JPS62123104A - Control of soil bright to solanaceous plant - Google Patents

Abstract

PURPOSE:To inhibit the growth of pathogenic germs invading later and suppress and control outbreak of blights, by inoculating bacteria of Pseudomonas solanacearum M4S into solanaceous plants, adding bacteriophage and migrating the bacteriophage while subjecting the bacteria of Pseudomonas solanacearum M4S to bacterriolysis. CONSTITUTION:Bacterial wilt of tobacco, tomato, eggplant, green pepper, potato, etc., is controlled. In the process, live bacteria of Pseudomonas solanacearum S4M (FERM-P No.700) are inoculated into the root part of solanaceous plants and a growth solution of bacteriophage (capable of subjecting both the live bacteria of the Pseudomonas solanacearum S4M and the pathogenic germs) is sprayed on the soil in the root part of the solanaceous plants within 7 days after the inoculation to supress growth and outbreak of the pathogenic germs and induce controlling reaction of the individual plants and control soil blights of the solanaceous plants caused by the parasitism of the bacteria of Pseudomonas solanacearum M4S.

Description

[Detailed Description of the Invention] [Industrial Application Field] This invention relates to Pseudomonas solanacearum (Pseudomonas fistula).
The present invention relates to a method for controlling soil diseases of Solanaceae plants caused by bacterial parasitism (domonas solamacearu++).

[Prior Art] Tobacco damp-off and bacterial wilt of solanaceous plants other than tobacco, such as eggplants, tomatoes, green peppers, potatoes, etc., are caused by parasitism of pathogenic Pseudomonas solanacearum bacteria. When plants are infected with these diseases, they wither and become unharvestable, causing great damage.

As a means of controlling tobacco damping-off and bacterial wilt of plants in the Solanaceae family other than tobacco (in this specification, these diseases are collectively referred to as soil diseases of plants in the Solanaceae family), the Patent Cooperation Treaty stipulates that An international application (International Publication No. WO35103519) published under the International Patent Publication No. WO35103519 discloses a method for inoculating live bacteria of Pseudomonas solanacearum M4S (Feikoken Article No. 700) strain into the roots of Solanaceae. .

[Problems to be solved by the invention] The effect of suppressing the onset of soil diseases by the method described in H has been confirmed in tree farms, but in order to use it as a soil disease control method in tree farms, it is necessary to further enhance the control effect. desired.

[Means for Solving the Problems] In the present invention, in order to further increase the effect of Pseudomonas solanacearum M4S live + 24 on controlling the solanaceous plant's solanaceous plant disease, live bacteria of Pseudomonas Φ solanacearum M4S are added to the solanaceous plant. The roots are inoculated, and within 7 days after the inoculation, a bacteriophage growth solution that lyses both the viable bacteria and the diseased Pseudomonas solanacearum is sprayed onto the soil of the roots.

Incidentally, attempts have been made for a long time to use bacteriophages (hereinafter simply referred to as phages) that lyse pathogenic bacteria in plants to control plant diseases caused by parasitism of pathogenic bacteria. However, at present, these attempts have not reached the level of practical use. The reason is thought to be as follows.

In other words, pathogenic bacteria that have grown inside the plant form clusters and are surrounded by a large amount of mucilage such as sugars, so that all bacterial cells are not infected by phages, and the distribution of phages is extremely localized. This is to be done. That is, it is thought that a major cause is that phages are distributed in large numbers in -f lesions of plants, and in small quantities in healthy parts.

Because of these reasons, phages cannot be expected to have a controlling effect, so in the present invention, phages are introduced into the plant and allowed to proliferate before the pathogenic bacteria proliferate within the plant. More specifically, Pseudomonas solanacearum M4 is a non-pathogenic strain of the Pseudomonas solanacearum strain, which is a soil pathogen of Solanaceae plants.
By inoculating S into the root of a Solanaceae plant, causing the clover to invade into the plant body, and dispersing the phage, the phage multiplies within the plant body before the pathogenic bacterium Pseudomonas solanacearum invades the plant body. Improves soil disease control effect.

Pseudomonas solanacearum M4ScM strain C (hereinafter simply referred to as M4S bacterium) used in the method of this invention is
As mentioned above, it has been deposited at the FIKEN (Deposit number: FIKEN Article No. 700), and its acquisition method, culture method, mycological properties, etc. are in the above-mentioned International Publication WO 3510.
This is as described in detail in 3519 Publication.

The phages used in this invention are isolated using the microbiological experimental method (
This can be done by applying the method described in Kodansha Scientific Fl, December 1975, edited by the Microbial Research Methods Council.

Specifically, 1 tobacco stems grown at Japan Tobacco Inc.'s Utsunomiya Experimental Station and infected with Riheko damping-off were cut into strips of 1 cm or less on each side, and 10g of each was suspended in 100ml of sterilized water. Shake for 1 hour.

The supernatant was centrifuged at 110,000 rpm for 10 minutes, and the supernatant was filtered through a Millipore filter (0.45 m). 0.2 ml of the filtrate was added to a known CP containing pathogenic tobacco blight bacteria.
G medium (ingredients: 1 g of casamino acid, 10 g of glucose, 10 g of peptone, 1 liter of water) was inoculated and cultured with shaking for 48 hours.

The supernatant of the culture filtrate was filtered with a Millipore filter, diluted one step, and 0.1 ml of the filtrate was clouded with 3 ml of CPG agar medium containing pathogenic damping-off bacteria, and this was poured into a Petri dish with a diameter of 9 cm. , and cultured at 30°C for 24 hours. Phage were isolated from the formed lytic plaques.

Cultivation of the phage follows the M4S quiet culture.

That is, in a liquid medium inoculated with phage and M4S at the same time, at 28 to 30°C, the sowing period is preferably 48 to 36°C.
Incubate for hours. The culture solution is centrifuged at high speed (eg, too many revolutions/minute) and the supernatant is used as a phage propagation solution. When actually spraying on soil, the number of phages per ml is set to 10 or more, preferably 10 or more JA9.

According to the present invention, in order to control soil diseases of Solanaceae plants, first, live M4S bacteria are preferably suspended in sterile water (bacteria concentration: 106 to 10 cells/ml,
Preferably 10 to 10 cells/ml) is inoculated into the roots of the plants 3 weeks before transplanting, including the day of Kibata transplanting, as described in the above-mentioned International Publication. The timing of this inoculation is preferably 1 to 4 days before transplantation.

In addition.

The root part is the part that exists in the soil and absorbs water and nutrients when cultivated using soil, and includes potato tubers and the like. For root inoculation, M4
This can be easily done by immersing the roots in a suspension of S bacteria.

The soaking time is usually 30 minutes to 3 hours, preferably 1
It's around the time.

One hour to 7 days after inoculation with the M4S bacteria, the above phage propagation solution with adjusted phage number is sprayed onto the root soil of the inoculated seedlings in an amount of 100 m or more per seedling. When transplanting seedlings to the main field (planting), 7 days after spraying the phage propagation solution, preferably 1
It is preferable to carry out this after ~4 days.

[Operations and Effects of the Invention] The mechanism by which soil diseases of Solanaceae plants, that is, tobacco damping-off and bacterial wilt of Solanaceae plants other than tobacco, are controlled by the method of the present invention is considered as follows.

That is, first, the M4S bacteria previously inoculated into the Solanaceae plant invade the plant. The phage added later moves into the plant body while lysing this M4S bacterium (it has been experimentally confirmed that phage moves into the plant body)
. This phage.

It inhibits the growth of pathogenic Pseudomonas solanacearum that invaded later and suppresses the onset of disease. In addition, the action of M4S bacteria induces the plant's own pesticidal response.

Thus, according to the present invention, soil diseases of Solanaceae plants are effectively controlled.

[Example] Example 1 Trees cultivated in vinyl chloride resin pots (25 plants) with a side of 28.5 cm and grown to a size that can be transplanted to a tree farm.
J9 cigarettes [A (variety: Hakuenshu No. 1)] were tested.

Each of these 20 seedlings was treated as follows.

(1) Treatment area with M4S bacteria and phage solution (invention):
M4S blank live bacteria were suspended in sterile water to a concentration of 10 cells/ml! 9. Prepare a suspension.

After soaking tobacco plants in this solution for 1 hour, they were potted in flower pots with a diameter of 15 cm. After 4 days, phage solution (number of phages: l
100 ml of Bacterium speriosa (8 cells/ml water) was added to each pot.

(2) M4SVi treatment group: The same procedure as treatment group (1) was performed except that no phage was added.

(3) Phage liquid treatment group: The same procedure as treatment group (1) was performed except that the treatment with M4S bacteria was not performed.

(4) Control group: Tobacco seedlings were immersed only in distilled water and then potted in the same flowerpot.

Four days after each treatment, an aqueous suspension containing pathogenic Pseudomonas solanacearum at a concentration of 10 cells/ml was prepared, and a knife was inserted into the roots of the above 80 fungi to injure them. Right after I put it on.

This suspension of pathogenic bacteria was irrigated in 10 ml portions. 10
The degree of 0 disease onset, which was observed after 1 day, was classified into 6 levels from O to 5 as shown in Table 1 below, and the average morbidity index was determined using the following formula and the control rate was calculated. The results are shown in Table 2.

Table 1 Disease onset 1-0 disease U O No disease 1 Part of the leaves withered 2 1 to 3 leaves! T is wilting 3 Wilting except for 2 to 3 leaves near the growing point 4 Wilting of all leaves However, N is the number of test individuals, no 5 is the number of individuals belonging to disease index 0 to 5 6 Average average of control plot Morbidity index table 2 P - 10 t”, disease Ft I/j = 4 (1)
O$ 0 100% (2) 16.7X 0.75 78.3% (
3) 83.3 3.67 04) (text!1r
(District 88.7$ 3.18 - As is clear from the results shown in Table 2, no disease onset was observed in the M4S bacteria and phage treated district (1), the effect of suppressing disease onset was the highest; In the phage-treated group (3), the disease onset rate was higher than in the control group, and no effect in suppressing the disease onset was observed.

Example 2 M4S bacterial suspension prepared in the same manner as in Example 1 (el 10
Tobacco W produced in the same manner as in Example 1 was soaked in 8 pieces/ml) and planted in pots, and 12 plants were treated in each section. That is, 30 minutes after potting (treatment area 5), 1 [after 1 (
After treatment area 6), 4B (treatment area 7), and 7 days after treatment (treatment area 8), loO+nl of 77-di solution (concentration [O pieces/ml) was added to each pot. Pathogenic Pseudomonas solanacearum was inoculated 10 days after potting, and the disease state was observed 14 days later. Note that the same observation was made for the treatment using only M4S (treatment area 9). The control group used sterile distilled water as in Example 1.

The results are shown in Table 3.

Table 3 (5) 25.0 0.75 78.6 (6) 8
．． 3 0.33 90.5 (7) 8.3 0.08
97.7 (8) 25.0 0.58 83.4 (
9) 50.0 1.75 50.0 sentences, open-83,
33,50- As can be seen from the results shown in Table 3, the effect of suppressing the onset of the disease was observed even if the phage solution was treated at any time within the period after the M4S bacteria incubation treatment. In particular, the effect of suppressing disease onset was high in the plots treated with phage culture 1 and 4 days after M45IJ treatment.

Example 3 Tobacco seedlings (variety Cobright Yellow No. 4) grown in the same manner as in Example 1 were immersed in the M4S bacterial suspension and potted in the same manner as in Example 1, and the phage solution was added 30 minutes later. (
Treatment area 10). In addition, the plot in which only M4S bacteria was added (treated plot 11) and the plot inoculated with only phages (treated plot 12)
) was also prepared. The control group was treated with sterilized water.

7]], pathogenic u-101A (which is lysed by phage) and 0K29 (which is not lysed by phage) were each inoculated, and the disease state was observed 10 days later. The results are shown in Table 4.

Table 4 (10) u-1021,40,4391,4(l l
) u-1064,31,9381,4(12) u
-1092,03,5030,0 control group u-101
00,05,0-(10) 0K29 57.0 1.
71 0 (11) 0K29 50.0 0.93 3
1.8 (12) 0K29 50.0 +, 93 0
Sentence r ``q-OK29 50.0 1.3B -Table 4
As can be seen from the table, when U-10 bacteria susceptible to phages was inoculated, similar to the results in the above examples,
The plot treated with M4S bacteria and phage (treated plot 10) had a high effect in suppressing the onset of the disease, and the eel was also highly effective.

In the plot to which only phage was added (treated plot 12), almost no effect on disease onset was observed.

When 0K29 bacteria, which is not susceptible to phages, is inoculated, no effect of suppressing the onset of damping-off disease is observed by adding the phage solution.

Example 4 Tomato sprouts (variety Nitoyokin) that were transplanted twice after sowing, cultivated using the vinyl chloride resin method, and grew to the size of seedlings planted in a tree field 40 days after sowing were used as samples. That is, the M4S bacterial suspension prepared in the same manner as in Example 1 (75 degrees 10" 1!/
m I) Nidomad [W was soaked and potted. After 4 days, the phage solution (10 C/ml) was added to loOm.
1 was added. After 2 u, pathogenic Pseudomonas solanacearum was inoculated, and the disease state was observed after 7 days. The control area was treated with sterile distilled water. The results are shown in Table 5. In addition, the results for the case of processing only with M4S (processing area 14) are also shown.

Table 5, -Incidence 8% -B disease prevention 2 (13) 0 0 to.

(14) 50.0 1.31 47.6 sentences・m×
62.5 2.50 - As can be seen from Table 5, no disease onset was observed in the plots in which the phage solution was added to m treated with M4S bacteria, showing a 100% control effect.

Example 5 Tobacco seedlings (Hakuenshu No. 1) grown in the same manner as in Example 1 were
We tested 160 bottles per ward. As in Example 1, M4
The roots of tobacco seedlings were immersed in a live bacterial suspension of SVi (concentration: 10 cells/m 1 ) for 1 hour. Three days later, 100 ml of y-y-shikaku'a solution (j5 degrees 2, 5 x 109 (14/ml) was added to the soil for each plant.
A few days later, on April 24th, a field contaminated with damping-off (the number of blight bacteria was 5.8x).
The cells were transplanted to 10 cells/g of dry cells and the disease state was observed. In addition, the control plot was transplanted on the same day without any treatment. Table 6 shows the results of the investigation of the disease onset status on July 16th and August 50th.

Table 6 Treatment area July 16th 14.5 0.73 37.
2 control group /l 18.4 1.18
- Treatment area August 5th 32.2 2.29 3
4.4 Civilized area /l 50.3 3.50
-As can be seen from Table 6, the disease incidence rate was lower in the treated plots and the average morbidity rate was also lower on all survey dates. The control rate was also over 30%.

Claims (1)

[Claims] A live bacteria of Pseudomonas solanacearum M4S is inoculated into the roots of a Solanaceae plant, and within 7 days from the inoculation, a propagation solution of a bacteriophage that lyses both the live bacteria and pathogenic Pseudomonas solanacearum is injected into the roots. A method for controlling soil diseases of solanaceous plants, the method comprising spraying the soil on the soil.