Preparation method and application of wettable powder of tea tree disease biocontrol bacteria

Abstract

The invention provides a preparation method and application of wettable powder of tea tree disease biocontrol bacteria, and belongs to the technical field of microbial bactericides. Comprising a brevibacillus brevis HNCS-1 fermentation broth, a carrier, an auxiliary agent and a protective agent, the spore rate of the brevibacillus brevis HNCS-1 fermentation broth is more than 90%, the mass percentage of the fermentation broth is 50-87.5%, the auxiliary agent is selected from at least one of a dispersant and a wetting agent, the mass percentage of the auxiliary agent is 4-16%, and the mass percentage of the protective agent is 1-10%. And the mass percentage of the carrier is 5-25%. The wettable powder of the tea tree disease biocontrol bacteria can be used for preventing and treating various tea tree disease biocontrol bacteria such as tea anthracnose, tea root rot, tea white spot, tea ring spot and tea round brown spot of tea trees.

Description

Technical Field

[0001] The present application relates to a method for preparing a wettable powder of a biocontrol fungus for tea tree diseases and its application, belonging to the technical field of microbial fungicides. Background Art

[0002] Tea trees are often attacked by various diseases, the most common of which include tea anthracnose, tea white spot disease, tea round red spot disease and tea ring spot disease. These diseases not only reduce tea yield by more than 10% to 50%, but also affect tea quality. Chemical pesticides are the main means of preventing and controlling tea garden diseases.

[0003] In recent years, chemical pesticides have been banned or restricted due to pesticide residues, increased resistance, environmental pollution and other issues. Microbial pesticides have become the most widely used type of biological pesticides due to their high efficiency, wide applicability, low toxicity to humans and good environmental compatibility. With the rapid development of biological pesticides and the gradual reduction in the use of chemical pesticides, people's demand for green and harmless agricultural products has gradually increased, bringing important opportunities for the further development of the biological pesticide industry. According to the "Pesticide Registration Information Requirements", the active ingredients of microbial pesticides are mainly active microorganisms such as natural or genetically modified bacteria, fungi and viruses. Its main mechanism of action on pathogens is to inhibit mycelial growth and interfere with pathogen signals. However, there is still room for improvement in research methods and improving technical benefits. For example, the current dosage forms and strain diversity of microbial pesticides are not sufficient, and the long-term use of a single pesticide may cause pathogens to develop resistance to drugs.

[0004] In disease management, excessive use of chemical pesticides limits the development of beneficial microorganisms in tea gardens, thereby accelerating the growth and reproduction of pathogens, making the originally minor diseases upgraded to major problems, and multiple infections are becoming more common. Biological control (i.e., biocontrol) is a key way to control tea tree diseases, and its development prospects are broad, but the current research on biological control of tea trees is still insufficient. Therefore, it is particularly important to develop biological control agents that are compatible with crops, have strong adaptability, and can resist adversity well. Summary of the invention

[0005] In view of this, the present application first provides a wettable powder of a biocontrol bacteria for tea tree diseases, which has a strong inhibitory effect on diseases such as tea anthracnose, tea white spot disease, and tea round red spot disease, and achieves a significant reduction in the incidence rate and disease index of tea anthracnose, etc., with an average field control rate of more than 50%.

[0006] Specifically, the present application is implemented through the following scheme:

[0007] A wettable powder of tea tree disease biocontrol bacteria, comprising Bacillus brevis HNCS-1 fermentation liquid, a carrier, an adjuvant and a protective agent, wherein:

[0008] The spore rate of the brevis bacillus HNCS-1 fermentation liquid is more than 90%, and the mass percentage of the fermentation liquid is 50-87.5%.

[0009] The auxiliary agent is selected from at least one of a dispersant and a wetting agent, and the weight percentage of the auxiliary agent is 4-16%.

[0010] The mass percentage of the protective agent is 1-10%,

[0011] The mass percentage of the carrier is 4-25%.

[0012] In the early research process, the applicant isolated Bacillus brevis HNCS-1 (CN116376780A, deposit number CCTCCNO: M2022713) from tea garden soil. The bacterium is a Gram-positive bacterium with strong antibacterial activity and broad-spectrum antagonistic activity. The bacterium can inhibit the growth of mycelium of pathogens such as tea white spot disease, tea round red spot disease, and tea anthracnose. It is safe and harmless to the environment and is a beneficial biocontrol bacterium with potential for biotechnology application. However, Bacillus brevis has not yet been registered as a pesticide product. Wettable powder has the advantages of easy use, good dispersibility, strong stability, wide adaptability, and environmental friendliness. Based on the above principles, this case chooses to prepare microbial pesticides in the form of wettable powders, and among many biological control methods, Bacillus HNCS-1 is used as the core active component to give full play to its strong environmental adaptability, excellent stress resistance, and obvious antibacterial and plant growth-promoting properties, so as to achieve simultaneous defense against tea white star disease, tea anthracnose, tea round red star disease and other tea tree diseases, and achieve effective biological control of common and susceptible diseases of tea trees.

[0013] Further, as a preference:

[0014] The carrier is any one of kaolin, diatomaceous earth, light calcium carbonate, bentonite and white carbon black.

[0015] The protective agent is any one of dextrin, ascorbic acid, sodium carboxymethyl cellulose and soluble starch.

[0016] The adjuvant can take three forms:

[0017] Method 1, only comprising a dispersant, wherein the dispersant is any one of sodium lignin sulfonate, hydroxypropyl cellulose, calcium lignin sulfonate, dispersant NNO, gum arabic, and polyethylene glycol.

[0018] Method 2, only including a wetting agent, wherein the wetting agent is any one of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, pull-opening powder, calcium chloride, penetrant T, Silok, and Sicare.

[0019] Mode 3, a compound auxiliary agent containing both a wetting agent and a dispersant. In this case, the mass ratio of the dispersant to the wetting agent is 1-9:9-1. More preferably, the wetting agent is calcium chloride or Sicare, and the dispersant is any one of hydroxypropyl cellulose, calcium lignin sulfonate, gum arabic, and polyethylene glycol.

[0020] In the above formula,

[0021] The mass percentage of the fermentation liquid of Bacillus brevis HNCS-1 is 70-83.2%, and the mass percentage of the carrier is 10-25%.

[0022] The percentage of additives is 4-5%.

[0023] In the above scheme, the applicant measured the biocompatibility of the carrier, dispersant, wetting agent, ultraviolet protective agent used in the Bacillus brevis wettable powder reported in the literature with Bacillus brevis HNCS-1, and selected suitable wettable powder formulation reagents therefrom. The suspension rate, wettability and bacterial content of the wettable powder with the above ratio were evaluated, and the preferred composition of the wettable powder was obtained: the carrier is white carbon black, the protective agent is sodium carboxymethyl cellulose, and the auxiliary agent is a compound of calcium chloride and gum arabic. When the mass ratio of calcium chloride to gum arabic is 4:6, the compatibility of each component with the strain Bacillus brevis HNCS-1 is good, the wetting time is short, the suspension rate is high, the spore content is high and stable, and it has good medicinal properties.

[0024] The wettable powder of the tea tree disease biocontrol bacteria can be prepared by the following steps:

[0025] Step 1: culturing Bacillus brevis HNCS-1 to obtain a fermentation broth.

[0026] The specific preparation process of the preferred fermentation broth is: the HNCS-1 strain stored at -80°C is transferred to the LB solid culture medium, cultured at 37°C, a single colony is selected and inoculated into the LB liquid culture medium, and the seed liquid is obtained by shaking and culturing at 37°C, and the obtained seed liquid is inoculated into the NB liquid culture medium, and the fermentation broth is obtained by shaking and culturing at 26°C.

[0027] Step 2: Mix the culture solution and the carrier in proportion, and spray-dry at 60° C. to obtain a mother powder.

[0028] Step three, adding the auxiliary agent and the protective agent to the mother powder respectively, mixing evenly, and obtaining a wettable powder.

[0029] The preparation method first forms a fermentation liquid with Bacillus brevis HNCS-1, then mixes the fermentation liquid with a carrier in proportion to form a mother powder, and then adds an auxiliary agent and a protective agent to complete the preparation of the wettable powder. The operation is simple and the obtained product meets national standards.

[0030] When the above-mentioned Bacillus brevis HNCS-1 wettable powder is applied, it can simultaneously prevent and control at least two of the tea anthracnose, tea root rot, tea white spot, tea ring spot and tea round red spot of tea trees. The wettable powder can be diluted with water and sprayed on the tea canopy surface, with a water consumption of 45L per mu. The dilution multiple is preferably controlled below 600 times, preferably below 400 times, and especially 200 to 300 times.

[0031] The wettable powder provided in the present application was used to verify its biological control effect. The wettable powder has a good antibacterial effect on the pathogen of tea anthracnose, and the incidence rate and disease index of tea anthracnose on tea trees are significantly reduced. The field control effects on tea anthracnose and tea white star disease are 56.52% and 33.85% respectively, which are similar to the control effects of chemical agents. It has good universality in the field of tea anthracnose prevention and control of tea trees. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0033] Figure 1 The figure is a comparison chart of the anti-ultraviolet protection effect of four protective agents on the wettable powder of the present application;

[0034] Figure 2 This is a comparison chart of the inhibitory effect of the wettable powder of this application on the mycelial growth of tea pathogens;

[0035] Figure 3 This is a comparison chart of the field efficacy of the wettable powder of this application against tea anthracnose;

[0036] Figure 4 This is a comparison chart of the field prevention effects of the wettable powder of this application and common pesticides on tea white star disease. DETAILED DESCRIPTION

[0037] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application clearer, the technical solutions in the embodiments of this application will be further described in detail below in conjunction with the drawings in the embodiments of this application. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit the technical solutions of this application. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0038] Unless otherwise specified, the reagents or equipment used in this example are all known products or commercially available products.

[0039] Example 1: Preparation of Bacillus brevis HNCS-1 fermentation broth

[0040] The HNCS-1 strain stored at -80℃ was transferred to LB solid medium, cultured at 37℃ for 12h, and then a single colony was selected and inoculated into LB liquid medium, and cultured at 37℃ with shaking at 200rpm for 12h to obtain the seed liquid. The seed liquid was inoculated into NB liquid medium at 2% of the total volume, and cultured at 26℃ with shaking at 140rpm for 3d to obtain a fermentation liquid with a spore rate of more than 90%.

[0041] LB solid medium formula: tryptone 10.0 g, yeast extract powder 5.0 g, sodium chloride 10.0 g, distilled water 1,000 mL, agar 15 g (solid), pH 7.0±0.1.

[0042] LB liquid medium formula: tryptone 10.0 g, yeast extract powder 5.0 g, sodium chloride 10.0 g, distilled water 1,000 mL, pH 7.0±0.1.

[0043] NB liquid culture medium formula: peptone 10g, sodium chloride 5g, beef extract powder 3g, distilled water 1000mL, pH about 7.0.

[0044] The above spore rate is determined by:

[0045] ① Smear and fixation: drop half a drop of saline in the center of the slide, pick a small amount of bacteria and mix with the saline and evenly spread, dry naturally at room temperature or blow dry with a hair dryer, with the smear facing up, fix by passing through a flame 2-3 times;

[0046] ② Primary staining: Add a few drops of 5% malachite green dye solution on the smear, hold the slide with a wooden clip and heat it on a low fire until the dye solution starts to steam, and then count for 5 minutes; (Note: Add dye solution in time during the heating process, and do not let the specimen dry up.)

[0047] ③ Washing: After the slide has cooled, rinse with slow-flowing tap water until the water is colorless;

[0048] ④Re-staining: add a few drops of 5% safranin staining solution and re-stain for 2 minutes;

[0049] ⑤ Washing: After the slide has cooled, rinse with slow-flowing tap water until the water is colorless;

[0050] ⑥ Drying: Dry the water on the slide naturally at room temperature or blow dry with a hair dryer (or use absorbent paper to dry it);

[0051] ⑦ Microscopic examination: After drying, observe under a microscope. Those stained green are spores, and those colorless are still spores. Then calculate the spore rate, spore rate (%) = number of spores / number of bacteria × 100.

[0052] Example 2: Biocompatibility determination of carriers, adjuvants and protective agents

[0053] Carrier: kaolin, white carbon black, light calcium carbonate, diatomaceous earth and bentonite.

[0054] Dispersants: sodium lignin sulfonate, hydroxypropyl cellulose, calcium lignin sulfonate, dispersant NNO, gum arabic, polyethylene glycol 6000.

[0055] Wetting agent: sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, pull-opening powder, calcium chloride, penetrant T, Silok, Sicare.

[0056] Protective agents: dextrin, ascorbic acid, sodium carboxymethyl cellulose, soluble starch.

[0057] The biocompatibility of the above-mentioned carriers, adjuvants (dispersants, wetting agents) and protective agents is verified.

[0058] The carrier, dispersant, wetting agent and protective agent were mixed with NA medium (15 g of solid agar was added to the above NB liquid medium) at concentrations of 50, 1.5, 0.25 and 0.05 mg / mL, respectively, and sterilized to prepare plates for later use.

[0059] Next, the fermentation broth obtained by the method of Example 1 was diluted 1000 times with sterile water. 100 μL was taken out from the diluted fermentation broth and applied on the culture medium plates with different additives (i.e., carrier, dispersant, wetting agent, protective agent). At the same time, NA culture medium plates without any additives were used as the control group. Each different treatment was repeated 3 times to ensure the reliability of the results. The plates were placed in an environment of 26°C and cultured for 24 hours, and the number of colonies on each plate was recorded afterwards. The results are shown in Table 1.

[0060] Table 1: Biocompatibility of different reagents with Bacillus brevis HNCS-1

[0061]

[0062]

[0063] As shown in Table 1, there are significant differences in the biocompatibility (i.e., the inhibitory effect on the growth of the strain) of different additives with Bacillus brevis HNCS-1. Among them, five carriers and four protective agents have good compatibility with strain HNCS-1; but only calcium chloride and Sicare, two wetting agents, have good compatibility with the strain; calcium wood sulfonate, hydroxypropyl cellulose, gum arabic, and polyethylene glycol also have good compatibility with the strain.

[0064] Example 3, selection of carrier

[0065] In this example, different carriers were mixed with the fermentation broth of Bacillus brevis HNCS-1 in Example 1 at a mass ratio of 1:7, spray-dried at 60° C., and sieved with a 200-mesh sieve to ensure uniformity, thereby completing the preparation of mother powder.

[0066] According to the national pesticide standards, the suspension rate, wettability (wetting time) and spore content of the mother powder of each mixture were measured. Each mixture was repeated three times to compare the effects. The results are shown in Table 2.

[0067] The method for determining the suspension rate refers to the "Method for Determining the Suspension Rate of Pesticides (GB / T 14825-2006)", the method for determining the wettability refers to the "Method for Determining the Wettability of Pesticide Wettable Powders (GB / T 5451-2001)", and the method for determining the spore content refers to the "Bacterial Microbial Pesticides Bacillus subtilis Part 1: Bacillus subtilis Mother (NY / T 2293.1-2012)".

[0068] Table 2: Effects of different carriers on wettable powders

[0069]

[0070] As shown in Table 2, the wetting time of diatomite is the shortest, which is 3.25s, significantly lower than other treatments, the suspension rate is low, which is 58.10%, and the spore content is relatively low, which is 0.84×10 7 CFU·g -1 ; The suspension rate of white carbon black was the highest, which was 70.40%, which was significantly different from other treatments; the spore content of white carbon black and kaolin was the highest, which was 1.36×10 7 , 1.33×10 7 CFU·g -1 There is no significant difference between them; the wetting time of kaolin is relatively long and the suspension rate is lower than that of silica, and the difference is significant.

[0071] Therefore, the effect of using white carbon black as a carrier of wettable powder is ideal. The best solution is to prepare mother powder with white carbon black as a carrier: HNCS-1 strains stored at -80℃ are transferred to LB solid medium, cultured at 37℃ for 12h, and then single colonies are selected and inoculated into LB liquid medium, and cultured at 37℃ with 200rpm for 12h to obtain seed liquid. The seed liquid is inoculated into NB liquid medium, and cultured at 26℃ with 140rpm for 3d to obtain fermentation liquid; white carbon black and fermentation liquid are mixed in a mass ratio of 1:7, spray dried at 60℃, and sieved with a 200-mesh screen to ensure uniformity, and mother powder prepared with white carbon black as a carrier is obtained.

[0072] Example 4: Selection of a single auxiliary agent

[0073] In this embodiment, each auxiliary agent (dispersant, wetting agent) was crushed and uniformly mixed through a 200-mesh screen, and added to the mother powder prepared with white carbon black as the carrier in Example 2. Based on the total mass of the substance after addition, the amount of auxiliary agent added was 5%, and the optimal dispersant and wetting agent were determined based on the measured suspension rate, wettability, and spore content data. The suspension rate, wetting time, and spore content were determined in the same manner as in Example 3. Each treatment was repeated three times, and the results are shown in Table 3.

[0074] Table 3: Effects of different adjuvants on wettable powders

[0075]

[0076] As shown in Table 3, the wetting time of polyethylene glycol 6000 was the shortest among the dispersants, which was 21.25s, significantly lower than that of other treatments; the suspension rates of polyethylene glycol 6000 and calcium lignin sulfonate were the lowest, which was 60.40%, and there was no significant difference between them; the suspension rate of hydroxypropyl cellulose was the highest, which was 82.00%; the spore content of hydroxypropyl cellulose and sodium lignin sulfonate was significantly lower than that of other dispersants, and there was no significant difference between them; the spore content of gum arabic was the highest, which was 1.57×10 7 CFU·g -1 , significantly higher than other dispersant treatments and its suspension rate as a dispersant is also relatively high.

[0077] Therefore, considering the wetting time, suspension rate and spore content, gum arabic is the best dispersant; among the wetting agents, calcium chloride has better results in wetting time, suspension rate and spore content than Sicare.

[0078] Embodiment 5, the composition of compound auxiliary agent

[0079] In this embodiment, gum arabic and calcium chloride are used as compounding auxiliary agents. After sieving gum arabic and calcium chloride through a 200-mesh sieve, they are mixed in a mass ratio of 1-9:9-1, and then added to the mother powder prepared with white carbon black as a carrier in Example 2 according to an auxiliary agent addition amount of 5%. The obtained product determines the optimal compound ratio based on the measured suspension rate, wettability, and spore content. The determination method is the same as that of Example 3. Each treatment is repeated three times, and the results are shown in Table 4.

[0080] Table 4: Effects of different adjuvant ratios on wettable powders

[0081] Calcium chloride: gum arabic Wetting time / s Suspension rate / % <![CDATA[Sporulation content / 10 9 cfu·g -1 > 1:9 46.50±7.05a 69.10±3.64ab 1.26±0.10ab 2:8 40.50±4.20b 79.45±17.33ab 1.11±0.11bc 3:7 37.50±2.65b 64.90±12.88b 1.10±0.11bc 4:6 23.00±3.16cd 87.05±7.01a 1.41±0.03a 5:5 24.75±3.59c 74.90±16.63ab 1.13±0.13bc 6:4 24.00±2.58c 76.45±19.61ab 1.33±0.08a 7:3 28.50±2.65c 71.25±13.01ab 1.04±0.03c 8:2 25.25±2.75c 79.25±8.85ab 1.01±0.09c 9:1 17.75±1.71d 72.85±16.93ab 0.98±0.07c .

[0082] According to Table 4, as the proportion of calcium chloride increases, the wetting time of the wettable powder of the present application gradually decreases. When the mass ratio of calcium chloride to gum arabic is 1:9, the wetting time is 46.50s, which is the longest and significantly higher than other treatments; when the mass ratio of calcium chloride to gum arabic is 9:1, the wetting time is the shortest, which is 17.75s; the suspension rate first increases and then decreases with the increase of the proportion of calcium chloride. When the mass ratio is 4:6, the suspension rate is the highest, which is 87.05%; the spore content results are consistent with the suspension rate results. When the mass ratio is 4:6, the spore content is the highest.

[0083] Therefore, when the mass ratio of calcium chloride to gum arabic is 4:6, the wettable powder prepared by using it as an adjuvant is the best solution, specifically: the HNCS-1 strain stored at -80°C is transferred to LB solid culture medium, cultured at 37°C for 12h, and then a single colony is selected and inoculated into LB liquid culture medium, and cultured at 200rpm at 37°C for 12h, which is the seed liquid. The seed liquid is inoculated into NB liquid culture medium, and cultured at 140rpm at 26°C for 3d to obtain fermentation liquid; white carbon black is mixed with fermentation liquid at a mass ratio of 1:7, spray dried at 60°C, and sieved with a 200-mesh sieve to ensure uniformity to obtain mother powder; 5% of the adjuvant (the adjuvant is a mixture of calcium chloride and gum arabic at a mass ratio of 4:6) is added to the mother powder, mixed evenly, and a wettable powder is obtained.

[0084] Furthermore, based on the “adjuvant selected as a wettable powder prepared at a mass ratio of calcium chloride to gum arabic of 4:6”, the addition amount of the adjuvant in the wettable powder was controlled to be 4%, 6%, 8%, 10%, 12%, 14% and 16%, respectively, and the effects of different addition amounts of adjuvant on the wettable powder were tested, and the results are shown in Table 5.

[0085] Table 5: Effect of different adjuvant addition amounts on wettable powder

[0086] Dosage Wetting time / s Suspension rate / % <![CDATA[Sporulation content / 10 9 cfu·g -1 > 4％ 29.00±2.94bc 86.75±2.47ab 1.90±0.19a 6％ 30.75±1.71b 88.25±5.62ab 1.06±0.15b 8％ 36.00±2.45a 84.25±12.48ab 0.87±0.13c 10％ 27.25±3.59bc 82.67±1.85b 0.28±0.02d 12％ 29.50±5.74bc 88.46±2.02ab 0.19±0.02d 14％ 31.00±3.27b 95.67±2.61a 0.26±0.04d 16％ 24.25±2.5c 85.11±10.03ab 0.21±0.00d .

[0087] It can be seen from Table 5 that different dosages of adjuvants have a great influence on the performance of the wettable powder of this application. As the dosage gradually increases, the wetting time generally shows a trend of first rising and then falling. When the dosage is 8%, the longest wetting time is 36s, which is significantly higher than other treatments. When the dosage is 16%, the shortest wetting time is 24.25s; the suspension rate generally shows a trend of first falling and then rising. When the dosage is 14%, the suspension rate is as high as 95.67%; the spore content gradually decreases with the continuous increase of the dosage. When the dosage is 4%, the spore content is the highest, which is significantly higher than other treatments. The spore content is an important indicator that affects the properties of pesticides. Considering the wetting time and suspension rate, the dosage of adjuvants is appropriately controlled at 4-5%.

[0088] Example 6: Screening of protective agents

[0089] The "wettable powder prepared when the auxiliary agent is selected from calcium chloride and gum arabic in a mass ratio of 4:6 and an addition amount of 4%" described in Example 5 is used, with the difference that: on the basis of the above-mentioned wettable powder prepared in Example 5, a protective agent is added, and the protective agents are selected from dextrin, ascorbic acid, sodium carboxymethyl cellulose, and soluble starch.

[0090] The specific method is as follows: add different protective agents to the wettable powder product at a dosage of 1%, apply it evenly on the culture dish after dilution, and irradiate it at a distance of 40cm from a 20W ultraviolet lamp (wavelength 254nm) for 30 minutes, and then count the number of colonies. Survival rate (%) = total number of viable bacteria after ultraviolet treatment / total number of viable bacteria in the sample before ultraviolet treatment × 100. Three repeated experiments were set for each treatment; at the same time, a control group without adding protective agent was set up, and the treatment method was the same. The results are as follows Figure 1 shown.

[0091] Depend on Figure 1 It can be seen that the protective effect of dextrin, ascorbic acid and sodium carboxymethyl cellulose on Bacillus brevis HNCS-1 is significantly better than that of soluble starch and the control group (CK). Among them, the spore survival rate of the wettable powder with sodium carboxymethyl cellulose added is significantly higher than that of other treatments, with a survival rate of 62.56%. Therefore, sodium carboxymethyl cellulose is the preferred protective agent for the wettable powder of this application.

[0092] Example 7

[0093] A wettable powder of tea tree disease biocontrol bacteria, its formula composition and preparation method are as follows:

[0094] (1) Formulation: The following composition by weight percentage

[0095]

[0096] (2) Preparation method

[0097] According to the above formula, the fermentation liquid and white carbon black are mixed evenly, spray dried at 60°C and sieved with a 200-mesh sieve to ensure uniformity to form a mother powder; then gum arabic, calcium chloride and sodium carboxymethyl cellulose are added, mixed evenly and sieved with a 200-mesh sieve to obtain a wettable powder.

[0098] Application Example 1

[0099] The wettable powder prepared in Example 7 was diluted 200, 400, 600, 800, 1600, 3200, 6400, and 12800 times with sterile water, respectively, and mixed with PDA medium sterilized at about 45°C at a ratio of 1:9, and made into a plate after shaking, and sterile water was used as a control. A 5mm diameter tea plate spore fungus (tea anthracnose fungal disease, pathogenic fungus scientific name: Gloeosporium theae-sinensis Miyake) cake was inoculated in the center of the plate, and each treatment was repeated 3 times. After culturing for 7 days at 26°C in the dark, the colony diameter of each treatment was measured by the cross method, and the inhibition rate was calculated. The results are shown in Table 6.

[0100] As a control example, the wettable powder prepared in Example 7 was stored at room temperature for 270 days, and then its antibacterial rate was tested using the above method.

[0101] Table 6: Inhibitory effect of wettable powder on mycelial growth of tea tray spore fungus

[0102]

[0103] From the above Table 6, we can see that:

[0104] (1) The wettable powder prepared in the present application exhibits a strong antibacterial effect against the tea tray spore fungus.

[0105] (2) The antibacterial rate of the wettable powder of the present application against Mycosporium spp. decreased with the increase of dilution concentration, but after dilution of 12,800 times, the antibacterial rate was still 52.90%.

[0106] (3) The wettable powder of the present application can be stored at room temperature for a long time, and compared with the newly prepared wettable powder (0d in Table 6), it still has a good antibacterial effect after storage for up to 270d.

[0107] Application Example 2

[0108] After the wettable powder prepared in Example 7 was stored at room temperature for 270 days, its inhibitory effect on the mycelial growth of five tea tree pathogens, including Pseudomonas aeruginosa, Pseudomonas syringae, Fusarium solani, Pseudomonas syringae and Pseudomonas syringae, was tested.

[0109] Test conditions:

[0110] The wettable powder stored for 270 days was diluted 200, 400, 600, 800, 1600, 3200, 6400 and 12800 times with sterile water, respectively, mixed with PDA culture medium sterilized at about 45°C in a ratio of 1:9, and made into plates after shaking, and sterile water was used as a control.

[0111] In the center of the plate, 5 mm diameter tea scab cysts, tea spot mold, tea Fusarium, and tea cercosporium were inoculated, and each treatment was repeated 3 times. Among them, tea scab cysts, tea spot mold, tea Fusarium, and tea cercosporium were cultured for 25 days under 26°C dark conditions, and tea pseudodisk multi-hairy spores were cultured for 7 days. Then the colony diameter of each treatment was measured by the cross method, and the inhibition rate was calculated. The results are shown in Table 7.

[0112] Table 7: Inhibition rate of wettable powder on mycelial growth of five tea pathogens after storage at room temperature for 270 days

[0113]

[0114] The above experimental process and Figure 2 The results show that after the wettable powder of the present application is stored for 270 days and diluted less than 400 times, it has a good inhibitory effect on the pathogens of various tea tree diseases such as tea white spot disease (tea scab cyst, tea spot mildew), tea root rot (tea Fusarium), tea round red spot disease (tea cercaria) and tea ring spot disease (tea pseudodisk multi-hairy spore), and the antibacterial effect on tea scab cyst, tea Fusarium, tea cercaria and tea pseudodisk multi-hairy spore is better, and the inhibition rate is all above 70%. It can be seen that the wettable powder of the present application can inhibit the growth of various tea tree pathogens.

[0115] Application Example 3

[0116] The wettable powder prepared in Example 7 was used for field control of tea anthracnose:

[0117] Three experimental groups and one control group were set up in a tea garden (tea trees 25 years old) with the same planting and management level. The experimental groups were sprayed with 200 times of wettable powder (that is, wettable powder diluted 200 times with sterile water), 600 times of wettable powder (that is, wettable powder diluted 600 times with sterile water), and 1000 times of carbendazim (that is, carbendazim diluted 1000 times with sterile water), and the control group was sprayed with sterile water. Each treatment was repeated 4 times, and the area of ​​each repeated plot was 15m 2(1.5m×10m), randomized block arrangement, adjacent plots set up alternate rows. After spraying, the tea anthracnose pathogen was inoculated by wound inoculation method. Seven days after inoculation, 35 pieces of tea leaves were randomly sampled in parallel in each plot, and their disease levels were investigated and recorded. The incidence rate, disease index and control effect were calculated (Table 8). The results are shown in Table 8. Figure 3 shown.

[0118] Table 8: Disease classification criteria

[0119]

[0120] The above field experiments and Figure 3 It shows that in the test area where the wettable powder of the present application was applied, different treatments had good control effects on tea anthracnose, and the incidence and disease index of tea anthracnose were lower than those of the control group treated with clean water. The average field control effects of the wettable powder diluted 200 times and 600 times on tea anthracnose were 56.52% and 43%, respectively, and the control effect of the positive control 1000 times dilution of carbendazim was 58.68%. The control effect of the 200 times dilution treatment was significantly better than that of the 600 times dilution treatment, and there was no significant difference in the control effect with the carbendazim treatment, indicating that the 200 times dilution of the wettable powder of the present application has a good biological control effect and can be used for field control of tea anthracnose.

[0121] Application Example 4

[0122] The wettable powder prepared in Example 7 was used for field control of tea white spot disease:

[0123] In the high mountain tea garden (tea trees 20 years old) with the same planting and management level, 27 plots were set up, each with an area of ​​325m 2 (6.5m×50m), with interval rows in the middle. In the early stage of the disease of Camellia oleifera, 7 registered fungicides for tea tree diseases, such as psoralea corylifolia seed extract, Bacillus subtilis, copper hydroxide, and the wettable powder of the present application (represented by a 300-fold sterile water dilution of the wettable powder prepared in Example 7) were sprayed. Each treatment was repeated 3 times, and clean water was used as the control. The spraying was performed twice with an interval of 7 days. 14 days after the first application of the pesticide, 100 pieces of tea leaves were randomly sampled in parallel in each plot, and the disease level was investigated and recorded respectively. The incidence rate, disease index and prevention effect were calculated (Table 10). The results are as follows Figure 4 and as shown in Table 9.

[0124] Table 9: Types of drugs and application concentrations

[0125] Drug Name Active ingredients and content Dosage form Drug concentration Psoralea corylifolia seed extract Propylene oxadiazine 0.2% Microemulsion 750 times liquid Bacillus subtilis Bacillus subtilis 100 billion spores / g Wettable powder 750 times liquid Copper hydroxide Copper hydroxide 46% Water dispersible granules 1500 times dilution Picoxystrobin Picoxystrobin 22.5% Suspension 1500 times dilution Difenoconazole Difenoconazole 10% Water dispersible granules 1500 times dilution Amino Acid Oligosaccharides Aminooligosaccharides 5% Soluble solution 450 times liquid This application wettable powder Bacillus brevis 1.9 billion spores / g Wettable powder 300 times liquid Mineral oil Mineral oil 99% EC 150 times liquid .

[0126] Table 10: Disease classification criteria

[0127]

[0128] Depend on Figure 4 It can be seen that the field control effect of the wettable powder of the present application on tea white star disease is comparable to that of psoralea corylifolia seed extract, Bacillus subtilis, difenoconazole and amino oligosaccharides, and is significantly higher than mineral oil. And the use of 300 times the wettable powder of the present application is sufficient to achieve field control of tea white star disease.

[0129] It can be seen from the above process that the wettable powder of the present application can simultaneously control a variety of pathogens common in tea gardens, including tea anthracnose, tea root rot, tea white spot disease, tea ring spot disease and tea round red spot disease, and has a significant control effect.

[0130] In the above embodiments, the preparation is in the form of wettable powder, which is convenient for transportation and storage.

[0131] The above-mentioned embodiments only express several feasible implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention, and the embodiments are not used to limit the scope of protection in the claims of the present invention. For ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and all equivalent implementations or changes that do not depart from the present invention should be included in the technology of the present invention.

Claims

1. A wettable powder of a tea tree disease biocontrol bacterium, characterized in that: It includes Bacillus brevis HNCS-1 fermentation liquid, carrier, auxiliary agent and protective agent, wherein: The spore rate of the Bacillus brevis HNCS-1 fermentation liquid is more than 90%, and the mass percentage of the fermentation liquid is 50-87.5%. The auxiliary agent is selected from at least one of a dispersant and a wetting agent, and the weight percentage of the auxiliary agent is 4-16%. The protective agent has a mass percentage of 1-10%. The mass percentage of the carrier is 5-25%.

2. The wettable powder of a tea tree disease biocontrol bacteria according to claim 1, characterized in that: The carrier is any one of kaolin, diatomaceous earth, light calcium carbonate, bentonite and white carbon black.

3. The wettable powder of a tea tree disease biocontrol bacteria according to claim 1, characterized in that: The protective agent is any one of dextrin, ascorbic acid, sodium carboxymethyl cellulose and soluble starch.

4. The wettable powder of a tea tree disease biocontrol bacteria according to claim 1, characterized in that: The auxiliary agent is a dispersant or a wetting agent, and the dispersant is any one of sodium lignin sulfonate, hydroxypropyl cellulose, calcium lignin sulfonate, dispersant NNO, gum arabic, and polyethylene glycol; the wetting agent is any one of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, pull-opening powder, calcium chloride, penetrant T, Silok, and Sicare.

5. The wettable powder of a tea tree disease biocontrol bacteria according to claim 1, characterized in that: The auxiliary agent includes a wetting agent and a dispersant at the same time, the mass ratio of the dispersant to the wetting agent is 1-9:9-1, the wetting agent is calcium chloride or Sicare, and the dispersant is any one of hydroxypropyl cellulose, calcium lignin sulfonate, gum arabic, and polyethylene glycol.

6. A wettable powder of a tea tree disease biocontrol bacteria according to any one of claims 1 to 5, characterized in that: The mass percentage of the Bacillus brevis HNCS-1 fermentation liquid is 70-83.2%, the mass percentage of the carrier is 10-25%, and the mass percentage of the auxiliary agent is 4-5%.

7. A method for preparing a wettable powder of the tea tree disease biocontrol bacteria according to claim 1, characterized in that: Step 1: culturing Bacillus brevis HNCS-1 to obtain a fermentation broth. Step 2: Mix the culture solution and the carrier in proportion, and spray dry at 60°C to obtain mother powder. Step three, adding the auxiliary agent and the protective agent to the mother powder respectively, mixing evenly, and obtaining a wettable powder.

8. The wettable powder of tea tree disease biocontrol bacteria according to claim 7, characterized in that: In step 1, the specific preparation process of the fermentation broth is as follows: the HNCS-1 strain stored at -80°C is transferred to the LB solid culture medium, cultured at 37°C, a single colony is selected and inoculated into the LB liquid culture medium, and cultured at 37°C by shaking to obtain the seed liquid, and then the seed liquid is inoculated into the NB liquid culture medium, and cultured at 26°C by shaking to obtain the fermentation broth.

9. An application of the wettable powder of the tea tree disease biocontrol bacteria according to claim 1, characterized in that: After diluting the wettable powder with water, spray it on the surface of the tea bush to prevent and control at least two of the tea tree diseases including tea anthracnose, tea root rot, tea white star disease, tea ring spot disease and tea round red star disease.

10. The use of a wettable powder of a tea tree disease biocontrol fungus according to claim 9, characterized in that: The dilution factor should not exceed 600 times.

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