato, peppers, and potatoes, may be infected with this bacterium that survives in the soil, causing a pair of plants to wither and die, and suffer significant damage. Many. The disease caused by this bacterium, namely tobacco wilt or bacterial wilt (hereinafter referred to as “this disease”), is extremely difficult to control because the pathogenic bacterium easily survives in soil for a long period of time.

In addition to the method of using chemicals, there are many methods of controlling the present disease that are currently used, such as resistant cultivars, application of organic matter, and tillage of soil. However, the control using chemicals has the problems of pollution due to irritating odors, the problem of animal and animal toxicity, and the killing of useful microorganisms in the soil, and other agricultural methods often do not show a stable effect. . Therefore, the development of a safer and more effective control method is strongly desired.

On the other hand, it is generally known that pathogenic bacteria and various microorganisms having antagonistic properties against them exist in natural soil and form an ecosystem by influencing each other. Today, research is being conducted to suppress disease outbreaks by using antagonistic microorganisms that suppress the activity of pathogenic bacteria among naturally occurring microorganisms, and after infecting plants with non-pathogenic or weakly pathogenic microorganisms, Research into suppressing infections and pathogenesis of pathogenic bacteria has become active.

In the above research, the most effective one for controlling the present disease at present is the M4S strain of the bacterium (hereinafter referred to as "M4S") (JP-A-60-186230).

[Problems to be Solved by the Invention] The control effect and stability of M4S in the field of the present disease are not sufficient, and a bacteriophage and M4S that lyse the present bacterium for use as a practical control agent Control method of this disease (Japanese Patent Application No. 60-261354) or a method of controlling this disease by immobilizing M4S with a polymer and applying the immobilized product in soil (Japanese Patent Application No. 60-288013). ) Has been disclosed.

The present inventors have further advanced research in this field, and have created a new strain of this bacterium that exhibits antagonistic activity against this bacterium by a mechanism different from that of M4S by mutation to complete the present invention.

[Means for Solving Problems] In the present invention, a strain having a high antagonistic activity against the present bacterium by a mechanism different from that of the conventional M4S was selected. That is, a new Pseudomonas solanasiarum OM2 strain (hereinafter referred to as "OM2") that produces an antibacterial substance called "bacteriocin" against this bacterium was produced by the method described below. The term "bacteriocin" used here means
It is an antibacterial substance produced by a certain type of microorganisms against microorganisms of the same or related species.

The present inventors isolated 70 pathogenic bacterial strains of this bacterium from tobacco fields all over the country where tobacco wilt disease occurred. Then, among these 70 strains, several strains producing bacteriocin against this bacterium other than the own strain were selected. Among them, the Ps48 strain had a remarkably broad antibacterial range and showed bacteriocin activity against 69 strains other than the Ps48 gold strain. OM2 was produced based on this Ps48 strain. That is, Ps48 strain was
YG medium (10 g tryptone, 1 g yeast extract, 10 g glucose,
1 liter of water: Journal of General Microbiology vol 7
6 pp.177-188 (1973)) and static culture was carried out in the dark at 35 ° C for 7 days. This is TZC plate medium (casamino acid 1g,
Glucose 5g, peptone 10g, agar 18g, water 0.
Add 005% 2,3,5-triphenyltetrazolium chloride: Phytopathology vol 44 PP.693-695 (1954)) and streak culture was performed using a sterilized platinum loop. Ten independent colonies were obtained from the individual colonies formed on the medium.

A sterile water suspension (concentration: 10 ⁹ cells / ml) of the 10 acquired strains was inoculated into the stem base of tobacco (12 true leaf seedlings) with a needle. As a result, unlike this pathogenic bacterium, none of the strains caused the wilting symptom of all tobacco strains,
Many of the lower leaves showed wilting and yellowing symptoms. Only one of these 10 strains showed no symptoms other than the lowest leaves. This least pathogenic strain is OM2 used in the present invention. In addition, as a result of examining the productivity of bacteriocin against the 69 strains of the bacterium of the present invention by a layered culture method using Ps48 strain and OM2, OM2 forms a clear inhibition circle for both strains as well as Ps48 strain. did.

That is, OM2 is an ideal strain for use because it has the ability to produce bacteriocin against this pathogenic bacterium and has extremely weak pathogenicity. In addition, as another feature, OM2 has a distinctly different colony type on TZC medium compared to the parent strain Ps48 strain, whereas the parent strain shows an irregular circular, white to light pink fluid colony. OM2 is a round, medium-high, buttery, non-fluid settlement.

The bacteriological properties of OM2 will be described in more detail below.

a) Morphological properties Gram-negative bacillus Approximately 0.5 to 0.7 μm × 0.9 to 1.8 μm Possessing monopolar hair Spores: Not formed Motility: Yes b) Growing state CPG medium 2 days later, round, smooth, buttery small Colony PSA medium Same as above c) Physiological properties Production of bacteriocin Yes Attitude toward oxygen Aerobic growth temperature 15 ℃ -38 ℃ Suitable growth temperature 26 ℃ -35 ℃ Growth pH 4.5-8.5 OF test 0 Nitrate reduction + starch Degradation-Degradation of gelatin-Use of carbon source: glucose, sucrose, lactose,
Gawactoose, glycerol, inositol, maltose, fructose, cellobiose, mannitol, sorbitol, dulcitol Not used: arabinose, malonate
rgey's Manual of Determinative Bacteriology 8th e
It is confirmed to be a homologous bacterium, which has the same properties as the bacterium described in d pp.231-233. OM2 is M
It is easily distinguished from 4S because it produces bacteriocin and decomposes inositol.

The OM2 used in this invention was deposited on June 5, 1987, at the Institute for Microbial Industry, Institute of Industrial Technology, under the deposit number Microorganism Research Institute No. 9399 (FERM P-9399).

There is no special condition for culturing OM2 of the present invention, and it is aerobically cultivated in a synthetic medium containing a carbon source, a nitrogen source and inorganic salts. The culture is carried out under aerobic conditions such as shaking culture and aeration-agitation culture, but at 25 to 35 ° C, preferably 28 to 32 ° C, pH 5
-8, preferably 6-7 are suitable.

The culture of OM2 in the present invention means a culture suspension of OM2, viable bacteria, culture filtrate or an extract thereof. The present disease control method of the present invention is applied to the roots of solanaceous plants to be protected by OM.
Consisting of introducing 2 cultures.

In this specification, the “root portion” is a portion that absorbs water and nutrients in soil or hydroponic liquid when a plant is cultivated. The introduction into the root can be easily performed by spraying or irrigating the culture, or by immersing the root in the culture.

When used as a control agent for Solanaceae plants according to the present invention, when OM2 viable bacteria or a culture suspension is used, OM2 is preferably present at a concentration of 10 ⁸ to 10 ⁹ / ml, including the time of transplantation in the present field. Introduce these to the root during the day. When the immersion treatment is performed as one method of introduction, the immersion time is 30 minutes to 3 hours, preferably about 1 hour. Further, in the case of irrigation and irrigation treatment, 20 to 40 ml per seedling is suitable.

In order to obtain even higher effect, transplantation to
After 30 to 30 days, preferably after 20 to 25 days, it is desirable to irrigate the above suspension with 200 ml of each suspension.

When the culture filtrate or its extract is used, it is introduced into the root as it is or together with various carriers, diluents, spreading agents and the like according to a method commonly used for agricultural chemicals.

[Operation and Effect of the Invention] The mechanism by which this method controls the disease of solanaceous plants is explained as follows.

OM2 introduced into the root part survives at a high concentration on the root surface, invades from the root, proliferates in the plant body, and produces bacteriocin having an antibacterial action against this bacterium. Therefore, the soil-injected bacteria are physically or antibacterially inhibited when invading from the root, and at the same time, they are similarly inhibited even after invading the plant. for that reason,
The control effect of OM2 is high.

In addition, when OM2 is introduced by irrigating the strain source after a predetermined period of time has elapsed, it protects the roots of the crop from new infection with pathogenic bacteria, making it more difficult for this bacterium to invade. Become.

Furthermore, when a culture containing bacteriocin produced by OM2 is introduced, the bacterium is killed by its direct antibacterial action.

Thus, according to the present invention, the disease caused by the present disease of Solanaceae plants can be effectively controlled.

Example 1 Example 1 CPG agar medium (1 g of casamino acid, 5 g of glucose, 1 peptone) mixed with a suspension of pathogenic tobacco wilt fungus in distilled water.
20 g of 0 g, agar 18 g, water 1 liter) in a petri dish with a diameter of 9 cm
Pour ml to make a plate medium and separate it with CPG liquid medium at 30 ° C 48
After impregnating a paper filter with the culture filtrates of OM2 (invention) and M4S, which had been shake-cultured for a period of time, the inhibition circles formed after 24 hours at 30 ° C. in the center of the dish were examined. As a control, a section containing only the CPG liquid medium was provided, and 5 petri dishes were used for each section. The results are shown in Table 1.

From the results shown in Table 1, no formation of blocking circles was observed in the control and M4S plots, but a clear blocking circle was formed in OM2 plot. That is, it is confirmed that OM2 has the ability to produce bacteriocin and that the culture has bacteriocin activity.

Example 2 Tobacco seedlings (cultivation: bright yellow No. 4) of seedlings (9 true leaves) sized to be cultivated in a vinyl chloride pot (25 plants) each side of which is 5 cm square and transplanted to the main field are in each ward. I tested 15 bottles each. OM2 was cultured in CPG medium at 30 ° C. for 40 hours, and the roots of the cultures or the seedlings of distilled water used as a control were immersed for 1 hour according to the following distinction. For immersion, put each liquid in the processing tank to a depth of 3 cm,
This was done by immersing the roots of the seedlings in this.

(1) OM2 culture filtrate group: filtrate after filtering OM2 in the culture suspension (2) Suspension group of OM2 viable bacteria: centrifugal collection of OM2 in the culture suspension and then in distilled water Resuspended (OM2 concentration: 10 ⁹ / m
l) (3) OM2 culture suspension group: (OM2 concentration 10 ⁹ cells / ml) (4) Control group: distilled water The treated tobacco seedlings are artificially contaminated soils of pathogenic tobacco wilt disease (bacterial wilt disease). It was transplanted to a clay pot with a diameter of 12 cm filled with several 1.5 × 10 ⁶ pieces / g dry soil). The illness was observed 20 days later. The degree of illness is 6 from 0 to 5 as shown in Table 2.
The average morbidity index was calculated by the following formula, and the control rate was calculated. The results are shown in Table 3.

As is clear from the results in Table 3, the morbidity rate of the treated group of the OM2 culture was lower than that of the control group, and the average morbidity index of the treated group was significantly lower (risk rate 1%). In addition, the effect of live bacteria or culture suspension treatment was the highest among the treatment groups.

Example 3 20 tobacco seedlings cultivated in the same manner as in Example 2 were immersed in the following 3 kinds of liquids for 1 hour. Example 2
The same method was used.

(1) Viable OM2 suspension: 10 ⁹ viable OM2 in distilled water /
(2) M4S viable cell suspension group: 10 ⁹ viable cells of M4S in distilled water /
Containing ml (conventional invention) (3) Control group: distilled water Each treated tobacco seedling was transplanted into a clay pot with a diameter of 12 cm, and after 10 days, the concentration of pathogenic tobacco wilt fungus was 10 ⁶
A suspension of distilled water (60 cells / ml) was prepared, and immediately after the roots of the above-mentioned 60 tobacco seedlings were injured by inserting a knife, 10 ml of this pathogen suspension was irrigated. After 10 days, the disease state was observed in the same manner as in Example 2. The results are shown in Table 4.

As is clear from the results in Table 4, the OM2 treatment group had the highest disease suppressive effect, and the effect was even higher than that of M4S. The average morbidity index was also the same, and the difference was statistically significant (risk rate 5%).

Example 4 Tobacco was cultivated in the same manner as in Example 2 and treated in the same manner as described above, and then artificially contaminated soil of pathogenic tobacco wilt disease (the number of wilt disease bacteria 3.0 × 10 ⁶ cells / g). It was transplanted to a clay pot with a diameter of 12 cm. After 20 days, the disease state was observed and the control effect was evaluated in the same manner as in Example 2. The results are shown in Table 5.

As can be seen from Table 5, the disease incidence was lower in the treated plots than in the control plots, and the average morbidity index was statistically significantly lower (1% risk rate) in the treated plots. In addition, OM2 processing area is M4S
The disease incidence was lower than in the treated plots, and the average morbidity index was also statistically significantly lower (risk rate 5%).

Example 5 Tomato seedlings (cultivar: Fukuju No. 100) that had been inoculated for 30 days in the vinyl chloride pot used in Example 2 were used for 20 days in each ward.
Samples were tested one by one, and the tomato seedlings were treated in the same manner as in Example 2. After the treatment, it was transplanted to an artificially contaminated soil (the number of bacterial wilt disease 2.2 × 10 ⁶ cells / g dry soil) in the same manner as in Example 2, and after 20 days, the disease incidence,
The average morbidity index and control rate were calculated. The results are shown in Table 6.

Both the treatment groups had lower incidence and average morbidity index than the control group. In particular, the OM2 treatment area has a lower disease incidence than the M4S treatment area, and the average morbidity index is also statistically significant (risk rate 1
%) Was low.

Example 6 Eggplant seedlings (variety: Nagaoka Nagase) of the size (7 true leaves) at the time of transplantation in the main field 40 days after sowing in a vinyl chloride pot
Was tested and treated and transplanted in the same manner as in Example 2. After 30 days, the disease state was observed and the control effect was evaluated in the same manner as in Example 2. The results are shown in Table 7.

The disease incidence was lower and the average morbidity index was lower in the treated plot than in the control plot. In addition, the OM2 treatment group had a significantly lower disease incidence than the M4S treatment group, and the average morbidity index was also statistically significantly low (risk rate 1%).

Example 7 Tobacco seedlings (cultivar: Bright Yellow No. 4) cultivated in the same manner as in Example 2 were tested for 10 cells per ward, and a viable bacterial suspension of OM2 (concentration: 10 ⁹ cells / ml) was introduced into the roots of tobacco seedlings. As a control section, a section in which distilled water was immersed was provided.

(1) Seedling root submerged area: A living bacterial suspension of OM2 was immersed in the root for 1 hour (the present invention).

(2) Irrigation area: The suspension was irrigated with a watering can (the present invention).

(3) Irrigation section: The same suspension was irrigated to the origin of the strain (the present invention).

(4) Control area: Immersed in distilled water for 1 hour.

The treated seedlings were transplanted to artificially contaminated soil (the number of bacterial wilt disease 2.2 × 10 ⁶ cells / g dry soil) in the same manner as in Example 2, and after 20 days, the disease incidence,
The average morbidity index and control rate were calculated. The results are shown in Table 8.

As can be seen from Table 8, the effect of suppressing the bacterial wilt disease was observed in all the treatment groups, and a significant difference (risk rate 1%) was observed between the average morbidity index of the treatment group and the control group. However, no significant difference was observed in the effect among the treated plots.

Example 8 72 tobacco seedlings (cultivar: Bright Yellow No. 4) cultivated in the same manner as in Example 1 were tested for each ward. Example 1
Similarly to the above, the roots of tobacco seedlings were immersed in a live OM2 suspension (concentration: 10 ⁹ cells / ml) for 30 minutes. The control plot was not treated at all. Immediately after the dipping treatment, the plant was transplanted to a field infected with wilt disease (the number of wilt disease bacteria: 2.9 × 10 ³ / g dry soil). Separately, 25 days after the transplantation, a section was provided in which the suspension of OM2 having the same concentration as in the case of the dipping treatment was irrigated with 200 ml of each tobacco strain. The results of investigating the disease state on July 7 are shown in Table 10. The disease onset survey was conducted based on the criteria shown in Table 9.

Both treatment groups had lower disease incidence and average morbidity index than the control group, and the control effect was recognized. Also, OM
The effect of the combined treatment of the immersion treatment at the time of transplantation and the strain irrigation in 2 was higher than that of the immersion-only treatment group.

Claims (3)

[Claims] 1. A Pseudomonas solanaciarum OM2 strain which has the ability to produce bacteriocin and forms a round, medium-high, buttery, non-fluidic community on TZC medium. 2. A control agent for tobacco wilt disease and solanaceous plant wilt disease, which comprises Pseudomonas solanaciarum OM2 strain as an active ingredient. 3. A method for controlling tobacco wilt and Solanaceae wilt, which comprises introducing Pseudomonas solanaciarum OM2 strain into the root of a Solanaceae plant.