CN102027998B - Bacillus spore and trichoderma chlamydospore compound preparation and preparation method and application thereof - Google Patents

Abstract

The invention provides a composite preparation for preventing and treating plant diseases. The composite preparation contains bacillus spores and Trichoderma chlamydospores. The invention also provides a method for preparing the compound preparation and its application.

Description

Composite preparation of Bacillus spores and Trichoderma chlamydospores, its preparation method and application

technical field

This application relates to the field of biotechnology, especially the field of agricultural biotechnology. Specifically, the application relates to a composite preparation of Bacillus spores and Trichoderma chlamydospores, its preparation method and application.

Background technique

Bacillus subtilis (Bacillus subtilis) is a mesophilic aerobic spore-forming rod-shaped bacterium with rich and diverse physiological characteristics, widely distributed, and easy to isolate and culture. The bacterium exists widely in nature, is non-toxic and harmless to humans and animals, does not pollute the environment, can produce a variety of antibiotics and enzymes, has broad-spectrum antibacterial activity and strong resistance to stress. Bacillus subtilis can not only widely exist in the external environment such as soil, plant rhizosphere, and body surface, but also is a common endophytic bacterium in plants, especially in the roots and stems of plants. At present, the fungus has shown good disease control effect on rice, pepper, cotton, wheat, cucumber, soybean, corn and other crops. The most prominent features of Bacillus subtilis are fast growth, simple nutrition, and the ability to produce heat-resistant and stress-resistant spores, which are not only beneficial to the production of biocontrol agents, formulation processing, and survival, colonization, and reproduction in the environment, but also in batches. The production process is simple, the cost is also low, the application is convenient, and the storage period is long. It is an ideal biocontrol microorganism and has broad application prospects.

Bacillus subtilis colonizes in the rhizosphere, surface or body of plants, competes with pathogenic bacteria for nutrients around the plant, secretes antibacterial substances to inhibit the growth of pathogenic bacteria, and induces the plant defense system to resist the invasion of pathogenic bacteria, thereby achieving the purpose of biocontrol. Most of the main control targets are plant diseases caused by filamentous fungi, such as rice sheath blight (Stagonospora curtisii), tomato leaf mold (Cladosporium fulvum), soybean root rot (Fusariumgraminerarum), apple mold core (Alternaria alternata), Cotton blight (Rhizoctonia solani), cotton blight (Fusarium oxysporum f.sp.vasinfectum), etc. The biocontrol mechanism of Bacillus subtilis mainly includes three aspects: competition, antagonism and resistance induction. In addition, the antibacterial substances with biocontrol activity produced by Bacillus subtilis include various compounds such as lipopeptides, peptides, proteins, phospholipids, polyenes, amino acids and nucleic acids, which are effective against fungi, bacteria, viruses, etc. etc. have an inhibitory effect.

Although Bacillus subtilis has made some achievements in the biological control of plant diseases as a biological fungicide, there are still many problems on the whole (R. campbell, 1993).

First of all, as a biofungicide, the effect of Bacillus subtilis in the field is not stable. This may be due to the fact that the strains stored indoors for multiple generations are usually used, which is quite different from the activity of wild strains, and indoor pot experiments are different from the complex micro-ecological environment in the field. The results are quite different, resulting in a large difference between the results of the indoor inhibition zone and the field results. Secondly, the toxicity and safety of microbial preparations are generally considered to be better than those of chemical agents, tending to be non-toxic and non-residual, but some microbial preparations may also cause pathogenic bacteria resistance, especially those strains that inhibit pathogenic bacteria with antibiotic secretions. In Europe, animal preparations of Bacillus subtilis, which produce the antibiotic bacitracin zinc, have been included in the prohibited list, on the grounds that it is prone to drug resistance and antibiotic residues. There are few studies on the safety of microbial pesticides in our country, and most of them have not attracted enough attention. Finally, as microbial pesticides, the control cost is generally higher than that of chemical agents, and they are mainly protective agents. They are used after plant disease, and the effect is poor, and some are even ineffective. This is the disadvantage of many microbial pesticides.

In order to solve the above existing problems, enhance the biocontrol effect of biocontrol bacteria, and improve their adaptability to environmental conditions, many laboratories at home and abroad have used cell engineering and genetic engineering technology to genetically improve biocontrol microorganisms, and constructed New and efficient bio-control bacteria (Wang Jinsheng, 2002). For example, Chen Zhongyi et al. (1999) introduced the Bt gene into rice biocontrol Bacillus B916 through a plasmid vector, and laboratory tests showed that the recombinant strain had good insecticidal and disease control effects and good genetic stability. However, because many antagonistic genes, especially antibiotic synthesis genes, are composed of gene clusters with complex structures, genetic manipulation is difficult. Therefore, the technology needs further research.

Trichoderma has been used to prevent and control plant diseases for more than 70 years, and its biocontrol effect has been recognized. It is a biocontrol fungus widely used in the world. Trichoderma belongs to the subphylum Deuteromycota, Hyphophyceae, Hyphospora, Myxosporaceae, and Myxospora. It is a type of fungus that commonly exists in soil and is an important community of soil microorganisms. It can parasitize plants On residues and animal feces, it can often be isolated from plant root circles, leaves, seeds, and bulb surfaces. Trichoderma can parasitize on a variety of soil-borne plant pathogens. Since the 1980s, with the rapid development of molecular biology, the research on Trichoderma has mainly focused on how to improve its biological control effect, such as by strengthening the expression of chitinase and glucanase genes, interspecies fusion and other means . Trichoderma PDA colonies are initially cotton-like or densely bundled, white to off-white in color, and have no fixed shape. When the conidia matured, the colonies gradually turned green in varying degrees from the center to the edge, and very few were white powdery. Conidiophores emerge from the side branches of the hyphae, erect branches, branchlets are often opposite, the top is not enlarged, and conidia clusters are born on the top. Conidia spherical, light or colorless. Most Trichoderma species are not strict on nutrients, they can grow under various carbon and nitrogen sources, and can transform and degrade some harmful or persistent harmful environmental pollutants; they can directly use various monosaccharides, monosaccharides derivatives, organic acids; significant degradation of various polysaccharides (cellulose, hemicellulose) and related polysaccharides (chitin); can also convert and degrade some pesticides, such as: malathion, thatch, pentachloronitrobenzene, etc. Trichoderma can use complex and simple nitrogen, and hydrolyzed casein amino acid mixture, aspartic acid, alanine, and glutamic acid can all be well utilized. Under high nitrogen conditions, it is beneficial for Trichoderma to produce some enzymes, such as fiber enzymes, lactase, etc. Trichoderma is an aerobic bacterium, suitable oxygen pressure is conducive to the growth of mycelium and spore production; the optimal pH is 4.0-6.5; the growth temperature varies with species, most of them grow very rapidly at around 25°C and self-regulate the environment pH to adapt the environment to its growing conditions.

The range of antagonism of Trichoderma has a broad spectrum. Related studies have shown that Trichoderma has antagonistic effects on at least 29 pathogenic fungi belonging to 18 genera. The plant pathogenic bacteria that Trichoderma can parasitize, that is, the antagonistic objects include Rhizoctonia, Sclerotium, Sclerotinia, Helminth osporium, Fusarium, Colletotrichum, Verticillium, Venturia, Endothia, Pythium, Phytophthora, Diaporthe) and Fusicladium, among others.

Trichoderma preparations currently on the market are all conidia preparations and mycelia preparations, most of which are fermented from solids. Solid-state fermentation is not only the best method for fungal culture to obtain a large number of conidia through aerial hyphae, but also its unique advantages have made it widely used and studied. In addition, the conidia produced by solid fermentation are of better quality than those produced by liquid fermentation, with strong desiccation resistance and long survival time. Moreover, when applied in the field, the solid-state fermentation product can be directly applied without making a preparation, and the post-treatment is simple and low in cost. But its production and application scale is not large, and the reason is that the most critical problem has not been solved: the shelf life of the product is short. Generally, for preparations with conidia as active ingredients, the time to ensure the content of viable bacteria will not exceed 3 months. The survival of Trichoderma in the preparations received in the later stage is difficult to meet the requirements, and the stability of its control effect has not been effectively guaranteed. In the control of plant diseases, it is still not possible to completely replace other control measures such as chemical control.

Contents of the invention

In order to solve the above problems, the present invention provides the following technical solutions.

The first aspect of the present invention provides a compound preparation for preventing and controlling plant diseases, the compound preparation comprises Bacillus spores and Trichoderma chlamydospores.

In a preferred embodiment, the composite preparation is a wettable powder, the composite preparation comprises a carrier and a wetting and dispersing agent, the carrier is selected from white carbon black or diatomaceous earth, and the wetting and dispersing agent is selected from Alkyl naphthalene sulfonates. In a more preferred embodiment, the content of the carrier is 5-30% (W/W), and the content of the wetting and dispersing agent is 0.5-5% (W/W), based on the weight of the fermentation broth Baseline (based on the weight of the fermentation broth before drying of the wettable powder). In another preferred embodiment, the content of the Bacillus spores in the composite preparation is 10 ⁸ -10 ¹⁰ cells/gram (preparation), and the content of the Trichoderma chlamydospores is 10 ⁸ -10 ⁹ live spores/gram (preparation).

Another aspect of the present invention provides a kind of method for preparing above-mentioned compound preparation, and this method comprises the following steps:

(1) providing a fermented liquid containing bacillus spores and a fermented liquid containing Trichoderma chlamydospores;

(2) Homogenizing the fermentation broth containing Trichoderma chlamydospores, and then mixing it with the fermentation broth containing Bacillus spores to obtain the composite preparation.

In a preferred embodiment, the fermentation broth containing Trichoderma chlamydospores in the step (1) is obtained by the following method:

In the fermentation medium, the temperature is 25-33°C, the seed inoculum size is 5-10%, the seed concentration is 10 ⁵ -10 ⁶ /mL, the aeration ratio is 1:2 to 2:1, and the tank pressure is 0.03-0.05MPa. After 84-96 hours, a fermentation broth containing Trichoderma chlamydospores was obtained, wherein the composition of the fermentation medium was: bran 0.4-0.6%, (NH ₄ ) ₂ SO ₄ 0.1-0.3%, KH ₂ PO ₄ 0.1 -0.2%, CaCO ₃ 0.1-0.2%, glucose 1-3%, soybean oil 0.2-0.5%, pH5.5-6.0.

In another preferred embodiment, the compound preparation is a wettable powder, which is obtained by further drying the compound preparation obtained in step (2), and in the step (2), it also includes mixing in The step of carrier and wetting and dispersing agent, the carrier is selected from white carbon black powder or diatomaceous earth, and the wetting and dispersing agent is selected from alkyl naphthalene sulfonate. In a more preferred embodiment, the content of the carrier is 5-30% (W/W), and the content of the wetting and dispersing agent is 0.5-5% (W/W), based on the weight of the fermentation broth .

Another aspect of the present invention relates to the application of the above-mentioned compound preparation of the present invention in the prevention and treatment of plant diseases. In a preferred embodiment, the plant disease is selected from watermelon wilt and wilt.

The compound preparation of the invention can be used to prevent and control various plant soil-borne diseases. At the same time, the bacterial agent can promote plant growth, improve fertilizer utilization rate, and induce plants to develop resistance to adversity and disease. The chlamydospore preparation produced by the invention has stronger vitality and stress resistance than the conidia preparation produced by the traditional method, has more functions and is more suitable for production and application.

Detailed ways

The first aspect of the present invention provides a compound preparation for preventing and controlling plant diseases, the compound preparation comprises Bacillus spores and Trichoderma chlamydospores. The bacillus is preferably Bacillus subtilis.

In a preferred embodiment, the content of the Bacillus spores in the composite preparation is 10 ⁸ -10 ¹⁰ cells/gram, and the content of the Trichoderma chlamydospores is 10 ⁸ -10 ¹⁰ viable spores /restraints.

The compound formulation of the present invention can adopt various pesticide formulations well known to those skilled in the art, which include but not limited to: powder, wettable powder, emulsifiable concentrate, granule, colloidal suspension, water solvent, aerosol, tablet, Smoke agent, fumigant, oil mist, sustained-release agent, microcapsule, suspoemulsion, solid emulsifiable concentrate, microemulsion, and flowable powder, among which wettable powder, microcapsule preparation, suspoemulsion, solid emulsifiable concentrate, and microemulsion are preferred and flowable powders. Wettable powders and microcapsules are the most preferred because they are convenient, safe, durable, and beneficial to the long-acting effect of biopesticides, and the auxiliaries used are harmless, do not contain organic solvents, and are environmentally friendly.

In the compound preparation (such as wettable powder) of the present invention, other carriers and auxiliary agents may also be included. In a preferred embodiment, the composite formulation of the present invention is a wettable powder, which contains a carrier and a wetting and dispersing agent. Inert carriers are often used to process solid dosage forms such as powders, wettable powders, and granules. Their functions are to dilute active ingredients for easy processing and improve physical and chemical properties for easy use. The inert material used as the carrier of the formulation will affect the stability of the spores in the formulation to some extent. Survival of the spores in the formulation is manifested by a certain level of metabolism. Even if the metabolic activity is very weak under certain storage conditions, the accumulated metabolic by-products can inhibit the spores themselves.

In the present invention, the preferred inert carrier is that the carrier is selected from white carbon black powder or diatomaceous earth.

A wetting and dispersing agent is a substance that can reduce the surface tension of water so that the active ingredient is quickly wetted by water. When processing wettable powders, suspension concentrates, dry suspension concentrates, water-dispersible granules, etc., add a wetting agent when mixing water into a suspension for spraying, so that the surface of the water-insoluble medicine particles is quickly wetted and dispersed Suspended in water. The liquid medicine is easy to spread when sprayed on the surface of the target body, and the coverage area is enlarged. Commonly used wetting agents include saponin, tea dry, lignosulfonate, alkylbenzenesulfonate, pull-off powder, and various non-ionic surfactants. In the present invention, preferred wetting and dispersing agents are alkyl naphthalene sulfonates.

The content of said carrier and wetting and dispersing agent is readily determined by a person skilled in the art based on limited experimentation. In a preferred embodiment, the content of the carrier is 5-30% (W/W), the content of the wetting and dispersing agent is 0.5-5% (W/W), the weight of the fermentation broth is benchmark.

In addition, other active components can also be added to the compound preparation of the present invention, which can be selected and determined by those skilled in the art through routine tests according to specific purposes.

Preparation

Another aspect of the present invention provides a method for preparing the compound preparation of the present invention, the method comprising the following steps:

(1) providing a fermented liquid containing bacillus spores and a fermented liquid containing Trichoderma chlamydospores;

(2) Homogenizing the fermentation broth containing Trichoderma chlamydospores, and then mixing it with the fermentation broth containing Bacillus spores to obtain the composite preparation.

Containing the fermented liquid of bacillus spore and the consumption (or its mixing ratio) of the fermented liquid containing Trichoderma chlamydospore can be determined by those skilled in the art according to the specific situation by routine test, it is usually preferred to hope that described in the compound preparation The content of Bacillus spores is 10 ⁸ -10 ¹⁰ cells/g, and the content of Trichoderma chlamydospores is 10 ⁸ -10 ¹⁰ viable spores/g. In the step (2), the purpose of the homogenization step is to fully break the mycelia, and various mechanical breaking methods well known to those skilled in the art can be used specifically.

In a preferred embodiment, the compound preparation is a wettable powder. In order to facilitate long-term storage, the fermentation broth can be mixed with carriers, wetting and dispersing agents or other additives in a certain proportion to obtain a liquid-solid mixture, and then dried by pressure spraying, centrifugal spraying, etc., so as to obtain the wettable powder. Therefore, in this embodiment, a step of mixing a carrier and a wetting and dispersing agent into the mixed liquid is also included. The order of adding the carrier and the wetting and dispersing agent is not particularly limited, one or both of them can be partially or completely added to the fermentation broth containing Bacillus spores and then homogenized with the fermentation broth containing Trichoderma chlamydospores The homogenate liquid is mixed to obtain a mixed liquid, and the homogenate liquid after being homogenized with the fermented liquid containing Trichoderma chlamydospores can also be partly or fully added and then mixed with the fermented liquid containing Bacillus spores to obtain a mixed liquid, or can be mixed with the fermented liquid containing Bacillus spores. The homogenate of the fermented broth containing Trichoderma chlamydospores is mixed with the fermented broth containing Bacillus spores, and then part or all of the carrier and/or wetting and dispersing agent are added. In a preferred embodiment, the carrier is preferably selected from white carbon black powder or diatomaceous earth, and the wetting and dispersing agent is preferably selected from alkylnaphthalene sulfonates. In a more preferred embodiment, the added amount of the carrier is 5-30% (preferably 10-20%) of the carrier and 0.5 - 5% (preferably 1-2%) of wetting and dispersing agents.

How to improve the tolerance of spores in existing preparations is also an important aspect to solve the problems in the prior art. The chlamydospore of Trichoderma is a dormant survival structure of Trichoderma against adverse environments. Chlamydospores are generally formed when the protoplasm in the mycelial cells shrinks and the cell walls thicken under adverse conditions. In non-septal hyphae, a part of the mycelium concentrates on storing nutrients, and at the same time produces a thick wall, forming a closed septum at both ends and cutting off other cells of the mycelium to form chlamydospores; in the older mycelium with septal mycelium Chlamydospores are often formed. Chlamydospores germinate to produce hyphae once they encounter suitable environmental conditions. Therefore, the biocontrol agents using chlamydospores as the main body may have the advantages of storage resistance and long shelf life, which can meet the requirements of biological pesticides. However, since chlamydospores require harsh conditions for artificial fermentation, it is an urgent problem to find suitable fermentation conditions to obtain chlamydospores with high biomass.

In the present invention, there is also provided a method for obtaining Trichoderma chlamydospores or Trichoderma chlamydospore-containing fermented liquid, which method comprises in the fermentation medium, the temperature is 25～33°C, and the seed inoculation amount is 5～10% , seed concentration 10 ⁵ -10 ⁶ /mL, ventilation ratio 1:2 to 2:1, under tank pressure 0.03-0.05MPa, carry out liquid fermentation to Trichoderma for 84-96 hours, thereby obtain rich Trichoderma chlamydospores of fermentation broth. The composition of the fermentation medium used is: bran 0.4-0.6%, (NH ₄ ) ₂ SO ₄ 0.1-0.3%, KH ₂ PO ₄ 0.1-0.2%, CaCO ₃ 0.1-0.2%, glucose 1-3%, 0.2-0.5% soybean oil, pH5.5-6.0.

In a more preferred embodiment, the method includes in the fermentation medium, the temperature is 28-30°C, the seed inoculum size is ^{6-9%, the seed concentration is 105-106 /} mL, and the aeration ratio is 1:2 to 2: 1. Under the tank pressure of 0.03-0.05MPa, carry out liquid fermentation on Trichoderma for 96 hours, so as to obtain a fermentation liquid rich in Trichoderma chlamydospores. The composition of the fermentation medium used is: bran 0.5%, (NH ₄ ) ₂ SO ₄ 0.2%, KH ₂ PO ₄ 0.1%, CaCO ₃ 0.1%, glucose 2%, 0.2-0.3% soybean oil, pH 5. 5-6.0.

Application in the control of plant diseases

The compound formulation provided by the invention can be used to effectively prevent and treat various plant diseases. In a preferred embodiment, the compound formulation of the present invention can be used to effectively prevent and treat bacteria including Rhizoconia, Macrophomina, Fusarium, Sclerotium ), Pythium, Phytophthora, Sclerotium, Helminth osporium, Colletotrichum, Verticillium, Black Star Plant diseases or soil-borne plant diseases caused by genus Venturia, Endothia, Diaporthe and Fusicladium.In addition, the compound preparation of the present invention can also prevent and treat strawberry It can control the powdery mildew and gray mold of plants, etc., and can also control various postharvest rot diseases of melons and fruits.

More specifically, the compound preparation of the present invention can be used for preventing and treating the following plant diseases: rice sheath blight, rice smut; notoginseng, soil-borne diseases of tobacco, flowers, wheat, cabbage, etc.; powdery mildew, gray spot, Anthracnose and other diseases; wheat sheath blight; rice blast, cabbage black spot; cucumber powdery mildew, strawberry powdery mildew and gray mold; damping-off (such as tomato, cucumber, eggplant, green pepper, watermelon, muskmelon, melon), Root rot, blight, blight and other diseases.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. All technical solutions of the present invention (including better, more or optimal technical solutions) can be combined arbitrarily to form new technical solutions. All these technical solutions are considered to be within the scope of the written description of this application. Unless otherwise described, the practice of the present invention will employ conventional techniques known to those skilled in the art. Alternatively, it can be performed according to the instructions provided by the reagent manufacturer.

Embodiment 1: the research of the 500L tank liquid fermentation of Trichoderma T4 producing chlamydospores

In this example, the production of chlamydospores of Trichoderma T4 in a 500L fermenter was studied. The bacterial strain used is Trichoderma viride (T.viride) T4, which is isolated from plant rhizosphere soil and can be purchased from the State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology. Bioengineering, "Studies on Solid Fermentation Process of Trichoderma viride T4 and Its Preparation Stability", 2008, Vol.25, No.12, pp. 52-56).

In the fermentation medium, the temperature is 28-30°C, the seed inoculation amount is 6-9%, the seed concentration is ^105-106 /mL, the air ratio is 1: ² to 2:1, and the tank pressure is 0.03-0.05MPa. The Trichoderma was subjected to liquid fermentation for 96 hours, thereby obtaining a fermented liquid rich in Trichoderma chlamydospores. The composition of the fermentation medium used was: bran 0.5%, (NH ₄ ) ₂ SO ₄ 0.2%, KH ₂ PO ₄ 0.1%, CaCO ₃ 0.1%, glucose 2%, soybean oil 0.2%, pH 5.5- 6.0.

It was found that Trichoderma T4 had better chlamydospore-forming ability in liquid fermentation in 500L fermenter. In the 500L fermenter, a large number of hyphae were formed in the first 24h without the formation of chlamydospores, and chlamydospores had begun to form in 36h, but the number was very small. At this time, the hyphae of Trichoderma formed abundantly and densely; Prolonged, chlamydospores gradually formed, and the amount of chlamydospores reached 1.5×10 ⁷ /mL at 72h, chlamydospores grew rapidly at 72h～84h, and there were 3.16×107 chlamydospores/mL at 84h, and chlamydospores reached 3.16× ¹⁰⁷ /mL at 96h The amount of chlamydospores increased slightly, 3.24×10 ⁷ /mL, and the mycelial material in the fermentation broth was clearly visible to the naked eye, and the fermentation broth became thinner, and the fermentation was terminated. It takes 6 days from the seed liquid to the end of fermentation, which is relatively less time compared with domestic and foreign researches, and the yield of chlamydospores has also reached a high level.

Embodiment 2: Vegetable oil is to the impact of Trichoderma T4 bacterial chlamydospore

The fermentation process of Example 1 was repeated, except that four concentrations of soybean oil were added to the medium, and then the chlamydospore yield of Trichoderma T4 was determined.

It was found that, compared with no soybean oil, the addition of soybean oil could significantly promote the production of Trichoderma T4 chlamydospores. With the increase of soybean oil concentration, the number of chlamydospores produced gradually increased. The number of chlamydospores was the most, reaching 3.3×10 ⁷ /mL, and the amount of chlamydospores produced at the concentration of 2g/L also reached 3.28×10 ⁷ /mL, which was almost close to the yield of 5g/L. Find under the concentration of 5g/L simultaneously, find that there is still big oil droplet to exist in the fermented liquid during microscopic examination counting, illustrate that the concentration of 5g/L is too much, can not be fully absorbed by Trichoderma, therefore, 2-3g/L (i.e. The addition of 0.2%-0.3%) soybean oil is most beneficial to the production of Trichoderma T4 chlamydospores.

Embodiment 3: the preparation of Trichoderma wettable powder

According to the description in Example 1, use a 500L fermenter to cultivate and obtain a chlamydospore fermentation broth, conditions: 30°C, 180r/min, 2.1L/min, the inoculum size is 6%, after 96 hours a large amount of chlamydospores are formed, ferment Finish. After the fermentation broth is homogenized at high speed (5000r/min homogenization for 3 minutes), add 10% (W/W) carrier and 1% (W/W) (based on the total weight of fermentation broth) wetting and dispersing agent, spray Dry (conditions: inlet temperature 155°C, outlet temperature 105-110°C, feed rate 4.5L/h) and make wettable powder after drying. It is tested according to wettable powder standards. Table 1 lists different carriers (light calcium carbonate (1250 mesh), kaolin (1250 mesh), white carbon black (500 mesh), talcum powder (800 mesh), diatomaceous earth (500 mesh), bentonite (325 order)) on the viability of Trichoderma.

Table 1

Test carrier Germination rate after 2d (%) 12h Germination rate (%) 24h after 60d Colony daily growth (mm/d) Colony daily growth (pH adjustment) (mm/d) PDA control 99.24±0.80A 98.36±0.58A 32.5±0.71 32.5±0.71 Kaolin 86.28±0.32D 68.72±0.20D 30.3±0.58 31.5±0.71 Silica 96.93±0.76B 91.79±0.32B 31.5±0.71 31.6±0.58 Talc powder 88.52±0.65C 85.22±0.64C 27.5±0.71 29.3±0.58 Diatomaceous earth 81.17±0.53E 38.48±0.37F 32.3±0.58 32.5±0.71 bentonite 62.45±0.56G 22.41±0.38G 22.6±0.58 20.3±0.58 Light calcium carbonate 76.34±0.81F 46.26±0.41E 7.3±0.58 7.5±0.71

As can be seen from Table 1, after the spore powder of the carrier was stored for a period of time, the germination rate of the spores changed, and the germination rate of the spores with white carbon black and talcum powder as the carrier changed relatively little; Postspore germination rate is the lowest. In addition, it was also found that the germination time of spores was generally prolonged after a period of time. It can be seen from the plate test of the colony daily growth of various test carriers that the pH value of the various carriers itself has little influence on the growth of Trichoderma, and the interference of the pH value can be eliminated. It can also be seen that light calcium carbonate can seriously Inhibit the mycelium growth of Trichoderma, white carbon black, diatomaceous earth and kaolin have almost no effect on the mycelium growth of Trichoderma, talcum powder and bentonite can inhibit the growth of Trichoderma mycelium to a certain extent. It can also be seen from the above studies that the same carrier has different effects on the spores and hyphae of Trichoderma.

The screening was based on the low change in germination rate and fast growth of mycelium in different treatments. After comprehensive analysis, the test samples can be used as carriers for Trichoderma wettable powder processing, including white carbon black, talcum powder, kaolin and so on. Among them, white carbon black has higher water absorption performance and lighter density, and is a more preferred carrier of Trichoderma chlamydospore fermentation liquid.

The following table 2 lists 5 kinds of wetting and dispersing agents: EFW (alkyl naphthalene sulfonate, Nanjing Jierun Technology Co., Ltd.), tea saponin (Ningbo United Biotechnology Co., Ltd.), Morwet D-425 (polycondensed naphthalene sulfonate salt, Nanjing Jierun Technology Co., Ltd.), T-type lignin (Jilin Tumen Shixian Jinjin Fuli Chemical Co., Ltd.), D110 (polycondensed naphthalene sulfonate, Nanjing Jierun Technology Co., Ltd.), MT-150 (naphthalene Sodium salt of sulfonic acid formaldehyde condensate, Japan Kao Stone Alkaline Co., Ltd.) on the wettability and dispersibility of powders with white carbon black as the carrier.

Table 2

The results showed that with the increase of the amount of wetting and dispersing agent, the wetting time of the powder decreased and the suspension rate increased. According to the principles of shorter wetting time, better wettability, higher suspension rate and better dispersibility, EFW is considered to be the best wetting and dispersing agent, with 1% and 3% addition, its wettability, Dispersion has been in line with national standards. Because the chlamydospores of Trichoderma chlamydoides are relatively large, they will settle in different degrees in the suspension. To increase its suspension force, it is more preferable to add a concentration of 3%.

The wettable powder of Trichoderma chlamydospore was prepared by spray drying process, and white carbon black was selected as the adsorption carrier, and the addition amount was 10%; EFW was the wetting and dispersing agent, and the addition amount was 3%, and the wettable powder was used after drying. The standard was tested and the results are shown in Table 3 below.

table 3

project measured value National standard Live spore content (pcs/g) 1.4×10 ⁸ ---- Moisture content (%) 4.6 5

pH value 6.95 6.0～7.5 Suspension rate (%) 48.3 34 Wetting time (s) 55 180 Fineness (through 44um sieve) (%) 97.25 95

In addition, during the 6-month storage, the germination rate of Trichoderma wettable powder remained above 90% at 4°C, which proved that low temperature storage is beneficial to maintain spore viability. The rate of viable spores decreased slowly with the prolongation of storage time, and the rate of viable spores was still as high as 62% after 6 months. Significantly higher than the fine granule of conidia (30%).

Example 4: Optimization of Bacillus subtilis fermentation process and development of wettable powder

Materials and Instruments

Strains:

Bacillus subtilis B99-2 (isolated from plant rhizosphere soil, can be purchased from State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology).

Medium:

LB plate medium: 200g of potatoes, peeled, sliced, boiled for 30min, filtered, added 20g of glucose to the filtrate, set the volume to 1000mL, 20g of agar, natural pH.

SDAY seed medium: glucose 20g, peptone 10g, yeast extract 5g, water 1000ml, pH7.0.

Reagent

Carbon sources: glucose, soluble starch, maltose, sucrose, corn flour, glycerin, brown sugar, lactose, bran, glutinous rice flour, sorghum flour, wheat flour

Nitrogen source: peptone, yeast extract, yeast powder, peanut powder, bean cake powder, soybean powder, casein, urea, ammonium sulfate, potassium nitrate

Inorganic salts: MgSO ₄ 7H ₂ O, MnSO ₄ H ₂ O, KH ₂ PO ₄ , K ₂ HPO ₄

Carrier: light calcium carbonate (1250 mesh), kaolin (1250 mesh), white carbon black (500 mesh), talcum powder (800 mesh), diatomaceous earth (800 mesh), bentonite (325 mesh).

Additives: D110 (polycondensed naphthalene sulfonate); T-type sodium lignosulfonate; CMC (sodium carboxymethylcellulose); PVA (polyvinyl alcohol); PEG 8000; gum arabic; sodium lauryl sulfate ( SDS); EFW (alkyl naphthalene sulfonate); Morwet D-425 (polycondensed naphthalene sulfonate); Sodium dodecylbenzene sulfonate; Tween-60; Tea saponin, My100

Protective agent: ascorbic acid (VC), carboxymethylcellulose (CMC), dextrin, SDS.

Stabilizers: Calcium Carbonate, Potassium Phosphate, Dipotassium Hydrogen Phosphate, Carboxymethyl Cellulose

Main equipment: biochemical incubator, drying oven, trinocular biological microscope, microwave oven, constant temperature water bath, precision acidity meter, constant temperature magnetic stirrer, small pulverizer.

Methods and Results

The initial fermentation medium: glucose, peptone, MgSO ₄ ·7H ₂ O, MnSO ₄ ·H ₂ O, KH ₂ PO ₄ , K ₂ HPO ₄ was used as the basic medium, and 11 kinds of carbons commonly used in industry and low in price were selected respectively. Source: Soluble starch, maltose, sucrose, corn flour, glycerin, brown sugar, lactose, bran, glutinous rice flour, sorghum flour, and wheat flour are used as carbon sources to replace glucose in the initial fermentation medium. A total of 12 groups were formed as culture media for different carbon sources. The medium without carbon source was used as the control, and each treatment was replicated three times, and samples were taken every 6 hours to determine the bacterial biomass and spore rate, so as to determine the influence of different carbon source medium on the bacterial biomass. The results showed that Bacillus subtilis B99-2 was most likely to utilize corn flour and wheat flour among the 12 carbon sources in terms of cell biomass (CFU). Judging from the time of spore formation, bran forms spores earlier, but the biomass of bran is lower; although wheat flour has higher biomass, the time of spore formation is later, so corn flour is selected as the carbon medium of the medium. source.

Replace the carbon source selected in the previous step with the carbon source of the initial fermentation medium, and select 9 kinds of nitrogen sources that are commonly used in industry and are cheap: yeast extract, yeast powder, peanut powder, bean cake powder, soybean powder, casein, urea , ammonium sulfate, and potassium nitrate as carbon sources to replace peptone in the initial fermentation medium. A total of 10 groups were formed as culture media for different nitrogen sources. The medium without nitrogen source was used as the control, and each treatment was repeated three times, and samples were taken every 6 hours to determine the bacterial biomass and spore rate, so as to determine the influence of different nitrogen source medium on the bacterial biomass. From the perspective of cell biomass (CFU), among the 10 nitrogen sources, Bacillus subtilis B99-2 was the most likely to utilize bean cake flour and soybean flour. From the perspective of the time of spore formation, soybean flour formed spores earlier, while bean cake flour formed spores later, so soybean flour was selected as the carbon source of the medium. Considering that corn flour and soybean flour are a late-acting carbon source and a late-acting nitrogen source, if a small amount of quick-acting carbon source is added, the quick-acting nitrogen source can promote the early growth of bacteria, so the medium formula retains part of the glucose and adds KNO ₃ .

Add 10g of glucose, 10g of KNO ₃ , 10g of glucose and 10g of KNO ₃ to the culture medium screened by the carbon source and nitrogen source screening test as quick-acting carbon and nitrogen sources, and analyze the effects of B99-2 in these three culture media The changes of CFU value and spore rate of B99-2 were compared with the culture medium without quick-acting carbon source and available nitrogen source, and the CFU amount and spore rate at different times were measured every 6 hours to determine the effect of B99-2 on the quick-acting and late-acting The growth of the medium with the combination of carbon and nitrogen sources.

Preparation of Wettable Powder

The carrier in the wettable powder has a great influence on the performance of the formulation. Choose a carrier that has no or little effect on bacterial growth, and determine the best carrier based on factors such as the adsorption capacity, fluidity, and price of the carrier. If the pesticide technical is a liquid preparation, the filler with high oil absorption rate should be selected. If it is a solid technical drug, the filler with general oil absorption rate should be selected. The sample made of the selected filler is a loose and flowing dry powder.

Choose white carbon black, kaolin, diatomaceous earth, bentonite, talcum powder, and light calcium as fillers, and use blank as a control to compare the adsorption capacity, price and influence of different carriers on spore content.

Table 4

As shown in Table 5, the properties of silica and diatomaceous earth are not much different, but the pesticide raw material is a liquid preparation, and a filler with a high adsorption rate should be selected. Silica has greater adsorption performance and better fluidity than diatomite. . Therefore, the samples made of white carbon black as filler are loose and easy to flow.

Analysis and Determination of Wettable Powder Quality Index

The wettable powder prepared in the previous test was measured according to the "National Standard of the People's Republic of China", wettability was measured in GB 5451-2001; suspension rate was measured in GB/T 14825-1993; fineness was measured in GB/T16150-1995; moisture Determination GB/T 1600-2001; pH determination GB/T 1601-1993; accelerated storage test GB/T19136-2003. Other indicators were tested according to relevant literature methods.

table 5

project National indicator value Actual measured value Active ingredient content (cfu/g) 1.8×10 ⁹ cells/g pH range 5-7 6.8 Moisture content (%) 4 3.7 Suspension rate (%) 70 88 Wetting time (s) 180 65 Fineness (%) 98 99

Thermal storage test (decomposition rate <5%) qualified cold storage stability qualified qualified room temperature qualified

Embodiment 5: the preparation of bacillus spore and trichoderma chlamydospore composite preparation

(1) Cultivate the spores of Bacillus by liquid fermentation

Bacterial strain used: Bacillus subtilis B99-2 (isolated from plant rhizosphere soil and can be purchased from State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology).

Fermentation medium: wheat flour 2%, soybean flour 2%, MgSO4·7H ₂ O 0.5%, MnSO4·H ₂ O 0.0003%, KH ₂ PO ₄ 0.05%, K ₂ HPO ₄ 0.2%, pH6.5-7.5.

Culture conditions: temperature 25-35°C, seed inoculation amount 5-10%, seed concentration 1×10 ⁶ /mL, aeration ratio 1:1, tank pressure 0.04MPa, fermentation time 28-36 hours.

(2) adopt the method for liquid state fermentation to cultivate the chlamydospore of Trichoderma

The strain used: Trichoderma viride (T.viride) T4, isolated from plant rhizosphere soil, and can be purchased from the State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, and can also be found in the literature (Xia Siqin, Wang Wei, Chemistry and Bioengineering, "Studies on Solid Fermentation Process of Trichoderma viride T4 and Its Preparation Stability", 2008, Vol.25, No.12, pp. 52-56).

Fermentation medium: bran 0.5%, (NH ₄ ) ₂ SO ₄ 0.2%, KH ₂ PO ₄ 0.1%, CaCO ₃ 0.1%, glucose 2%, soybean oil 0.2%, pH 5.5-6.0.

Culture conditions: temperature 25-33°C, seed inoculation amount 5-10%, seed concentration 1×10 ⁶ /mL, aeration ratio 1:1, tank pressure 0.04MPa, fermentation time 96 hours.

(3) Fermentation broth treatment

After the high-speed homogenization (5000r/min 3 minutes) of the Trichoderma chlamydospore fermented liquid obtained by the above-mentioned step (2), with the spore fermented liquid of the bacillus obtained by the above-mentioned step (2) 1: 1 (V/V )mix. Add 10% (W/W) carrier white carbon black powder (500 mesh), and 1% (W/W) wetting and dispersing agent alkylnaphthalene sulfonate, and mix thoroughly.

(4) dry

The mixed fermented liquid obtained in step (3) is spray-dried, and the specific process is as follows: the inlet temperature is 130-170° C., the outlet temperature is 80-120° C., and the composite wettable powder is made after drying. The total content can reach 2×10 ⁹ cells/gram wettable powder as measured by dilution plate method.

Embodiment 6: biological control test

Materials and Methods

Test agents: Bacillus, Trichoderma single preparation and composite wettable powder (prepared in the examples); the control agent is 50% carbendazim wettable powder.

Test crops: greenhouse watermelon, variety 8424, Huinan Town, Nanhui District, Shanghai.

Trial Design and Administration Methods

The wettable powder treatment was carried out in the continuous cropping greenhouse; 50% carbendazim wettable powder was used as the chemical control, and clear water was used as the blank control. Arranged in random blocks and repeated 4 times, each plot was treated with 2 rows (24 strains).

The application time is when the watermelon field is prepared and one week after colonization, and the pesticide is applied twice, sprinkled on January 30 and root irrigation on March 1, respectively, and the wettable powder is diluted 500 times (10 ⁵ /mL). Ling diluted 1000 times. 100mL was applied to each watermelon, and the control was irrigated with clear water.

Condition investigation

In the early stage of harvest, the incidence of fusarium wilt and creeping blight were investigated respectively, and the relative control effect was calculated. For fusarium wilt, the rate of diseased plants and the control effect are calculated by the number of diseased plants. The disease severity of vine blight is measured by the condition index of the vine.

Grading standards for vine blight stems:

Grade 0 - no disease;

Grade I - Fusiform or round lesions appear on the nodes;

Grade II - the epidermis of the lesion is ruptured, with amber gel substance overflowing, and 1/3 of the stem is dead;

Grade III - the epidermis of the lesion is ruptured, with amber gel substance overflowing, and 1/2 of the length of the stem is dead;

Grade IV-the epidermis of the lesion is ruptured, with amber gum substance overflowing, and 2/3 of the length of the stem is dead; Grade V-the whole plant is dead.

Disease index = [∑ (number of diseased plants at each level × representative value of this level) ÷ (total number of plants under investigation × representative value of the highest level)] × 100.

Control effect (%)=[(disease finger in control area-disease finger in control area)÷disease finger in control area]×100.

results and analysis

Control effect of preparation on watermelon wilt in greenhouse

The test results with the preparation show that it has obvious control effect on Fusarium wilt in watermelon growth period, and the control effect of the composite wettable powder reaches 84.02%, which is better than the control effect of the single Trichoderma preparation and the single Bacillus subtilis preparation. During the test, it was found that the growth of watermelon plants treated with the initial formulation was better than that of the control treatment.

Table 6 The disease control effect of preparations on watermelon wilt

Pharmacy treatment Number of investigated plants Number of diseased plants Diseased plant rate (%) Relative control effect (%) Trichoderma wettable powder 94 8 8.51 77.14 Bacillus subtilis wettable powder 96 16 16.67 55.22 Composite preparation of spores and chlamydospores 95 5 5.26 85.87 Carbendazim 1000 times solution 95 17 17.89 51.95 Clear water control 94 35 37.23 --

Control Effects of Preparations on Watermelon Blight in Greenhouse

The investigation of the disease found that the disease index of the clear water control was significantly higher than that of the four kinds of agents, the disease index of the compound preparation of spores and chlamydospores was the lowest, only 9.36%, the Trichoderma preparation was 16.29%, and the Bacillus subtilis preparation was 16.29%. 14.22%, and the disease index of bacterazim treatment also has 54.73%. It can be seen that the compound wet powder has the highest control effect on vine blight, which is 86.67%, which is significantly higher than the single Trichoderma and Bacillus subtilis preparations.

Table 7 The control effect of preparations on watermelon blight

Pharmacy treatment Number of investigated plants Disease index (%) Relative control effect (%) Trichoderma wettable powder 94 16.29 78.33 Bacillus subtilis wettable powder 95 14.22 81.08 Composite preparation of spores and chlamydospores 92 9.36 87.55 Carbendazim 1000 times solution 95 54.73 27.18 Clear water control 94 75.16 --

It can be seen from the greenhouse test that the compound wettable powder has good control effect on watermelon wilt and wilt, and the control of wilt is better than fusarium wilt. Carbendazim has a certain control effect on fusarium wilt, but the control effect on wilt is relatively low. It is also found in the experiment that the watermelon seedlings treated with the preparation are obviously robust, with well-developed root system and dark green leaves, which can obviously promote the growth of watermelon plants.

The experimental greenhouse is a field where melons have been cropped repeatedly for 2 years, and the rate of diseased plants in the previous year can sometimes reach more than 50%, and the yield is low. It can be seen from the test results that several wet powders have good control effects on the control of watermelon wilt and creeping blight, which are significantly higher than chemical agents; the preparations have obvious growth-promoting effects on watermelon seedlings.

Fusarium wilt and wilt of watermelon are relatively common and serious diseases in watermelon cultivation. Fusarium wilt is a typical systemic soil-borne disease, and it is more likely to develop in the case of watermelon heavy-cropping cultivation. The incidence rate of heavy-cropping land is generally 30% About, up to more than 80% in severe cases. Commonly known as "vine cutting disease", vine blight occurs in watermelon growing areas in my country. According to reports, the incidence rate of watermelon vine blight is generally 15% to 25%, and it is as high as 60% to 80% when it is severe. When the disease is prevalent, a large number of dead vines will appear in the melon field, and the yield will be reduced by more than 30%. The occurrence of the disease seriously affected the yield and quality of watermelon.

The results of this experiment showed that the wettable powder of Bacillus and Trichoderma combined with chlamydospores had a good inhibitory effect on watermelon wilt and wilt. In addition, compared with the wettable powder of Bacillus subtilis and the wettable powder of Trichoderma chlamydospore alone, the result of using the wettable powder of Bacillus and Trichoderma composite chlamydospore of the present invention is significantly improved.

In addition, it was found that the control effect of chlamydospore wettable powder was better than that of conidia fine-grained agent. The reason may be that chlamydospores have better survival and colonization ability in soil than conidia, and generally at least 20 spores can survive. 1 month; Even if the same strain has different types and sizes of spores, the degree of sensitivity to the antifungal effect of the soil is also different, and the smaller spores are more sensitive to the antifungal effect of the soil.

Although specific examples have been described herein, it will be apparent to those skilled in the art that various changes and modifications can be made in the present invention without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes which are within the scope of this invention. All publications, patents, and patent applications cited herein are hereby incorporated by reference.

Claims (6)

1. a compound preparation for preventing and treating plant diseases, is characterized in that, described compound preparation comprises bacillus spore and Trichoderma chlamydospore, and described compound preparation is wettable powder, and described compound preparation comprises carrier and wetting Dispersant, the carrier is selected from white carbon black or diatomaceous earth, the wetting and dispersant is selected from alkylnaphthalene sulfonate, the content of the bacillus spores in the composite preparation is 10 ⁸ -10 ¹⁰ cells per gram, and the content of Trichoderma chlamydospores is 10 ⁸ -10 ⁹ viable spores per gram. 2. composite preparation as claimed in claim 1, is characterized in that, described wettable powder is made with following method: (1) providing a fermented liquid containing bacillus spores and a fermented liquid containing Trichoderma chlamydospores; (2) Homogenizing the fermented liquid containing Trichoderma chlamydospores, then mixing with the fermented liquid containing Bacillus spores to obtain a composite preparation; The composite preparation obtained in step (2) is further dried to obtain the wettable powder, And in said step (2), also include the step of mixing into carrier and wetting and dispersing agent, the content of said carrier is 5-30% (W/W), the content of said wetting and dispersing agent is 0.5-5 % (W/W), based on the weight of the fermentation broth before the wettable powder is dried. 3. a method for preparing the described compound preparation of claim 1, the method may further comprise the steps: (1) providing a fermented liquid containing bacillus spores and a fermented liquid containing Trichoderma chlamydospores; (2) homogenate the fermented liquid containing Trichoderma chlamydospores, then mix with the fermented liquid containing Bacillus spores to obtain the composite preparation, The compound preparation is a wettable powder, which is obtained by further drying the compound preparation obtained in step (2), and also includes the step of mixing in a carrier and a wetting and dispersing agent in the step (2), so The carrier is selected from white carbon black powder or diatomaceous earth, and the wetting and dispersing agent is selected from alkylnaphthalene sulfonate. 4. the method for claim 3 is characterized in that, the fermented liquid containing Trichoderma chlamydospore in described step (1) obtains with following method: In the fermentation medium, the temperature is 25～33℃, the seed inoculation amount is 5～10%, the seed concentration is 10 ⁵ -10 ⁶ /mL, the aeration ratio is 1:2 to 2:1, and the tank pressure is 0.03-0.05MPa. After 84-96 hours, a fermentation liquid containing Trichoderma chlamydospores was obtained, wherein the composition of the fermentation medium was: bran 0.4-0.6%, (NH ₄ ) ₂ SO ₄ 0.1-0.3%, KH ₂ PO ₄ 0.1 -0.2%, CaCO ₃ 0.1-0.2%, glucose 1-3%, soybean oil 0.2-0.5%, pH5.5-6.0. 5. the method for claim 3 is characterized in that, the content of described carrier is 5-30% (W/W), and the content of described wetting and dispersing agent is 0.5-5% (W/W), Based on the weight of the fermentation broth. 6. the application of the compound preparation described in claim 1 in the prevention and treatment of plant diseases, said plant diseases are selected from watermelon wilt and creeping blight.