JPH0358903A - Removal of disease of plant and storage disease of plant - Google Patents

Abstract

PURPOSE: To control plant diseases and storage disease of plants by adding Streptomyces griseoviridis actinomycete fungi, which produce effective agents, to seeds, ground substance or vegetation to treat it. CONSTITUTION: Streptomyces griseoviridis actinomycete fungi in the form of a dispersion is added to seed, ground substance or vegetation to control diseases of plants, etc. The above fungi produce an antibiotic having a structure of the formula (R is CH3 , OH, H or a keto group; R' is CH3 or H), and the fungi are formulated as a suspension containing 10<3> spores/ml. The suspension is preferably applied by dipping roots of a plant using at the minimum ratio of 1dl/m<2> to the ground substance, or by spraying, etc. The above suspension exhibits its effectiveness only by adding it to a plant in an extremely low concentration and at a low frequency, and accordingly the treatment is easy and economical.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention provides a streptomycin that produces an effective drug! Sls
The present invention relates to a method for eliminating plant diseases and plant storage diseases by treating seeds, substrates or vegetation with microorganisms of the genus Treptomyces.

(Problems to be solved by the prior art and the invention) In order to prevent plant diseases in greenhouses, disinfection methods and replacement of substrates are used.
Efforts have been made to try various cultivation methods, to suppress the disease with chemicals, etc. However, these methods are inconvenient and expensive, and introduce unsuitable substances into the environment, especially when suppressed by drugs. For this reason, much attention has been focused on biological control methods. In particular, control of plant diseases is difficult due to restrictions on the use of chemical inhibitors, the long period of damage caused by residues, and the lack of sufficient preliminary research on effectiveness. There were so many things going on.

Finnish Patent No. 821,099 describes the use of S1-leptomyces actinomycetes in soil to eliminate soil-borne or seed-borne diseases.
ycete fungi) is used. These actinomycetes of the genus Streptomyces not only suppress disease activity in seeds, but also suppress the transmission of diseases from diseased seedlings to healthy seedlings.

British Patent No. 1,357,538 discloses that aureofacients microL+ of the genus Strebtomyces has an apparent effect on plant diseases.
discloses a new antibiotic produced by e).

The effective agent is isolated pure from the mycelium of the microorganism.

U.S. Pat. No. 2,992,162 discloses that Streptomyces griseus
describes a method for producing candicidin, an antibiotic that inhibits bacterial growth, by using

Streptomyces griseoviridis (gri seov-i) produces antibiotics with aromatic heptaene structure.
It is still surprisingly recognized that the addition of strains of actinomycetes (rjdis) to seeds, substrates or plants is effective in controlling plant diseases.

S (Streptomyces). Griseoviridis actinomycin strains are described, for example, in Antibiotics and Chen+the
rapy 6 (1956) 100-115.

Previously, S. Griseoviridis was not recognized to produce butaene to aromatics.

According to the literature, macrocyclic aromatic heptaenes as antibiotics have the following types of general structures:

(Left below) Sugar derivative, where R is C
HJ, O}I, H or keto group, R
' is CH or I-1. It is known that aromatic heptaene has the effect of inhibiting bacterial growth.

(Means and effects for solving problem a) The method for eliminating plant diseases and plant storage diseases of the present invention includes adding Streb 1 hemyces griseoviridis actinomycin strain to seeds, substrates, or vegetation. The above-mentioned bacterial strain produces an antibiotic having an aromatic hebutaene structure having the above formula, and the antibiotic has an aromatic hebutaene structure of about 368, 388 and 412 nm (r
The antibiotic is characterized by having a maximum value of ultraviolet absorption at a wavelength of The gist is that it is soluble in polar organic solvents containing water and dimethyl sulfoxide.

In the method of the present invention, the effective agent does not need to be isolated pure from the Streptomyces griseoviridis mycelium that produces it; instead, the effective agent is isolated from the mycelium of the Streptomyces griseoviridis that produces it; Griseoviridis suspensions are added to seeds, substrates or vegetation at extremely low concentrations of 104 spores/m Q and at extremely low levels of 1 dQ/rn, making it economical and easy to use. 9 producing aromatic heptaene. Griseoviridis is known to be effective against at least the following pathogens: That is, Alternaria brassicicol.
a), Rhizoctonia asorani
solani) % Fusarium carmorum (Fu
sarium culmorum). F. Oxysporum (F. oxysporulI1), Agronobecterium tumfaciens (Agroaocte)
rium tumefaciens), Pizium S
P (Pythium SP.) Botrytis cinerea, Phomopsis sclerothioides
The strain of Streptomyces griseoviridis used in the above example was isolated from peat.

The present invention will be described in more detail below based on examples.

Ya41 Raw L Pure isolation of active drugs For the pure separation of active drugs and antibiotics, a commonly used method is to extract them directly from the cell mass and/or the culture medium, and then extract the extract. It is a method of concentrating and purifying. S. Griseoviridis is grown in culture medium containing glucose 4g/Q, yeast extract 4
g/fl and malt extract was fermented on 10 g/Q and the cell mass was separated.

0.5-1 volume part of acetonitrile was added to the cell mass or culture solution, and the mixture was shaken. The obtained S suspension was centrifuged at 3.00 rpm for 10 minutes.

The supernatants were combined and evaporated to dryness. After the syrupy residue was suspended in water and the pH was adjusted to 3.0 by HCR, the same volume of acetonitrile was added thereto and the mixture was shaken. 80 g/Q of NaCQ was added to this mixture and then most of the acetonitrile was separated as supernatant. The lower phase was further extracted using 0.5 parts by volume of acetonitrile, and the supernatants were combined. Depending on need, N a
The supernatant was washed with O.L. Supernatant liquid (PH7)
was evaporated to dryness in a circular evaporator, and methanol (10-2
0 mQ). The resulting solution was transferred to a test tube and concentrated to approximately 1 mQ under nitrogen flow, after which a precipitate formed at the bottom of the tube. lmk of methanol was added to the tube, after which most of the active drug dissolved and most of the precipitate remained undissolved. The bright dark brown solution was placed in a Sephadex L H20 gel column for chromatographic analysis.

Purification of active drugs The purified and separated active drugs are purified using Saphadex L H
It was purified by column chromatography using a -20 column. The eluent used was phosphate buffer (0.
06M, pH 7.2), to which methanol (0-100%) was added at each step. 0.4-0.6 mixture
Elution was performed at a flow rate of mQ/+nin. 2 or 4 mQ fractions were collected and tested for efficacy against Fusarium or Alterinaria. Furthermore, the absorbance of the fraction was measured at a wavelength of 254 nI1 for impurities and at a wavelength of 380 nm for active drug. Then, plot the chromatograph l2, A2. Or A3. . is shown as a function of fraction number (Figure 1).

Analysis by thin-layer chromatography (TLC) to evaluate the purity of the pure separated fractions, while for bioautography and HPLC analysis, separation by thin-layer chromatography (TLC) method was used. Thin layer chromatographic analysis is
silica 6 as phase n + material)
0 and chloroform, ethanol and water as mobile phase 50:50:
The experiment was conducted using 8 solutions. thin layer
r) is tested in the visible and ultraviolet regions, and the R of the active drug is
The f value was determined by bioautography method. In bioautography, nutrient agar containing the test organic matter (Alternaria brassicicola) was used.
agar) on the surface of the phase, followed by 2
The effective drug was segregated by culturing at 8°C. The results were known after 3-4 days, at which time the location of the spot of active agent was observed as an area where no B sample had grown. Samples of active drug were transferred to Sephadex L H
- The Rf value obtained when passing through the 20 column is 0.
．． It was 33.

For liquid chromatographic analysis of active drugs, adjustment layer (pr
Separate layer chromatography was performed in the same manner as analytical layer chromatography. Measure the Rf value of the separated fraction, remove the sample corresponding to each Rf value region,
The active agent was extracted from the sample with a mixture of tetrahydrofuran or acetonitrile and water (50:50). The extract was subjected to efficacy testing and liquid chromatography evaluation.

High pressure liquid chromatography (HPLC) liquid chromatography. Bischoff ODS}1yps
It was performed using a rsil R P =18 reverse phase column and isocratic elution. As an eluent, EDTA aqueous solution or acetic acid
- Ammonium acetate mixed with nitrile or tetrahydrofuran was used. Amphotericin B was used as a comparative drug. This is a compound similar to the active drug produced by actinomycetes, albeit of the non-aromatic heptaene type. The chromatogram of the active drug is shown in FIG. The main components of active drugs are designated by symbols A and B. Indicated by C. The content is a mixture of polyene fractions, all of the separated polyene fractions being heptaene according to the UV spectrum.

The active agent degrades upon treatment with tJV light.

The distinct polyene peak in HPLC disappeared in the analysis of the UV-treated sample. That is, upon UV treatment, the active drug lost its activity, and as a result, the ineffective drug did not show a characteristic peak like the active drug. From this, it can be concluded that the efficacy is related to the structure of the boriene.

UV and IR spectra of active agents In the ultraviolet spectrum of active agents, the characteristic curve of heptaene polyene is prominent. A mixture of active agents extracted from actinomycete samples (4% in dimethyl sulfoxide)
The UV spectrum of 0 μg/mQ) is shown in FIG. Judging from this UV spectrum, the active drug ingredient contains seven conjugated double bonds forming a typical heptane structure, and their maximum wavelengths are approximately 368, 388 and 412 in λ value ( using DMSO as the solvent).

Characterization of active agents using IR was carried out on crystalline material obtained by concentrating oily solutions from cell masses and purified by extraction with various solvents.

The infrared spectrum is carbonyl region @1,750-1,
It showed absorption at 500 am' (Fig. 4).

Confirmation that the effective drug is aromaticAccording to an article published in a scientific journal, whether or not hebutaene is aromatic can be determined at least by hydrolyzing heptaene with an alkali to liberate it. This can be demonstrated by detecting para-aminoacetophenone (PAAP) or by detecting para-aminobenzoic acid (PABA) biosynthetically linked to the active drug molecule. P
ABA labeling (PABA-label
ling) can be detected, for example, using radioactive PABA and detecting radioactivity from the active drug molecule. The results of hydrolysis tests and the radioactivity of the active agent labeled with radioactive PABA are discussed below.

Identification principle of para-aminoacetophenone in heptaene complex: A small amount of pure isolated heptaene complex was subjected to alkaline hydrolysis and extracted using chloroform 11. A semi-prepared version of para-aminoacet-1-phenone (PAAP)
+e-preparative) The PAAP was isolated from the chloroform extract using a HPLC power ram, and the PAAP was purified using gas chromatography and mass spectrometry techniques by selectively performing IlIO of the signal corresponding to a molecular weight of 135. Confirmed in separated components. Analytical results showed that alkaline treatment of the active agent caused para-aminoacetophenone to separate into solution. This could be detected on the basis of the retention time versus mass spectrum (Figure 5). Fig. 5 shows a GC (gas chromatography) mass chromatogram (A) of a precipitate corresponding to para-aminoacetophenone purified using HPLC, and a PAAP
Mass vector 1 ゛ (B) corresponding to the GC retention time of .

Detection principle of radioactive “C-PABA” in effective drugs: Strain 6
1 (strain 61) Y M G (yea
steextract, malt extract, g
lucose medium) culture medium (same composition as in Example 1), and "C-PAB" at the beginning of culture.
A500.000-1,500, OOO dpm/culture (dpm/culture) was added. The test tubes were shaken at 28° C. for approximately 50 h, after which the active drug was extracted from the cell mass. The active drug content in cell mass extracts and culture media was determined. HP that measures radioactivity to measure the transfer of radioactivity to active drugs during biosynthesis.
This was verified by determining the radioactivity of the active agent with an LC detector (Radiomatic). The labeling results are shown in Table 1. Figure 6 shows a typical radiochromatogram, in which the first peak is 14C.
-PABA, the next broad peak indicates the active drug. This chromatogram shows that the radioactivity is incorporated into the active agent, which also suggests that the active agent is aromatic. The chromatogram also shows that all active agents are labeled and aromatic.

However, the rate is the activity of the nutrient base, a part of which is mixed with the active drug. Labeling efficiency (labeliB
efficacy) was calculated based on the radioactivity ratio of the active agent produced to the radioactivity added.

yield of effective drug
e agent) = μg/g of active drug in cell mass Specific activity =
dpm amount/effective drug amount for 1 gram of cellsRadioactivity (activity dpm) CPM display frequency (readin) using the calculated efficacy (65.2%)
g) The results of the calculated dpi+display frequency labeling test indicate that some of the radioactivity has entered the active drug. According to the radiochromatogram, the active drug was identified by 14C-radioactivity, but PA
A portion of BA remained intact and was extracted from the cell mass along with the active drug. Thus, the ratio of the radioactivity of 14C-PABA to the radioactivity of the identified active agent in the cell mass extract was approximately 0.56. In addition, approximately 1.7 times more unmixed PABA was detected in the culture medium than in the extract. A total of 622,000 dpi+ (sample 2) and 840,000 dpm (sample 3), i.e. 61% and 55% of the radioactivity added to the culture, were detected in the samples analyzed by the above method.Radioactivity balance of the
activity) remained mostly in unextracted material in the cell mass (its radioactivity was not measured).

The results show that an active drug has been identified and, based on the existence of published research data, its exfoliating ability is similar to that of the aromatic group (para-aminoacetophenone).
dication), it can be concluded that it is related to the active drug molecule. Thus, it is clear that the idle strain of Actinomycin-1, which was the subject of research, produces aromatic heptaene.

Daikoku ffi Solubility of active drug The 18 solubility in various solvents of the evaporation residue extracted from the cell mass containing the active drug was determined by adding 2■ precipitate to the solvent considered for the 0.1 + nQ batch. This was considered.

The active agent was dissolved in a polar organic solvent containing acetonitrile, methanol, acetone, and tetrahydrofuran, as well as water, such as an aqueous solution of DMS○.

Efficacy of pure isolated active drug precipitates The pure isolated and lyophilized active drugs were dissolved in dimethyl sulfoxide (DMSO) lmg/1+o (1) and their MuC values (minimum inhibition 05 degrees, minimum
al inhjbitory concentration
on) by diluting the solution widely! The results are shown in Table 2.

The solubility of each blunt-isolated heptaene T4 body and reference drug was evaluated using the K-primary MIC value (μg/mu).
B represents amphotericin B.

According to the solution dilution test described above, active drug samples TA54 and TA541.1 (both obtained from the alkaline washed organic phase) are almost as effective as candicidin and somewhat more effective than amphotericin B.

The MIC values of pure isolated precipitates as a function of polyene content (sum of peak heights in HPLC analysis) suggest the idea of purity of the sample compared to candicidin (Figure 7). The figure shows that polyene content increases efficacy (
It clearly shows how the MIC value increases as the MIC value decreases). Pure isolated active drug TA54
The purity of T A 5411 and T A 5411 is almost on the order of candicidin, which was used as a reference drug.

Effects of S. griseoviridis and other actinomycin strains (1-10) on Alternaria causing wilting of cauliflower on Ibumi Salmon Ubito substrate, and the effectiveness of these strains in liquid culture. We considered the situation regarding the birth of a tree. Seeds were contaminated with Alternaria and various actinomycetes strains (10"spores/m Q
) was treated with an aqueous suspension. The results are shown in Table 3. Reference strain 8-10 is S. It may also be a strain of Griseoviridis actinomycinto.

(Margin below) One sho table S. Griseoviridis Reference strain here, O in ne is healthy; 1 is Having become ill, 2 means it has withered.

The disease onset suppressive effect was calculated based on the following formula.

The table shows that strains that produce effective drugs offer high inhibitory efficacy.

Rhizoc.
tonia) was investigated on steam-treated soil substrates. The substrate was contaminated with Rhizoctonia and the seeds were added to an aqueous suspension of actinomycetes (concentration 10 spores/m
Q). The results are shown in Table 4.

Isamu 4 It is difficult to suppress Rhizoctonia, which causes oilseed oilseed rape to wither, and there is no suitable method for chemically suppressing it. The results obtained by the method of the invention are therefore very good.

Control by chemical agents has been used on carnations, but this has not been satisfactory. Fusarium damping-off of carnations is caused by S. Suppression by B. griseoviridis (strains 1 and 2) was investigated. The experiment was carried out in a commercially available greenhouse by culturing P1 to Ii on substrates. The results are shown in Table 5.

Table 5 Regarding treatment Nα: 1; Non-treated example 2; 1% actinomycete suspension (
10′ spores/rgQ) were sprayed twice.

3; Roots were soaked in a 1% suspension before planting, and 0.0% during the growing season.
A 1% suspension (104 spores/IIQ) was sprayed twice.

4; Roots were soaked in 1% suspension, and during the growth period, 0.1% suspension (
104 spores/mQ) were sprayed twice.

Roots were soaked and sprayed once a month with a 5:1% suspension.

The numerical effects of various Streptomyces griseoviridis on the yield of oilseed oilseed rape were investigated through field tests. The results are shown in Table 6.

(The following is a blank space) Table 6 However, the numbering of the actinomycin strains described in Examples 10-12 corresponds to the numbering in Table 3 of Example 9.

(Effects of the Invention) In the method of the present invention, S. A suspension of griseoviridis is used, which is economical and simple because it only needs to be added to the species, substrate or vegetation at very low concentrations and infrequently to be effective.

[Brief explanation of drawings]

Figure 1 is a chromatogram spotted as a function of fraction number, Figure 2 is a chromatogram of the active drug, Figure 3 is the UV spectrum of the active drug mixture, Figure 4 is the infrared spectrum, and Figure 5 is the infrared spectrum. A diagram showing the relationship between retention time and mass spectrum, FIG. 6 is a typical radiochromatogram, and FIG. 7 is a diagram showing the relationship between effective drug concentration and MIC value. one or more one

Claims (1)

[Claims] 1. The method is characterized in that seeds, substrates, or vegetation are treated by adding a Streptomyces griseoviridis actinomycete strain, and the above-mentioned strain has the formula ▲ mathematical formula, chemical formula, table, etc. ▼ (wherein R is any one of CH_3, OH, H or keto group, and R' is CH_3 or H), which produces an antibiotic having an aromatic heptaene structure, and the antibiotic has about characterized by having ultraviolet absorption maxima at wavelengths of 368, 388 and 412 nm (using DMSO as solvent) and absorption in the carbonyl range 1750-1500 cm^-^1 in the infrared spectrum; and a method for eliminating plant diseases and plant storage diseases, characterized in that the antibiotic is soluble in a water-containing polar organic solvent and dimethyl sulfoxide. 2. In species, substrates and vegetation, S. 2. A method according to claim 1, characterized in that the G. griseoviridis strain is added as a suspension. 3.S. 3. A method according to claim 1 or 2, characterized in that the griseoviridis strain is added as a suspension containing at least approximately 10^3 spores/ml. 4.S. At least 1 liter of griseoviridis strain to the substrate
Claim 3 characterized in that it is applied at a ratio of d/m^2.
Method described. 5.S. Claim 1, characterized in that the griseoviridis strain is added by soaking the roots of the seedlings in an actinomyce sheet suspension before planting and/or by spraying the vegetation with an actinomyce sheet suspension,
The method described in 2 or 3.