JPH0699260B2 - Plant active foliar spray - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a spraying agent which is spread on the leaf surface of a plant to enhance resistance to pests and diseases and to enhance activity such as harvesting of plants.

[0002]

2. Description of the Related Art Heretofore, foliar sprays for controlling pests and diseases mainly consist of chemical agents, and there has been no effective spray for reducing pests and diseases and improving plant activity by using microorganisms.

[0003]

The foliar sprays using conventional chemical agents are problematic because their side effects and their accumulation in plants adversely affect foods and humans. In addition, it was not possible to sufficiently enhance the activity of plants by the foliar spray using a chemical agent. The present invention solves these conventional problems, and provides a safe plant active foliar spray that utilizes microorganisms and is highly resistant to pests and diseases and that can enhance plant activity.

[0004]

Means for Solving the Problems The gist of the present invention which has solved the above problems is as follows: 1) Clostriduim thermocem SK522 (Clostriduim thermocem)
llum biovar.SK522, Micro Engineering Research Center No. 3459)
And Thermus Aquatics SK542 described in 2 below
(Thermus aquaticus biovar SK542, Mikokenjojo 3382 No.) and a mixed culture obtained by symbiotic mixed culture under a thermophilic environment of 60 ° C. or higher, or a plant active foliar spreading agent containing the mixed culture 1 It has lignin solubilizing ability, the optimum growth temperature is 65-72 ° C,
A thermophilic fibrin-degrading bacterium that grows in the temperature range of 40 to 80 ° C. and ferments actively, Clostriduim thermocellum biovar.S
K522, Mikori Kenjo No. 3459) Note 2 Absolute aerobic, optimum growth temperature is 72 to 76 ° C, 40 to 82 ° C.
It grows in a medium of normal concentration in the temperature range of
Thermos aquaticus SK542 (Thermus aquaticus biovar S) which produces a proteolytic enzyme and a carotenoid-type yellow pigment with a thermophilic broad-range action hydrogen ion concentration of 85 ° C and a action hydrogen ion concentration pH = 4.0 to 11.3
K542, Micro Engineering Research Center No. 3382). In addition, the thermophilic fibrin-degrading bacterium Clostridium
Thermocellum SK522 (Clostriduim thermocellum b
iovar.SK522, Microtech Research Institute No. 3459) and Thermus aquaticus bi
ovar SK542, Micro Incorporated Research Article No. 3382) are all related to the discovery of the present inventors, and are hereinafter simply referred to as SK522 strain and SK542 strain, respectively.

[0005]

The present invention provides useful new strains of SK522 and SK5.
Effectively use a mixed culture of 42 strains. The synergistic effect of symbiotic mixed culture of SK522 strain and SK542 strain is
It not only enhances the solubilization of lignin, but also enhances the ability to decompose fibrin and other functions. The present invention is a liquid spray containing the symbiotic mixed culture as an effective main component, which is diluted to a required concentration and sprayed on the leaf surface of a plant for use. The symbiotic mixed culture of the SK522 strain and the SK542 strain of the present invention promotes the growth of useful foliar bacteria, suppresses plant pathogens, and imparts strong resistance to pests and diseases. At the same time, it produces useful growth micro-factors for plants, performs nitrogen fixation, enhances plant activity, and increases yield.

The functions of each new strain will be described below.
The SK522 strain has a strong fibrin-decomposing ability and a weak or weak lignin-solubilizing ability. The SK542 strain is an absolutely aerobic and moderately thermophilic bacterium, and produces a strong proteolytic enzyme having a thermophilic broad-acting hydrogen ion concentration activity and a carotenoid yellow pigment. Physicochemical properties of proteolytic enzyme produced by SK542 strain

[Table 1] It is a complex of several unstable enzymes in addition to protease A and protease B, which are considered to be novel enzymes having the above physicochemical properties. Physicochemical Properties of Pigment Produced by SK542 Strain The yellow pigment produced by this bacterium is a carotenoid yellow pigment, which is insoluble in water and soluble in the following solvents. Methanol, ethanol, petroleum ether, benzine,
A mixture of acetone, chloroform, acene + methanol (7: 3) and acetone + ethanol (1: 1).
Also, the infrared absorption maximum value is 450 nm. Other 430,
There is a small peak at 435,470 nm.

The thermophilic symbiotic mixed culture of these two new strains is not an additive action of the functions of the respective strains, but a synergistic effect produced by the interaction, and enhances the functions of each other. It strongly and stably decomposes organic components such as lignin, fibrin, and proteins quickly and strongly. A plant active foliar spray can be obtained by using the mixed culture of the present invention as an effective main component. This plant-active foliar spray enhances its effect further by mixing useful microorganisms in addition to the SK522 strain, the SK542 strain depending on its use, purpose, climate, climate, soil, and obtains a new useful effect. it can.

[0008]

EXAMPLES The present invention and examples of use thereof will be described in detail below. The microorganism used in the present invention is a new SK.
522 strain and SK542 strain. Properties of both new strains are as follows. In addition, all the tests and classification and identification of mycological properties described below are carried out by the Burgers Manual of Determinative Bacteriology 7th Edition (19
57), 8th Edition (1974) and Burgers Manual of Systematic Bacteriology No. 1
Volume (1984), Volume 2 (1986) (Bergey's Manu
al of Determinative Bacteriology 7th. Ed. (1957), 8t
h.Ed. (1974) .Bergey's Manualof Systematic Bacterio
logy Vol.1 (1984), Vol.2 (1986).).

[I] SK522 strain (Clostridium thermocellum biovar SK522, Mikori Kenjojo No. 3459, FERM BP-3459) Mycological properties It is impossible to grow alone without a concomitant bacterium. However, since the associated bacteria are easily isolated and purely cultured, it is the result of testing various properties of the isolated associated bacteria based on which they were co-cultured. Shape There are also straight rod shape and slightly curved shape. Alone, sometimes two. 0.3-0.5 × 2.2-4.0 μ. When the culture becomes old, it extends into filaments, long-chain, and lengths of 5.7 to 12.8.
μ. Motility is not observed in pericardium and room temperature hanging drop specimens. Oval or circular spores are formed at the ends to expand the cells and form a club. Gram negative.

Culture Properties (1) Plate Culture It does not grow on plate culture using broth agar or other common medium. However, while the medium presented by Viljoen et al., Tetrault circular filter paper agar plate forms colonies with associated bacteria and yellow spots, it cannot be produced by this bacterium alone. (2) Slope culture, stab culture Addition of filter paper treated with Schweizer reagent to filter paper Viljoen et al. Medium Agar slope culture and stab culture do not grow. (3) It does not grow on potato culture, gelatin culture surface, or puncture. (4) Liquid culture Viljoen et al. Medium, fibrin gravy,
Growth is observed only in a medium containing fibrin such as fibrin peptone water (poor growth). If a substance other than fibrin such as glucose or xylose is used as a carbon source, the degrading power of fibrin is lost.

Physiological and biochemical properties 1. Enzymatic action, etc. (1) Fibrinolysis The fibrinolytic enzyme possessed by this bacterium is essentially composed of β
It is a complex of -1,4-glucanase. Secreted extracellularly, action temperature 80 ℃ or more, same hydrogen ion concentration pH = 1
It was confirmed that several kinds of enzymes having heat resistance and alkali resistance of 0.0 or more were contained. It cleaves from the ends of macromolecules of fibrin forming a micellar structure to produce glucose, cellobiose, cellooligosaccharides and the like. When the substrates that act on these enzymes are mainly shown, it decomposes filter paper FP-ase: Positive Avicel is decomposed Avicelase: Positive Cellobiose is decomposed into two molecules of glucose Cellobiase: Positive (2) Lignin-decomposed Inawala lignin solubilization : Positive (weak, weak) (3) Proteolytic enzymes: Negative peptidase: Positive (4) Starch hydrolysis test: Slightly positive (5) Hemicellulose, xylan, pectin hydrolysis test : Negative (6) Invertase, Maltase: Positive (7) Lipolysis test: Negative (8) Oxidation reaction Hydroquinone reaction: Positive Tyrosine reaction: Negative (9) Reduction action: Positive 2. Product Test (1) Fibrin Fermentation Fermentation rate of 78-91%, vigorous fermentation of fibrin, ethanol, methanol, acetaldehyde, acetic acid, lactic acid, formic acid, lactic acid, succinic acid, fumaric acid, tartaric acid, gluconic acid, It produces glucose, cellobiose, cellooligosaccharides, cellodextrin, large amounts of carbon dioxide, hydrogen, and hydrogen sulfide. (2) Produced acid from other sugars and alcohol Produced acid from glucose, sucrose, maltose, and cellobiose. (3) Gas generation test Although gas is generated more violently than fibrin, no gas generation is observed from glucose, sucrose, maltose and cellobiose. (4) Peptone water test Ammonia: Slight reaction Indole: Positive skatole: Positive Hydrogen sulfide: Positive (5) Pigment production Usually, carotenoid yellow pigment production 3. Growth conditions (1) Growth temperature Optimum temperature is 65-72 ° C, temperature range is 40-80 ° C, 4
It does not grow below 0 ° C. (2) Hydrogen ion concentration Optimum hydrogen ion concentration pH = 6.7-8.0, the range is 5.6-9.6. (3) Nitrogen source Peptone is the best, and urea, uric acid, asparagine, sodium glutamate, etc. are also good. Ammonium salts are also good sources of inorganic nitrogen. (4) Carbon source When the subculture is continued with carbohydrates other than fibrin, its growth and fermenting power are lost. (5) Relationship with oxygen Eh = 200 to −250 mV, which is an anaerobic bacterium. (6) Requirement of micronutrients Micronutrients such as biotin, pyridoxamine, vitamin B ₁₂ and _p -aminobenzoic acid are required. 4. Content of G + C of DNA mol% of G + C = 38-40 (Tm) is estimated.

Taxonomic position: SK522 which actively ferments fibrin at an optimum growth temperature of 65 to 72 ° C. and a temperature range of 40 to 80 ° C.
The following thermophilic fibrinolytic bacteria are similar to the strains. Clostridium ther
mocellum Viljoen, Fred and Peterson, 1926 .: Jour. A
gr. Sci. (London), 16,7 (1926).) C. dissolvens
Bergey et al. 1925, aka Bacillus cellulose dessolve
ns Khouvine, 1923 .: Ann. Inst. Past.37,711 (1923); Be
rgey's Manual, 2nd.Ed. (1925) p.344.) C. thermocellulas
eum Enebo, 1951.: Bergey's Manual, 7th.Ed. (1957) p.68
9.) Bacillus thermocellolitex
ellulolyticus Coolhaas, 1928.: Cent Bakt.II, 75,101
(1928), 76,38 (1929).) Bacillus thermofibrinco
lus Itano and Arakawa, 1929: Farming, 5,816,921 (1929); 6,
248,257 (1930).) Among these, Clostridium disorbens is a bacterium that is very similar to this strain in morphological, culture, and physiological biochemical tests except for the following four differences. (1) It has a lignin-solubilizing ability although it is weak or weak. (2) Suitable growth temperature of 65 to 72 ° C, growth temperature range of 40 to 80
Does not grow at temperatures below 40 ° C. (3) Actively ferment fibrin, but not hemicellulose, xylan, pectin, etc. (4) Lignin solubilization ability is the genus Thermus
It is prominently symbiotically emphasized by certain types of bacteria, and at the same time has a positive effect on the fibrinolytic activity and other action functions of this bacterium. But now, the Burgers Manual (Be
rgsy's Manual of Systematic Bacteriology Vol.2 (19
86) p.1104, p.1141.), Only Clostridium thermocellum is described as a thermophilic degrading bacterium. All of them are treated as subspecies or variants of Clostridium dissolvens, or incompletely studied. Therefore, for the classification and identification of this strain, it was decided to compare and examine Clostridium thermocellum and various mycological properties, and at the same time, other thermophilic fibrinolytic bacteria were also compared and used as a reference.

As described above, the results of comparative examination of various mycological properties are summarized as follows. The characteristic properties of this strain and known thermophilic fibrinolytic bacteria including Clostridium thermocellum are listed. The differences are the above-mentioned four items, and they can be enumerated in various ways such as the action on various other carbohydrates, the requirement of micronutrients, etc., but what I would like to emphasize especially is the problem with lignin. This strain has a lignin solubilizing ability, and the solubilizing ability is thermus aquaticus SK542 (Thermus aquaticus bi
It is significantly enhanced by the symbiotic mixed culture with ovar SK542). In contrast, there is no description about lignin of other thermophilic fibrinolytic bacteria including Clostridium thermocellum. If so, it is about its inhibitory effect on fibrin degradation. For this reason, a new strain (Strain) was established based on the physiological and biochemical properties of the lignin-solubilizing ability of this strain as one basis, and Clostridium thermocellum SK522 (Clostridium thermocellum biova
r SK522).

Microorganism Accession Number The microorganism accession number of this strain is Microtechnology Research Institute No. 3459 (FERMBP-3459). This strain is the standard strain, and Clostridium thermocellum (Clostrid
ium thermocellum Viljoen, Fred and Peterson, 192
It has the ability to solubilize lignin among bacterial species belonging to 6.), and significantly enhances the ability to solubilize lignin by symbiotic mixed culture with certain bacteria belonging to the genus Thermus Brock and Freeze1969. It is a novel strain with physiological and biochemical properties that characterizes the SK522 strain and natural and artificial mutants.

Screening of SK522 strain Soil, beach sludge, manure / manure, human / livestock faeces are used as separation sources, and concentrated culture is repeated several times at 55 to 65 ° C., then presented by Viljoen et al. Medium Tetrault circular filter paper Agar plates are used for isolation according to standard methods. The bacterium was able to be isolated substantially purely. The salt composition suggested by Viljoen et al. Is suitable as the medium, but if necessary, a trace amount of inorganic metal salts, vitamins, growth promoting factors such as yeast extract, etc. may be added. Viljoen's medium (Viljoen, Fred and Peterson 1926) Peptone 5.0 g Calcium carbonate Excess ammonium phosphate sodium 2.0 g Potassium acid phosphate 1.0 g Magnesium sulfate 0.3 g Calcium chloride 0.1 g Ferric chloride Trace fiber Element (filter paper) 15.0 g Imizu 1000 ml

Utility of SK522 strain The present SK522 strain is a fibrin-degrading bacterium that is originally active. The present inventors have found that this strain has a lignin solubilizing ability, although this strain is weak or weak, and the solubilizing ability of the strain is symbiotic mixing of this strain with a certain bacterium of the genus Thermus (genus Thermus). It was remarkably enhanced by culturing, and also had a favorable effect on the fibrinolytic activity and other action functions of this strain. In addition, the success of this mixed culturing enables the decomposition and utilization of many types of persistent organic materials, improves their productivity, and ensures the nodoko composting method. The manufacturing method was established. And SK
The formation of a healthy active soil microbial flora mainly consisting of the 522 strain and the SK542 strain stabilized the growth and action function of effective bacteria against changes in environmental factors, and eliminated or overcame soil diseases and continuous cropping disorders. In addition, it is expected that new technology of microbial utilization such as new technology of direct spraying on soil or leaf surface of plant and deodorization of human waste and decomposition of hard protein will be developed.

[II] Strain SK542 (Thermus aquaticus biovar SK542, Microtechnical Lab Article 3382, FERM BP-3382) Mycological properties Long rod-shaped, 0.4-0.6 × 3.0-5 0.0μ. Under certain conditions, for example, when culture becomes old, filamentous, length 20 to 130
μ. No flagella and no motility in room temperature hanging drops.
No endospores. Gram negative.

Cultural properties Proliferation is active. Generation time is 20 to 50 minutes. (1) Plate culture 3% agar, 60 ° C culture: yellow, relatively dense, small round colonies. (2) Agar stab culture Only surface growth, yellow slightly expanded. (3) Liquid culture Grows in a film on the surface (static culture).

Physio-biochemical properties 1. Enzyme action, etc. (1) Proteolytic proteolytic enzyme: Positive (strong) (proteolytic enzymes) Gelatin hydrolysis: Positive peptidase: Positive (2) Starch hydrolysis: Slightly positive (3) Pectin degradation: Positive (weak) (4) Degradation of fibrin: Negative (5) Degradation of lignin: Negative Degradation of both fibrin and lignin is negative, but symbiotic mixed culture with thermophilic fibrinolytic SK522 strain allows lignin digestion of SK522 strain. It significantly enhances the solubilizing ability, and at the same time has a favorable effect on the fibrin decomposing power and other action functions. (6) Lipolysis: Negative (7) Invertase, maltase: Positive (8) Catalase, oxidase reaction: Positive (9) Hydrogen sulfide production: Positive (weak) (10) Sulfate reduction reaction: Negative 2. Product test (1) Raw acid and gas generation from sugar and alcohol No gas generation. A raw acid from glucose, galactose, maltose, lactose, and glycerol. (2) Formation of indole and skatole: Negative (3) Pigment production Yellow pigment (carotenoid pigment, maximum absorbance 450n
m, others with small peaks at 430, 435, 470 nm). However, it is scarcely produced in a synthetic medium containing glucose and other sugars as a carbon source.

3. Growth conditions (1) Medium concentration Bacteria of the genus Thermus are generally sensitive to organic matter concentrations. It is said that the less nutrients and the lower the concentration, the better. However, this strain is different from these in that it grows well even at normal concentrations. It grows even with salt NaCl 5% or more. Optimal concentration 2.
0-3.0%. In general, Genus Thermus bacteria do not grow in the presence of more than 2% NaCl. It grows even with sodium glutamate of 3% or more. It grows on sucrose or maltose 5% or more. Its growth is not inhibited even in the presence of 2% peptone + 1% yeast extract. Generally the genus Thermu
For the bacteria of s), trypton and yeast extract concentrations of about 0.1% are suitable, and they do not grow when they exceed 1%. (2) Growth temperature Optimum temperature is 72-76 ℃, growth temperature range is 40-82 ℃,
It cannot grow below 40 ° C. (3) Hydrogen ion concentration Optimal hydrogen ion concentration pH = 6.0 to 10.0. Growth hydrogen ion concentration range pH = 4.0 to 11.0. (4) Nitrogen source Proteins such as gelatin, glutamate, urea, and ammonium salt are often used as an inorganic nitrogen source. (5) Carbon source Glucose, sucrose, maltose, galactose, cellobiose, raffinose, stachyose, starch, glycerol, acetic acid, lactic acid, malic acid, glutamate are used. (6) Relationship with oxygen Absolutely aerobic. (7) Demand for micronutrients This strain has high demands for micronutrients. In addition to vitamins such as biotin, nicotinamide, and thiamine, a relatively high concentration of metal ions such as iron, manganese, and calcium is required for good growth of this strain. It is also sensitive to the amount of these minerals. (8) Susceptibility to antibiotics Like general Thermus genus (genus Thermus) bacteria,
It shows high sensitivity to penicillin G, chloramphenicol, tetracycline, streptomycin, kanamycin and other antibiotics. 4. Content of G + C of DNA mol% of G + C = 68 (Tm) is estimated.

Taxonomic position The bacterium does not grow in absolute aerobic condition, optimum growth temperature of 72 to 76 ° C, growth temperature range of 40 to 82 ° C, 40 ° C or lower. Since it is a non-spore-free, Gram-negative long bacillus, etc., in consideration of other mycological characteristics, the genus Thermus
k and Freege 1967). And, as a similar bacterium of the genus Thermus, Thermus aquaticus Broc
k and Freege 1969.:Bergey's Manual (1984) Vol.1, P.3
37.) Thermus thermophilus Osh
ima and Imahori 1974.:Int.J.Bacteriol.,24,102(197
4)) Thermus Flavus Saiki, Kimura an
d Arima 1972.:Agr.Biol.Chem.,36,2357(1972)), etc., but currently Bersey's Manual
s Manual of Systematic Bacteriology Vol.1 (1984) P.33
3) Genus Thermus Brock and Freeg 196
Bacterial species belonging to 7) are Thermus aquatics (The
rmus aquaticusBrock and Freege 1969),
Moreover, since it has the characteristic properties that cannot be found in the known bacterial species as described below, this strain is used as a new strain of Thermus aquaticus (strain), and Thermus aquaticus biovar SK54
We conclude that it is appropriate to name it 2).

(1) The lignin-solubilizing ability is remarkably enhanced by symbiotic mixed culture with the thermophilic fibrinolytic bacterium SK522 strain. At the same time, it has a positive effect on the ability to decompose fibrin and other functions. (2) It has a strong proteolytic activity with a wide range of working hydrogen ion concentration and working temperature. (3) The genus Thermus (genus
(Thermus) bacteria are very weak and grow well in medium of normal concentration. (4) Strong demand for micronutrients, demand for various vitamins and metal ions, and sensitivity to their mineral contents. (5) Yellow pigment (carotenoid pigment, maximum absorbance 45)
0 nm and others have small peaks at 430, 435 and 470 nm).

Microorganism Accession Number The microorganism accession number of this strain is Microtechnology Research Article No. 3382 (FERMBP-3382). The SK542 strain is a standard strain of this strain, and by symbiotic mixed culture with a thermophilic fibrinolytic bacterium (SK522 strain) among bacterial species belonging to Thermus aquaticus Brock and Freege 1969, A new strain with a physiological and biochemical property that significantly enhances its lignin solubilizing ability and at the same time has a favorable effect on the fibrin degrading power, and has physiobiochemical properties that embraces the SK542 strain and natural and artificial mutants ( strain).

Screening of SK542 strain Isolate sources are hot spring sources in various places in Kyushu, soils near hot spring sources, humus and the like. The separated sample is precultured at 55 to 60 ° C. and then isolated by 3% agar plating according to a standard method. The bacterium was able to be isolated substantially purely. Separation medium composition (Castenholz medium: Castenholz, RW; Bacteriol.
Rev., 33, 467 (1969)) Nitrilotriacetic acid 100 mg CaSO ₄ .2H ₂ O 60 mg MgSO ₄ .7H ₂ O 100 mg NaCl 8 mg KNO ₃ 103 mg NaNO ₃ 689 mg Na ₂ HPO ₄ 111 mg FeCl ₃ 0.28 mg MnSO ₄ .H ₂ O _{2.2mg ZnSO 4 · 7H 2 O 0.5mg} H 3 BO 3 0.5mg CuSO 4 0.016mg Na 2 M 0 O 4 · 2H 2 O 0.025mg ( yeast extract 5000 mg) (tryptone 5000 mg) (sucrose 10000mg ) Total volume (with pure water, pH = ~ 8) 1000 ml Note: The composition in () is modified by the inventors.

Utility of SK542 Strain The utility of this new strain is characterized by the following three functions. (1) By symbiotic mixed culture with a thermophilic fibrinolytic bacterium (SK522 strain), the lignin degrading ability of the bacterium is remarkably enhanced. At the same time, it also has a positive effect on its fibrin decomposing power and other action functions. (2) It has a strong proteolytic activity with a wide range of action hydrogen ion concentration and action temperature. (3) It produces an insoluble yellow carotenoid pigment. This yellow pigment in the fungus body is decomposed by other microorganisms and becomes a water-soluble low molecular weight substance, which is absorbed by the plant,
It helps improve the quality of fruits such as taste, color and storability. When the SK542 strain is applied, it promotes the growth of general heterotrophic microorganisms and soil-effective bacteria that propagate using this strain as a substrate. The above is the known genus Thermu (genus Thermu
In combination with the extremely weak sensitivity of organic matter concentration unlike bacteria of (s), in natural ecology and agricultural ecosystem, various kinds of persistent organic substances, hard proteins and surplus sludge, and various other organic materials are widely used. Is now available. Furthermore, this leads to the development of a new dimension of microbial utilization, such as productivity improvement, overcoming soil diseases and continuous crop damage, and deodorizing and decomposing human waste, and developing new technology for direct spraying on soil or plant foliage. Connect.

[III] Mixed culture of SK522 strain and SK542 strain 1. Large-scale culture of SK522 strain Using the medium of Viljoen et al., 1926 described above, under anaerobic or semi-anaerobic conditions, 65-70 ° C, 48-
After culturing for 60 hours, the culture solution is used as a culture containing cells. 2. Large-scale culture of SK542 strain Using the culture medium of Castenholz (1969), 70-75 under aerobic conditions such as aeration or shaking
Cultivate at 24 ° C for 24 hours, and use the culture solution as a culture.

3. Mixed culture of SK522 strain and SK542 strain Using the following medium, appropriate amounts of large-scale cultures of the SK522 strain and the SK542 strain were inoculated, respectively, to obtain 65-70.
Cultivate at 24 ° C for 36 to 36 hours to obtain a mixed culture of both strains. Then, this is made into a powder or granular material by using a shaping agent, and a product having a water content of 5 to 7% or less can be stored for 2 years or more. In addition, just before application, a large amount of the cultures of both strains are mixed in equal amounts and mixed well, and immediately applied, or even if it is manufactured into a powder or granule using a excipient, the quality performance of the final product is completely Unchanged. Further, the following E-medium for mixed culture is simply referred to as E-medium. E-medium for co-cultivation mixed culture K ₂ HPO ₄ 5 kg (NH ₄ ) ₂ SO ₄ 2 kg Urea 2 kg Peptone 5 kg Yeast extract 5 kg Filter paper (fibrin) 15 kg CaCO ₃ excess water (~ pH 7.0) 1000l 4. Utility of symbiotic mixed culture of both strains Under the condition of high temperature environment, symbiotic mixed culture with SK542 strain of absolutely aerobic Genus Thermus is anaerobic thermophilic fibrinolytic bacterium SK522. It remarkably enhances the decomposition and fermentation of natural refractory organic materials, which is almost impossible with the strain alone. First, the growth of the SK542 strain is S
It complements the proteolytic activity of the K522 strain.
In addition, decomposition and fermentation of persistent organic materials such as waste and manure, waste wood, and municipal sewage sludge are all in solid-state fermentation, and are in a well-ventilated environment. And
The growth of the SK542 strain in the early stages is transformed into a high temperature anaerobic environment, which allows the SK522 strain to grow in nature.

(1) Solubilization of lignin Here, it is particularly noted that the SK542 strain exerts a remarkable lignin-solubilizing ability of the SK522 strain. The persistent organic material actually exists in a state in which the fibrin is strongly bound to lignin and other organic components. Such natural lignocellulose can hardly be decomposed by the single application of the SK522 strain having a strong fibrinolytic activity. Although the chemical structure of lignin has not been completely clarified yet, phenylpropane, which has a side chain with three carbon atoms in the benzene ring, serves as the basic unit, and this unit is formed by a radical reaction catalyzed by peroxidase. It is a polymer compound that is randomly and three-dimensionally polymerized. It is a consensus view of many researchers and engineers that resistance to microbial decomposition is extremely strong and extremely difficult to decompose. However, S used in the present invention
The K522 strain, although weak or weak, solubilizes lignin. And, this lignin solubilization ability is SK542
It has now been clarified for the first time in the present invention that, for example, 50% or more of Inawara lignin is solubilized in a short time of about 10 days by symbiotic mixed culture with a strain. Its practical application is an important characteristic aspect of the present invention, and its action and effect are 50 to 8
It is exerted at a high temperature stage of 0 ° C or higher, and humification progresses rapidly to decomposition fermentation.

(2) Production of yellow pigments genus Thermu (genus Thermu) including SK542 strain
The yellow pigment produced by s) is a carotenoid insoluble pigment. This yellow pigment in the bacterial cells is decomposed by other microorganisms to become a water-soluble low molecular weight substance, which is absorbed by the plant body and transferred to a necessary site, which is just a convenient precursor. That is, such a yellow pigment not only promotes the growth and proliferation of rhizomes along with other lysed cell contents and secretions, and fermentation products, but also causes the formation of flower buds, fruit set, fruit enlargement, etc. It has been clarified by research conducted at the molecular biological level that it is deeply involved in the metabolic system of reproductive growth, and it has been developed into its application and has already been improved in quality such as fruit taste, color and storage quality. Is useful to.

(3) Mixing and complexing of microbial ecosystem When the bacterial cells of the SK542 strain containing the yellow pigment are applied, the growth of general heterotrophic microorganisms, soil effective bacteria and the like that propagate using the yellow pigment as a substrate is promoted. More importantly,
In the field such as compost production in the natural world, thermophilic microorganisms such as the SK522 strain do not immediately proliferate and activate. Initially, general room temperature heterotrophic microorganisms grow,
Fermentation heat due to these actions is accumulated to help increase product temperature, and only a single strain of thermophilic fibrinolytic bacteria and a small number of microorganisms are involved in the humification of natural persistent organic materials. Do not mean. It is required to mix and diversify the microbial flora. It is desirable to have a complex content that there are many effective bacteria of various types and that "feed (substrate)" is also appropriate. Thus, the high-temperature decomposition of persistent organic matter is
The bacterial flora centering on the K522 strain is formed first, and the humification process not only involves simple changes in the constituent components, but also the extremely complex interactions and transitions of the microbial flora, which are most efficiently and closely related. Of. Other effective bacteria to be mixed with the SK522 strain and the SK542 strain include the following.

(1) Culture of Hemicellulose Degrading Bacteria Hemicellulose forms a plant (cell wall) with fibrin, and is called xylan, araban, dextran, mannan, galactan, etc. depending on the sugars that compose this. As a culture of hemicellulose-degrading bacteria, Iwata's medium (1936) added to a concentration of about 1% of rice straw xylan is used, and it is anaerobically cultivated at 30 to 38 ° C. Usually Bacterium vulgatus, Bac. Prodigiosum, Bacterium mesentericu
s ruber), Microspyra Agariquefisense (M
A mixed culture of one or more strains such as icrospira ager-liquefaciens) is obtained. (2) Cultures of pectin substance-degrading bacteria Many of the bacteria that strongly decompose pectin substances belong to the Bacillus subtilis group bacteria and ethanol / acetone bacteria when they are aerobic, and to the lactic acid bacteria when they are anaerobic. In the present invention, using a Morish medium (Molisch 1939), soil, compost,
Separation sources such as horse manure, bagasse and chomp are 27
The inoculum is obtained by collecting culture several times in a thick layer and a thin layer at ˜35 ° C. for 3 to 5 days of culturing. In the present invention, cultures of one or more strains are used.

(3) Soil actinomycete culture It is generally difficult to say about the action of actinomycetes (Actinomycetales Buchanan, 1917) in soil. But,
It decomposes various organic substances, especially incombustible fibrin and lignin, together with other microorganisms, and plays an important role in the formation of humus, which is the source of soil fertility. Certainly, it has important significance in terms of microflora control. The culture of actinomycetes is Waxman's medium (Waksman 1919), fertilized soil as a separation source,
In addition, strong bacteria are collected by using the manure and manure. (4) Cultures of soil filamentous fungi and yeasts The most abundant locations of soil filamentous fungi are soils like bacteria and actinomycetes, and filamentous fungi in soils are naturally large in cultivated soil with plant roots, especially rhizosphere. Then, the work is also active. It is involved in the decomposition of organic substances such as plant remains and is related to soil fertility. It is considered that filamentous fungi are mainly active in the initial stage of decomposition. Next, there are many unclear points about the function of soil yeast. However, there are quite a few yeasts in the soil,
In addition, it is certain that it will affect coexistence with other microorganisms, soil activity, etc., regarding the abundant vitamins and growth factors contained therein. Soil filamentous fungi and yeast cultures are separated and cultured from soil or manure / manure using a Tuapeck Dox medium (Czapek & Dox, 1910).

(5) Culture of thermophilic Bacillus genus Bacillus which is generally aerobic, motile, and has endospores, and is a group of bacteria widely distributed in the natural world such as soil, leaf surface, and hay.
It is also reported that most of the bacterial strains have strong heat resistance and can grow even at 55 ° C. or higher, and thus are major microorganisms during compost production. In addition, secreting antibacterial substances such as bacitracin and basilicin has attracted attention in the field of soil plant pathology in recent years. From this, it is possible to try to utilize this bacterial group for crop production, and fermented soil or a suspension of manure / manure is heat treated at 80 ° C. for 10 minutes to be used as a separation source. Waxman's medium (Waksman, 19
22), and aerobically collect the bacterial group at 50 to 60 ° C. In the present invention, single or mixed culture of these bacteria is used. (6) Culture of yellow pigment-producing bacteria Yellow pigment produced by bacteria is a carotenoid pigment. There are many types of ripe compost, and many types. It is also often found on the leaves of plants. Using these as a separation source, a normal meat extract medium or peptone-yeast extract medium,
Incubate and separate at 25-35 ° C under aerobic conditions. It is easily identified by colors such as yellow-green, yellow, yellow-brown, and red, and is generally flavobacterium (Flavobacterium), chromobacterium (Chromobacterium), Pseudomonas, serratia, phototrophic bacteria. A culture of one or more strains belonging to, etc. is obtained.

Example of production of foliar spray and its application effect Test purpose: The effect of application of the foliar spray of the present invention to cultivated plants is examined. Foliar spray: A liquid product containing the following raw materials. Blending of raw materials Mixed culture of SK522 strain and SK542 strain 100 kg (mixed culture described in 0028) Culture of yellow pigment-producing bacteria 100 kg (culture described in 0034) Yeast extract 50 kg Trace element complex 2 kg (B, Mg, Mn)

1. Test method (1) Test cultivar: Shilocennari (soybean) (2) Test area: Yokote City, Akita Prefecture (3) Scale: 5a for both test and control areas (4) Soil: Kuroboku soil (5) Fertilization (kg) / A) No. Ward name Chemical fertilizer (464) Remarks 1. Test section 10 No. 1, 2 and both wards have the same season last year 2. Control area 10 Sukikomu. (6) Spread on leaves: Immediately before spraying, 500-1000 sprays
Dilute twice and once every period, 50k per 5a as follows
g to 100 kg (100 g of foliar spray raw material) is sprayed. 1 month after sowing, early growth, once a day, 3 times a day. Early flowering, once a day, three times a day. Fruit set, once or twice a day. (7) Growth item Seeding Harvest Remarks No. Ward name Cultivated soil, crushed soil, medium tillage, 1. Test area June 5th, October 30th Other shelling, 1. Control section June 5th, October 30th Simultaneously with the test section and control section.

2. Results and Discussion The results of the effect of foliar spraying on the prototype soybean are shown in Table 2. And Table 3. It is as follows. The soybean yield was 43% higher than that of the control plot, and strong resistance to pest damage could be seen. In particular, Table 3. The growth of yellow pigment-producing bacteria in is remarkable. Although there are many unclear points regarding the relationship between foliar bacteria and plants, the results of this test suggest that they are antagonistic to potential pathogens, produce trace growth factors, and fix nitrogen.

3. Main test data Main test data are shown in Table 2. And Table 3. It is as follows.

[Table 2]

[Table 3]

[0038]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, microorganisms that are harmless to humans and large animals are used to suppress plant pathogens to enhance resistance to pests, and to enhance plant activity. It can be increased to increase the yield. Also, unlike chemical pesticides, it does not accumulate and can be applied naturally and safely.

[Equation 1]

[Equation 2]

[Equation 3]

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Claims (1)

[Claims] 1. A thermophilic fibrinolytic bacterium as described in 1 below, Clostridium thermocellum SK522 (Clostriduim).
thermocellum biovar.SK522, Micro Engineering Kenjiro 345
9) and Thermus Aquatics SK described in 2 below.
542 (Thermus aquaticus biovar SK542, Mikori Kenjo No. 3382) and a mixed culture obtained by symbiotic mixed culture in a thermophilic environment of 60 ° C. or higher, or a plant active foliar spreading agent containing the mixture as an effective main component . Note 1 having a lignin solubilizing ability, a suitable growth temperature is 65 to 72 ° C.,
A thermophilic fibrin-degrading bacterium that grows in the temperature range of 40 to 80 ° C. and ferments actively, Clostriduim thermocellum biovar.S
K522, Mikori Kenjo No. 3459) Note 2 Absolute aerobic, optimum growth temperature is 72 to 76 ° C, 40 to 82 ° C.
It grows in a medium of normal concentration in the temperature range of
Thermos aquaticus SK542 (Thermus aquaticus biovar S) which produces a proteolytic enzyme and a carotenoid-type yellow pigment with a thermophilic broad-range action hydrogen ion concentration of 85 ° C and a action hydrogen ion concentration pH = 4.0 to 11.3
(K542, Micro Engineering Research Center No. 3382)