A complex microbial agent containing bacillus simplex and bacillus megaterium and its application in tea planting

Abstract

The application belongs to the field of agricultural microorganisms, and discloses a compound microbial agent containing bacillus simplex and bacillus megaterium and application of the compound microbial agent in tea planting. The compound microbial agent is composed of bacillus simplex CYG04 and bacillus megaterium CY32. The preservation number of the bacillus simplex is CCTCC NO:M20231901, and the preservation number of the bacillus megaterium is CCTCC NO:M20211409. Compared with a single strain, the compound strain has the effects of promoting growth and increasing yield on tea, and also has the effects of preventing and controlling tea cake disease, gray mold and / or target spot. The compound strain has a certain application prospect in the aspects of preventing diseases and increasing yield of tea.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology, specifically relating to a compound microbial agent containing Bacillus simplex and Bacillus megaterium and its application in tea cultivation. Background Technology

[0002] Plant rhizosphere microorganisms are considered the plant's second genome, playing a crucial role in nutrient absorption by the host and assisting plants in coping with various biotic and abiotic stresses. Applying rhizosphere growth-promoting bacteria to improve crop yield and quality is one of the important trends in modern green agriculture. Currently, the main bottleneck restricting the development of the bio-fertilizer industry is the instability of its field application effects. Functional microorganisms in fertilizers face rejection and competition from native microorganisms after being applied to the soil, making it difficult to guarantee the stable functioning of microbial fertilizers. Scientific research shows that improving the species diversity of functional microorganisms, i.e., applying compound microbial agents, can enhance the functioning and stability of various biological functions of microbial fertilizers. This can effectively improve crop quality and yield, as well as disease resistance, thereby achieving the effect of reducing fertilizer and pesticide use, and contributing to intensive agricultural production and sustainable development.

[0003] Tea trees mostly grow in warm, humid tropical and subtropical regions, where the climate is conducive to the development of tea diseases. Tea cake disease and ring spot disease are two important tea diseases, reported in tea-growing areas worldwide, including my country, where they can cause significant yield reductions, quality declines, and severely impact tea production. The pathogen of tea cake disease is *Exobasidium vexans*, an obligate parasite that cannot be cultured in vitro, posing a challenge to research. Currently, *Pseudomonas fluorescens* Pf1 has been reported to have some control effect on tea cake disease in the field; however, these biocontrol bacteria exist in vegetative form without spores, resulting in a short shelf life and hindering widespread application. Additionally, *Bacillus subtilis* B-9 has been reported to have some inhibitory activity against the pathogen of tea cake disease. Currently, only three pesticides are registered in China for controlling tea leaf spot: polyoxin, Bacillus subtilis, and psoralea seed extract. Control agents are relatively scarce. Tea ring spot is mainly caused by fungi of the genus *Pestalotiopsis* spp. Sanjay et al. isolated *Trichoderma harzianum*, *Gliocladium virena*, and *Pseudomonas fluorescens* from tea garden soil, but their field control efficacy was all below 40%, indicating unsatisfactory results. Additionally, *Bacillus belyi* CY30 has been reported to have some control effect on tea ring spot, but there are currently no registered agents for this disease. The use of chemical fungicides remains the main method for controlling tea leaf spot and tea leaf spot; however, the application of chemical fungicides easily leads to fungal resistance and also causes pesticide residues and environmental pollution.

[0004] Fertilization is one of the key agricultural measures affecting tea quality and yield, as well as sustainable soil utilization. Fertilizers have a significant effect on improving the quality and efficiency of tea. Traditional tea gardens mainly use fast-acting chemical fertilizers such as urea and compound fertilizers, and the amount of chemical fertilizers used is relatively large, which causes soil acidification and compaction, leading to a decline in tea quality. These problems urgently need to be solved in production.

[0005] Current reports on the role of simple Bacillus in disease control show that strain P10 has good inhibitory effects against *Phytophthora infestans*, the pathogen causing potato late blight; strain STY005 has good control effects against potato scab; and strain FJAT-47158 has good inhibitory effects against *Fusarium oxysporum*. However, the causal agent of tea scab is a basidiomycete, which differs significantly from the aforementioned plant pathogens in taxonomy. Furthermore, none of these strains were isolated from the rhizosphere soil of tea trees and are not native microorganisms of tea gardens. Their colonization ability in the roots and tea garden soil differs from that of native microorganisms, thus affecting their effect on promoting tea growth and increasing yield.

[0006] To address the aforementioned issues, this invention isolates and screens a simple Bacillus CYG04 from the rhizosphere soil of tea trees. This Bacillus has control effects against both tea leaf spot and tea leaf blight, and also possesses the ability to colonize tea trees in the rhizosphere and promote growth in tea trees. When combined with the applicant's prior application strain Bacillus megaterium CY32 (CN114085797B), it exhibits a synergistic effect, significantly improving both its disease prevention and growth promotion capabilities. Summary of the Invention

[0007] The purpose of this invention is to provide a compound microbial agent containing Bacillus simplex CYG04 and Bacillus megaterium CY32, with preservation numbers CCTCC NO:M 20231901 and CCTCC NO:M 20211409, respectively. This compound Bacillus agent can be used to promote tea growth and yield, and to control tea cake disease, gray mold, and / or ring spot disease.

[0008] Another object of the present invention is to provide the application of compound microbial agents in tea cultivation.

[0009] To achieve the above objectives, the present invention adopts the following technical solutions.

[0010] A Bacillus compound inoculant, comprising Bacillus simplex CYG04 and Bacillus megaterium CY32, with accession numbers CCTCC NO:M 20231901 and CCTCC NO:M20211409, respectively.

[0011] Preferably, the above-mentioned compound bacterial agent is prepared by mixing Bacillus simplex and Bacillus megaterium at an effective bacterial concentration ratio of 1:0.5-2.

[0012] The scope of protection of this invention includes:

[0013] The scope of protection of this invention also includes the application of the above-mentioned compound microbial agent in tea cultivation varieties, including its use in preventing and controlling tea cake disease, ring spot disease, gray mold, and / or promoting tea growth.

[0014] When used for biocontrol, it is applied by spraying.

[0015] The scope of protection of this invention also includes the use of the above-mentioned compound microbial agent in the preparation of antibacterial agents, wherein the antibacterial agents inhibit *Pestalotiopsis theae* and / or *Botrytis cinerea*.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] (1) This invention provides a compound microbial agent, which is reported for the first time to be used to promote the growth and increase the yield of tea and to prevent and control tea cake disease and ring spot disease, and has no cross-resistance with existing bactericides.

[0018] (2) Compared with the current use of chemically synthesized pesticides for pest control, it has the advantages of being highly efficient, low in toxicity, not polluting the environment, and not easily developing resistance, because it comes from the natural environment.

[0019] (3) The compound Bacillus obtained in this invention has a synergistic effect on promoting growth and increasing yield compared with a single strain, and is particularly suitable for use in tea production.

[0020] (4) The present invention provides giant and simple Bacillus strains, which are highly resistant to stress, easy to ferment in large quantities, and have a broad antibacterial spectrum. (5) The compound microbial agent provided by the present invention has a growth-promoting effect on tea trees, can colonize the roots of tea trees, is easy to use, leaves no residue or pollution, and can effectively reduce the application of chemical fertilizers and pesticides. Attached Figure Description

[0021] Figure 1 Colony morphology of strains CYG04 and CY32.

[0022] Figure 2 Schematic diagram of the antibacterial activity of CYG04.

[0023] Figure 3 Schematic diagram of the antibacterial activity of CY32.

[0024] Figure 4 A schematic diagram illustrating the effect of compound microbial agents in controlling gray mold in tea. Detailed Implementation

[0025] To better explain the present invention, the main contents of the invention are further illustrated below with reference to specific embodiments, but the content of the present invention is not limited to the following embodiments. Unless otherwise specified, the technical solutions described in this invention are conventional techniques in the art, and the reagents or materials described, unless otherwise specified, are all from commercial sources. The Bacillus megaterium CY32 involved in this invention has been disclosed in CN114085797B.

[0026] Example 1:

[0027] Obtaining and identifying simple Bacillus CYG04:

[0028] Bacillus simplex CYG04 was isolated from the rhizosphere soil of tea trees in Maoba Town, Lichuan City, Enshi Prefecture, Hubei Province. Figure 1This strain was obtained through screening for antibacterial activity against the pathogens of tea leaf spot and gray mold. Its taxonomic position was further determined by 16S rDNA analysis combined with physiological and biochemical identification (Table 1). It was deposited at the China Center for Type Culture Collection (CCTCC) on October 16, 2023, with the following classification: Bacillus simplex CYG04; accession number: CCTCC NO: 20231901; location: Wuhan University, Wuhan, China.

[0029] In this invention, Bacillus simplex CYG04 is referred to as CYG04.

[0030] Table 1 Physiological and biochemical characteristics of CGY04

[0031]

[0032]

[0033] Example 2:

[0034] Fermentation of Bacillus simplex CYG04 (all percentages in the following culture medium formulations are by mass):

[0035] 1) Primary seed culture: 100 ml of primary seed culture medium was placed in a 500 ml Erlenmeyer flask and sterilized by moist heat at 121℃ for 30 min. The CYG04 lyophilized tube inoculum was inoculated into the primary seed culture medium and cultured at 35℃ and 180 rpm for 10 h. The raw materials and quantities of the culture medium used for primary seed culture were: glucose 4.0%, beef extract 2%, peptone 1.5%, sodium chloride 0.5%, and the pH of the culture medium was 7.

[0036] 2) Secondary seed culture: 200L of secondary seed culture medium was placed in a 500L fermenter and sterilized at 121℃ for 30 minutes. 200ml of primary seed was inoculated into the secondary seed culture medium. The culture was carried out at 35℃ for 12 hours. The pressure of the fermenter was controlled at 0.05Mpa, the stirring speed at 200rpm, and the aeration ratio at 1:0.5. The raw materials and quantities of the culture medium used for secondary seed culture were as follows: glucose 0.5%, yeast extract 0.05%, fish meal 2%, potassium dihydrogen phosphate 0.001%, magnesium sulfate 0.01%, and the pH of the culture medium was 7.

[0037] 3) Fermentation: A 10-ton fermenter was filled with 5 tons of CYG04 fermentation medium and sterilized by moist heat at 121℃ for 30 minutes. 200L of secondary seed liquid was transferred into the fermentation medium. The tank pressure was controlled at 0.02Mpa, the stirring speed at 150rpm, the aeration ratio at 0.5-1.0, and the culture was carried out at 35℃ for 24 hours. The raw materials and dosages of the fermentation medium were: corn flour 11g / L, soybean meal 6g / L, yeast powder 10g / L, potassium dihydrogen phosphate 0.3g / L, magnesium sulfate 0.5g / L, and the pH of the medium was 7.

[0038] 4) Fermentation is stopped when 20-40% of the spores have detached. After being placed in a tank, the fermentation broth contains 12 billion CFU / mL of spores. This is used in the following examples.

[0039] Example 3:

[0040] Obtaining and identifying Bacillus megaterium CY32:

[0041] Bacillus megaterium CY32 was isolated from the rhizosphere soil of tea trees in Wangjiapo Village, Hefeng County, Enshi Prefecture, Hubei Province. Figure 1 The taxonomic position of this strain was further determined by 16S rDNA analysis combined with physiological and biochemical identification (Tables 2 and 3). It was deposited at the China Center for Type Culture Collection (CCTCC) on November 15, 2021, with the following classification: Bacillus megaterium CY32; accession number: CCTCC NO: 20211409; location: Wuhan University, Wuhan, China.

[0042] In this invention, Bacillus megaterium CY32 is referred to as CY32 for short.

[0043] Morphological characteristics: Colonies are round and milky white. Figure 1 ).

[0044] Physiological and biochemical characteristics of the strain:

[0045] Table 2 Physiological and biochemical characteristics of strain CY32 - enzyme activity, carbon source oxidation

[0046]

[0047] +: Positive reaction; -: Negative reaction;

[0048] Table 3. Physiological and biochemical characteristics of strain CY32 - Acid production using carbon sources

[0049]

[0050]

[0051] +: Positive reaction; -: Negative reaction;

[0052] Example 4

[0053] Fermentation of Bacillus megaterium CY32 (all percentages in the following culture medium formulations are by mass):

[0054] 1) Primary seed culture: 100 ml of primary seed culture medium was placed in a 500 ml Erlenmeyer flask and sterilized by moist heat at 121℃ for 15-30 min. The CY32 lyophilized tube inoculum was inoculated into the primary seed culture medium and cultured at 35℃ and 180 rpm for 10 h. The raw materials and amounts of the culture medium used for primary seed culture were: glucose 3%, beef extract 1.5%, peptone 0.8%, sodium chloride 0.5%, and the pH of the culture medium was 7.

[0055] 2) Secondary seed culture: 200L of secondary seed culture medium was placed in a 500L fermenter and sterilized at 121℃ for 15-30 minutes. 200ml of primary seed was inoculated into the secondary seed culture medium. The culture was carried out at 35℃ for 10 hours. The pressure in the fermenter was controlled at 0.05Mpa, the stirring speed was 200rpm, and the aeration ratio was 1:0.5. The raw materials and quantities of the culture medium used for secondary seed culture were as follows: glucose 0.5%, yeast extract 1%, peptone 3%, potassium dihydrogen phosphate 0.001%, magnesium sulfate 0.01%, and the pH of the culture medium was 7.

[0056] 3) Fermentation: A 10-ton fermenter was filled with 6 tons of CY32 fermentation medium and sterilized at 121℃ for 30 minutes. 200L of secondary seed culture was transferred into the fermentation medium. The tank pressure was controlled at 0.02Mpa, the stirring speed at 150rpm, and the aeration ratio at 0.5-1.0. The fermentation was carried out at 35℃ for 24 hours. The raw materials and quantities of the fermentation medium were as follows: corn flour 2.0%, molasses 1.0%, peanut meal 1.5%, yeast extract 1.5%, potassium dihydrogen phosphate 0.015%, magnesium sulfate 0.04%, calcium carbonate 0.15%, and the pH of the medium was 7.

[0057] 4) Fermentation is stopped when 20-40% of the spores have detached. After being placed in a tank, the spore count in the obtained fermentation broth is 5 billion CFU / mL. This is used in the following examples.

[0058] Example 5:

[0059] In vitro antibacterial activity of Bacillus simplex CYG04 and its application in the prevention and control of tea leaf spot, ring spot and gray mold:

[0060] The tested fungi *Pestalotiopsis theae*, the pathogen causing tea leaf spot, and *Botrytis cinerea*, the pathogen causing tea gray mold, were isolated from leaves infected with these pathogens and stored at 4°C on PDA slants. Other pathogenic fungi included *Phytophthora capsici*, the pathogen causing blight of pepper; *Sclerotinia sclerotiorum*, the pathogen causing sclerotinia rot of lettuce; *Rhizoctonia solani*, the pathogen causing sheath blight of rice; *Colletotrichum graminicola*, the pathogen causing anthracnose of maize; *Fusarium oxysporum*, the pathogen causing wilt of watermelon; and *Alternaria alternata*, the pathogen causing red spot disease of tobacco. All of these fungi were stored at 4°C on PDA slants.

[0061] The slant culture strains were transferred to PDA plates for activation and incubated at 30°C for 6 days. Sterile filter paper discs were placed at both ends of the PDA plate, followed by 7 μL of CYG04 fermentation broth. After 24 hours, the fungal pathogens were inoculated into the plates using a 4 mm punch, with sterile water serving as a blank control. The distance between the pathogens and the filter paper discs was 25 mm. Each setup was repeated three times. After the control and plate were fully colonized, the colony diameters for each treatment and the control were recorded, and the inhibition rate was calculated.

[0062] Inhibition rate = [(Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelial cake diameter)] × 100%

[0063] In vitro antibacterial experiments showed that CYG04 exhibited good antibacterial activity against a variety of plant pathogens, with inhibition rates of 88.21% and 85.37% against *Botrytis cinerea* and *Botrytis cinerea*, respectively. Figure 2 (Table 4).

[0064] Table 4. Antibacterial activity of CYG04 against pathogens

[0065]

[0066]

[0067] *Pseudomonas aeruginosa* and *Botrytis cinerea* were activated and cultured on PDA plates for 5 days. Uniform tea leaves (Zhongcha 108) without fungicide treatment were collected for in vitro leaf experiments. CYG04 fermentation broth was prepared according to Example 2, with approximately 12 billion cfu / mL spores. -1The fermentation broth was diluted 10 times and 20 times with sterile water for later use. Using an electric sprayer, the fermentation broth was evenly sprayed onto tea leaves, 2 mL per leaf. A blank control was sprayed with sterile water. After each treatment was air-dried, tea leaf spot and gray mold fungal cakes were inoculated using the needle-punch inoculation method. The mixture was incubated at 28℃ for 12 days. The diameter of lesions in the control and treatments was recorded, and the control efficacy was calculated. Each treatment was repeated three times, with 15 leaves per replicate. Control efficacy = (control lesion diameter - treatment lesion diameter / control lesion diameter) × 100.

[0068] Experiments showed that Bacillus simplex CYG04 had good control efficacy against tea leaf spot and gray mold. The diameter of lesions after treatment with 10-fold and 20-fold fermentation broth was significantly lower than that of the control. The control efficacy against leaf spot was 75.93% and 56.19%, respectively, and the control efficacy against gray mold was 70.93% and 56.83%, respectively (Tables 5 and 6).

[0069] Table 5. Effects of CYG04 fermentation broth on tea leaf spot control

[0070]

[0071]

[0072] Table 6. Effects of CYG04 fermentation broth on control of gray mold in tea.

[0073] deal with Lesion diameter (mm) Preventive efficacy (%) CYG04 10x fermentation broth 6.82 70.93 CYG04 20x fermentation broth 10.17 56.83 Comparison 23.56 -

[0074] The field efficacy trial for tea cake disease was conducted in Zouma Town, Hefeng County, Enshi Prefecture. A 100-fold diluted CYG04 fermentation solution was sprayed before the disease appeared, applied every 7 days for a total of 3 applications. A control group was sprayed with plain water. The trial consisted of 4 replicates, each 32 m². 2 After sufficient disease development in the control group, the disease index was statistically analyzed and compared with the control group. Five points were selected from each plot, totaling 50 leaves, to investigate the disease index and calculate the control efficacy. Disease index grading standards: Grade 0: No lesions; Grade 1: Lesion area less than 1% of leaf area; Grade 3: Lesion area 2%-10% of leaf area; Grade 5: Lesion area 11%-25% of leaf area; Grade 7: Lesion area 26%-50% of leaf area; Grade 9: Lesion area more than 50% of leaf area.

[0075] Disease index = ∑(number of plants at each level × level) / (total number of plants surveyed × highest representative level) × 100;

[0076] Prevention and control effect = (Control disease index - Treatment disease index) / Control disease index × 100%

[0077] Experiments showed that CYG04 had good field control efficacy against tea scab, with a control efficacy of 65.59% in 100-fold fermentation solution (Table 7).

[0078] Table 7. Effects of CYG04 fermentation broth on tea cake disease control

[0079] deal with Disease index Preventive efficacy (%) CYG04 100x fermentation broth 14.65 65.59 Comparison 42.57 -

[0080] Example 6:

[0081] In vitro antibacterial activity of Bacillus megaterium CY32:

[0082] The tested tea leaf spot pathogen (Pestalotiopsis theae) was isolated from tea leaf diseased with tea leaf spot and stored at 4°C on a PDA slant. Other pathogenic fungi included *Colletotrichum gloeosporioides* (the pathogen of walnut anthracnose), *Sclerotinia sclerotiorum* (the pathogen of lettuce sclerotium rot), *Botrytis cinerea* (the pathogen of strawberry gray mold), *Rhizoctonia solani* (the pathogen of rice sheath blight), *Alternaria solani* (the pathogen of tomato early blight), *Fusarium solani* (the pathogen of solanaceous plant disease), and *Fusarium oxysporum* (the pathogens of tomato early blight), all stored at 4°C on PDA slant. *Pythium aphanidermatum* was stored at 10°C on PDA slant. The tested pathogenic bacteria was *Xanthomonas campestris* (the pathogen of walnut black spot), stored at -20°C in glycerol tubes.

[0083] The slant culture strains were transferred to PDA plates for activation and incubated at 30°C for 6 days. Sterile filter paper discs were placed at both ends of the PDA plate, followed by 7 μL of CY32 fermentation broth. After 24 hours, the fungal pathogens were inoculated into the plates using a 4 mm punch, with sterile water serving as a blank control. The distance between the pathogens and the filter paper discs was 25 mm. Each setup was repeated three times. After the control and plate were fully colonized, the colony diameters for each treatment and the control were recorded, and the inhibition rate was calculated.

[0084] Inhibition rate = [(Control colony diameter - Treated colony diameter) / (Control colony diameter - Mycelial cake diameter)] × 100%

[0085] Single colonies of the bacterial pathogen *Xanthomonas brassicae* were obtained by streaking with glycerol tubes and then transferred to NB liquid medium. The culture was incubated at 37°C for 48 hours with shaking at 180 rpm. NA medium was melted and poured into plates at approximately 45°C. *Xanthomonas brassicae* bacterial suspension was then added at a ratio of NA to bacterial suspension of 50:1, and thoroughly mixed. Sterilized Oxford cups were then used to punch wells on the inoculated plates, and 200 μL of CY32 fermentation broth was added to each well. The plates were incubated at 37°C for 48 hours to observe for the formation of inhibition zones.

[0086] In vitro antibacterial tests showed that CY32 exhibited high antibacterial activity (above 50%) against *Polytrichum jasminoides*, *Sclerotinia sclerotiorum*, *Colletotrichum gloeosporioides*, *Botrytis cinerea*, *Rhizoctonia solani*, and *Alternaria alternata*, with all showing above 50% inhibition. The highest antibacterial activity was observed against *Polytrichum jasminoides*, the pathogen of tea leaf spot. It also demonstrated good antibacterial activity against the pathogen *Xanthomonas auricula-judae*, exhibiting a distinct inhibition zone. However, its antibacterial activity against *Pythium spp.*, *Fusarium oxysporum*, and *Fusarium oxysporum* was relatively poor. (Table 8) Figure 3 ).

[0087] Table 8. Antibacterial activity of CY32 against pathogens

[0088]

[0089]

[0090] Pestalotiopsis theae was activated and cultured on PDA plates for 5 days. Uniform tea leaves (Zhongcha 108) that had not been treated with fungicides were collected for in vitro leaf experiments. CY32 fermentation broth was prepared according to Example 3, with approximately 5 billion cfu / mL spores. -1 The fermentation broth was diluted 10 times and 20 times with sterile water for later use. Using an electric sprayer, the fermentation broth was evenly sprayed onto tea leaves, 2 mL per leaf. A blank control was sprayed with sterile water. After each treatment was air-dried, tea leaf spot pathogen cakes were inoculated using the needle inoculation method and cultured at 28℃ for 12 days. The diameters of lesions in the control and treatments were counted, and the control efficacy was calculated. Each treatment was repeated three times, with 15 leaves per replicate. Control efficacy = (control lesion diameter - treatment lesion diameter / control lesion diameter) × 100.

[0091] Experiments showed that Bacillus megaterium CY32 had a good control effect on tea leaf spot disease. The diameter of the lesions after treatment with 10-fold and 20-fold fermentation broth was significantly lower than that of the control, with control effects of 76.88% and 56.38%, respectively (Table 9).

[0092] Table 9. Effects of CY32 fermentation broth on tea leaf spot control

[0093] deal with Lesion diameter (mm) Preventive efficacy (%) CY32 10x fermentation broth 6.25 76.88 CY32 20x fermentation broth 11.79 56.38 Comparison 27.03 -

[0094] Example 7:

[0095] Colonization ability of Bacillus simplex CYG04 in tea tree rhizosphere soil

[0096] Single colonies of *Bacillus simplex* CYG04 were screened for resistance using medium containing different concentrations of rifampicin to obtain rifampicin-resistant mutant strains (these strains, apart from exhibiting rifampicin resistance, showed no significant differences in other physiological and biochemical characteristics compared to the original strain). These strains could be cultured in medium containing 300 μg / mL rifampicin. -1 It grows normally in LB with rifampicin.

[0097] The resistant mutant CYG04 was fermented according to Example 2. The spore count of the fermentation broth was adjusted to 100 million CFU / mL with water and sprayed onto the roots of tea plants at a rate of 2 L / plant. Tomato plants were sprayed as a control. After 30 days, rhizosphere soil was obtained by shaking and diluted using a serial dilution method. The diluted soil was then spread onto a substrate containing 300 μg / mL of the nutrient solution. -1 The colonization capacity of CYG04 in the rhizosphere soil of tea and tomato was determined by counting rifampicin on LB plates at 37°C for 24 hours.

[0098] Experiments showed that CYG04 had a stronger and more specific colonization ability in the rhizosphere soil of tea trees, with a colonization rate of 4.16 × 10⁻⁶. 6 cfu / g, while the tomato colony was only 4.23×10 4 cfu / g.

[0099] Example 8:

[0100] Colonization ability of Bacillus megaterium CY32 in tea tree rhizosphere soil

[0101] Single colonies isolated from Bacillus megaterium CY32 and commercially available Bacillus megaterium bacterial fertilizer were screened for resistance on media containing different concentrations of rifampicin. Rifampicin-resistant mutant strains were obtained (these strains, apart from exhibiting rifampicin resistance, showed no significant differences in other physiological and biochemical characteristics compared to CY32). These strains can be cultured on media containing 300 μg / mL rifampicin. -1 It grows normally in LB with rifampicin.

[0102] The resistant mutant CY32 and a commercially available Bacillus megaterium were fermented according to Example 2. The spore count of the two fermentation broths was adjusted to 100 million CFU / mL with water and sprayed onto the roots of tea trees, ensuring full contact between the roots and the fermentation broth. After 30 days, rhizosphere soil was obtained by shaking, diluted using a serial dilution method, and then spread onto a substrate containing 300 μg / mL of the fermentation broth. -1 The colonization capacity of CY32 in the rhizosphere soil of tea was determined by counting rifampicin on LB plates at 37°C for 24 hours.

[0103] Experiments showed that CY32 had a stronger ability to colonize the rhizosphere soil of tea trees, with a colonization rate of 3.85 × 10⁻⁶. 6 The concentration of cfu / mL was higher than that of commercially available Bacillus megaterium, which was only 5.19 × 10⁻⁶. 4 . ,

[0104] Example 9

[0105] Effects of compound Bacillus on the control of tea diseases

[0106] Pestalotiopsis theae and Botrytis cinerea were activated and cultured on PDA plates for 5 days for later use. Uniform tea leaves (Zhongcha 108) without fungicide treatment were collected for in vitro leaf experiments. The fermentation broths of CYG04 and CY32 prepared in the previous example were mixed at an effective concentration ratio of 1:1 to obtain a compound Bacillus strain (effective bacterial concentration of 2.5 billion CFU / mL for both strains) for later use.

[0107] Treatment 1: CYG04 fermentation broth (5 billion CFU / mL); Treatment 2: CY32 fermentation broth (5 billion CFU / mL); Treatment 3: Compound microbial agent fermentation broth (5 billion CFU / mL); Treatment 4: Water control. The fermentation broth was diluted 10 times and sprayed evenly onto tea leaves using an electric sprayer, 2 mL per leaf. The blank control was sprayed with sterile water. After each treatment was air-dried, it was inoculated with tea leaf spot fungus cakes using the needle inoculation method and with gray mold using the fungal patch inoculation method. The tea leaves were incubated at 28℃ for 12 days. The diameter of lesions in the control and treatments was recorded, and the control efficacy was calculated. Each treatment was repeated 3 times, with 15 leaves per replicate. Control efficacy = (control lesion diameter - treatment lesion diameter / control lesion diameter) × 100.

[0108] Experiments showed that the compound microbial agent had good control efficacy against tea leaf spot and gray mold, with control efficacy reaching 79.05% and 80.34% respectively. The compound microbial agent was more effective than the single microbial agent (Table 10).

[0109] The field efficacy trial for tea cake disease was conducted in Zouma Town, Hefeng County, Enshi Prefecture. Fermented inoculant broth was sprayed before the disease appeared. Treatment 1: CYG04 fermented broth (5 billion CFU / mL); Treatment 2: CY32 fermented broth (5 billion CFU / mL); Treatment 3: Compound inoculant fermented broth (5 billion CFU / mL); Treatment 4: Water control. Applications were given every 7 days for a total of 3 applications. The control was sprayed with water. The experiment had 4 replicates, each 32 m². 2After sufficient disease development in the control group, the disease index was statistically analyzed and compared with the control group. Five points were selected from each plot, totaling 50 leaves, to investigate the disease index and calculate the control efficacy. Disease index grading standards: Grade 0: No lesions; Grade 1: Lesion area less than 1% of leaf area; Grade 3: Lesion area 2%-10% of leaf area; Grade 5: Lesion area 11%-25% of leaf area; Grade 7: Lesion area 26%-50% of leaf area; Grade 9: Lesion area more than 50% of leaf area.

[0110] Disease index = ∑(number of plants at each level × level) / (total number of plants surveyed × highest representative level) × 100;

[0111] Prevention and control effect = (Control disease index - Treatment disease index) / Control disease index × 100%

[0112] Experiments showed that the compound microbial agent had good field control efficacy against tea cake disease, with a control efficacy of 68.43% in 100-fold fermentation liquid, which was better than that of single microbial agents (Table 10).

[0113] Table 10. Effects of compound microbial agent fermentation broth on tea disease control

[0114]

[0115] Example 10

[0116] The effect of compound Bacillus on tea yield increase

[0117] The experimental field is located in Zouma Town, Hefeng County, Enshi Prefecture, Hubei Province. Ten-year-old tea trees (Zhongcha 108) were selected for yield increase experiments.

[0118] The CYG04 and CY32 fermentation broths prepared in Examples 2 and 4 were mixed at an effective concentration ratio of 1:1 to obtain a compound Bacillus (the effective bacterial concentration of each was 2.5 billion CFU / mL) for later use.

[0119] Treatment 1: CYG04 fermentation broth (5 billion CFU / mL); Treatment 2: CY32 fermentation broth stock solution (5 billion CFU / mL); Treatment 3: Compound microbial agent fermentation broth stock solution (5 billion CFU / mL); Treatment 4: Water control.

[0120] Dilute 20 times and apply to the roots of tea trees for root irrigation, using 600 catties of water per mu (approximately 0.067 hectares). Use plain water as a control. Apply once every 7 days, for a total of three applications. After 20 days, at the one bud and two leaf stage, count the bud density (within a 33.3cm x 33.3cm square) and measure the weight of 100 buds.

[0121] The experimental results show that the compound microbial agent has a better growth-promoting effect than the single strain (Table 10).

[0122] Table 11 Effects of compound microbial agents on promoting growth and increasing yield in tea trees

[0123] deal with Germination density (number of germinated germs) Weight of 100 sprouts (g) CYG04 140.42 37.91 CY32 135.47 38.43 Compound microbial agent 155.67 40.56 CK 109.34 39.88

Claims

1. A Bacillus compound inoculant, comprising Bacillus simplex (Bacillus simplex) Bacillus simplex ) and Bacillus megaterium ( Bacillus megaterium The preservation number of the simple Bacillus is CCTCC NO: M20231901, and the preservation number of the giant Bacillus is CCTCC NO: M 20211409.

2. The compound microbial agent according to claim 1, wherein the simple Bacillus and the megaterium are mixed at an effective bacterial concentration of 1:0.5-2.

3. The application of the compound microbial agent according to claim 1 in the preparation of an antibacterial agent, wherein the antibacterial agent inhibits *Pterygodium japonicum* (a type of fungus). Pestalotiopsis theae ) and / or Botrytis cinerea ( Botrytis cinerea ).

4. The application of the compound microbial agent according to claim 1 in the prevention and control of tea leaf spot, gray mold and / or ring spot disease.

5. The application of the compound microbial agent according to claim 1 in promoting tea growth.

6. The application according to claim 4, when used for prevention and control, is by spraying.

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