Gastrodia elata continuous cropping obstacle antagonistic composite bacillus inoculant and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a gastrodia elata continuous cropping obstacle antagonistic composite bacillus inoculant and application thereof, specifically, the inoculant provided by the invention is microbial fermentation liquor, and microorganisms are bacillus amyloliquefaciens SWFU000435 and bacillus velezensis SWFU000440, and the bacillus amyloliquefaciens SWFU000435 and the bacillus velezensis SWFU000440 are bacillus amyloliquefaciens SWFU000435. The bacillus amyloliquefaciens SWFU000435 is classified and named as bacillus amyloliquefaciens, and the preservation number of the bacillus amyloliquefaciens SWFU000435 is CGMCC (China General Microbiological Culture Collection Center) NO.33089; the bacillus velezensis SWFU000440 is classified and named as bacillus velezensis, and the preservation number of the bacillus velezensis is CGMCC NO.33088. By additionally applying the microbial agent disclosed by the invention, not only can the disease incidence rate of gastrodia elata be reduced, but also the yield of gastrodia elata can be effectively increased, and economic loss caused by continuous cropping obstacles can be relieved.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to an antagonistic composite bacillus agent for Gastrodia elata continuous cropping obstacle and its application. Background Art

[0002] Gastrodia elata ( Gastrodia elata BI.) is one of the traditional Chinese medicinal materials in China and is cultivated in most parts of China. Especially, Gastrodia elata produced in Yiliang County, Zhaotong City, Yunnan Province is most famous and has extremely high economic value, medicinal and edible value. With the continuous expansion of the cultivation area of Gastrodia elata, the problem of continuous cropping obstacle has gradually emerged. Replanting Gastrodia elata will cause serious soil-borne diseases of Gastrodia elata, resulting in slow growth and development of Gastrodia elata and a significant decrease in its yield. The prevention and control of continuous cropping obstacles of traditional crops mainly rely on physical or chemical control, which not only has a serious impact on the ecological system safety, but also can lead to the deterioration of soil physical and chemical properties. In addition, as a traditional Chinese medicinal material, if chemical pesticides are used for prevention and control, its quality and safety will be seriously affected. Therefore, taking the diseases of Gastrodia elata as a starting point to alleviate and prevent the continuous cropping obstacle of Gastrodia elata is a key problem that urgently needs to be solved in the Gastrodia elata industrial chain. In recent years, biological control technology has shown remarkable effects in alleviating the problem of continuous cropping obstacles of crops due to its high efficiency, low toxicity and environmental friendliness. The research and development and application of microbial agents at home and abroad have become a competitive trend.

[0003] In recent years, the artificial cultivation industry of Gastrodia elata in Yiliang County, Zhaotong City, Yunnan Province has developed vigorously and has become a major economic pillar industry in the local area. As a perennial parasitic plant, due to its special planting mode, Gastrodia elata is often damaged by pathogenic bacteria during the growth process, seriously affecting its yield and quality. According to statistics, the root rot disease of Gastrodia elata caused by fungi causes at least a reduction of more than 20% in yield every year. The most important problem in Gastrodia elata cultivation is the continuous cropping obstacle problem. With the increasing demand of people, the planting area of Gastrodia elata has also been expanding year by year, the phenomenon of replanting is becoming more and more common, and the diseases of Gastrodia elata are becoming more and more serious, resulting in a decrease in yield and deterioration of quality, seriously affecting the economic benefits of hemp farmers. Pathogenic microorganisms are one of the important factors leading to the occurrence of diseases and the reduction of yield of Gastrodia elata. Therefore, the main purpose of the present invention is to analyze the main types of pathogenic bacteria causing the diseases of Gastrodia elata, find out the widely distributed pathogenic bacteria, and use the isolated pathogenic bacteria to screen antagonistic bacteria for the prevention and control of Gastrodia elata diseases and continuous cropping obstacles. Summary of the Invention

[0004] The main purpose of the present invention is to provide an antagonistic composite bacillus agent for Gastrodia elata continuous cropping obstacle and its application for the prevention and control of Gastrodia elata diseases and continuous cropping obstacles. Specifically, the present invention provides the following technical solutions: On the one hand, the present invention provides a special composite microbial agent for Gastrodia elata, and the agent is a microbial fermentation broth, and the microorganisms are Bacillus amyloliquefaciens SWFU000435 and Bacillus velezensis SWFU000440; The Bacillus amyloliquefaciens SWFU000435 is classified and named as Bacillus amyloliquefaciens Bacillus amyloliquefaciens , and the preservation number is: CGMCC NO.33089; The Bacillus velezensis SWFU000440 is classified and named as Bacillus velezensis Bacillus velezensis , and the preservation number is: CGMCC NO.33088; The special compound microbial inoculant for Gastrodia elata is prepared by mixing the fermentation broth of Bacillus amyloliquefaciens SWFU000435 and the fermentation broth of Bacillus velezensis SWFU000440 according to a volume ratio of 1:1. The effective viable count of the fermentation broth of Bacillus amyloliquefaciens SWFU000435 ≥ 6.9 × 10 8 CFU / g; the effective viable count of the fermentation broth of Bacillus velezensis SWFU000440 ≥ 7.1 × 10 8 CFU / g.

[0005] Furthermore, the fermentation broth medium of Bacillus amyloliquefaciens SWFU000435 is: glucose 20.59 g / L, yeast extract powder 5.55 g / L, peptone 10.26 g / L, and potassium chloride 2 g / L; the culture temperature is 31 °C, the initial pH of the medium is 6.0, and the shaker speed is 180 r / min; The fermentation broth medium of Bacillus velezensis SWFU000440 is glucose 11.3 g / L, beef extract powder 5.43 g / L, peptone 10.28 g / L, yeast extract powder 5.87 g / L, and potassium chloride 5.05 g / L; the culture temperature is 28 °C, the initial pH of the medium is 7.0, and the shaker speed is 180 r / min.

[0006] On the other hand, the present invention provides a special compound microbial fertilizer for Gastrodia elata, which includes microorganisms, carriers or auxiliaries, and is characterized in that the microorganisms are the compound microbial inoculant.

[0007] Furthermore, for the compound microbial fertilizer, the auxiliaries include one or more of wetting agents, dispersants, disintegrants, binders, synergists or protectants.

[0008] Furthermore, for the compound microbial fertilizer, the carrier includes talc powder, kaolin, bentonite, diatomaceous earth or light calcium carbonate; and / or the wetting agent includes sodium dodecyl benzene sulfonate (SDBS), sodium dodecyl sulfate (SDS), polyvinyl alcohol (PVA) or sodium carboxymethyl cellulose (CMC-Na); and / or the dispersant includes sodium carboxymethyl cellulose, sodium hexametaphosphate, polyvinylpyrrolidone or sodium polyphosphate, and / or the disintegrant includes sodium chloride, calcium chloride, ammonium sulfate or sodium alginate; and / or the binder includes added dextrin, polyethylene glycol (PEG) or soluble starch; and / or the synergist includes potassium humate from mineral sources, chitosan or amino acids; and / or the protectant includes ascorbic acid VC, humic acid or cyclodextrin.

[0009] Furthermore, for the compound microbial fertilizer, the dosage form of the fertilizer includes liquid agent, powder, granule, wettable powder, suspension or water dispersible granule.

[0010] Furthermore, for the compound microbial fertilizer, when the fertilizer is in powder form, it is obtained by fully mixing the compound microbial inoculant and the carrier in equal mass, drying the mixture, and then grinding it.

[0011] Furthermore, for the compound microbial fertilizer, when the fertilizer is in water dispersible granule form, the water dispersible granule, by mass percentage, is composed of 10%-80% of the compound microbial inoculant, 5% of the wetting agent, 6% of the dispersant, 4% of the disintegrant, 4% of the binder, and the carrier is made up to 100%. They are mixed to prepare a preparation, and then based on the preparation, 18% - 20% of distilled water is added, mixed evenly and granulated, and dried to obtain the water dispersible granule.

[0012] Furthermore, for the compound microbial fertilizer, when the fertilizer is in water dispersible granule form, the water dispersible granule, by mass percentage, is composed of 10%-80% of the compound microbial inoculant, 5% of the wetting agent sodium dodecyl benzene sulfonate, 6% of the dispersant sodium hexametaphosphate, 4% of the disintegrant calcium chloride, 4% of the binder polyethylene glycol, and the carrier diatomaceous earth is made up to 100%. They are mixed to prepare a preparation, and then based on the preparation, 18% - 20% of distilled water is added, mixed evenly and granulated, and dried to obtain the water dispersible granule.

[0013] Furthermore, the present invention provides the application of the inoculant or any of the compound microbial fertilizers in biological control of Gastrodia elata diseases or continuous cropping obstacles, or in the preparation of agents for preventing and treating Gastrodia elata diseases or continuous cropping obstacles.

[0014] Furthermore, the present invention provides a method for preventing and treating continuous cropping obstacles of Gastrodia elata, which is to apply the inoculant or any of the compound microbial fertilizers to the land with continuous cropping obstacles of Gastrodia elata.

[0015] The technical effects achieved by the present invention are as follows: The test results of the present invention show that the additional application of microbial inoculants can not only promote the reconstruction of the soil microbial community structure, reduce the incidence of Gastrodia elata diseases, but also effectively increase the yield of Gastrodia elata and alleviate the economic losses caused by continuous cropping obstacles. Compared with the single Bacillus amyloliquefaciens inoculant, the application of B. amyloliquefaciens SWFU000435 and B. velezensis SWFU000440 The preventive effect of the 300-fold Xinanlin 0001 compound bacteria water dispersible granule is significant, the incidence of Gastrodia elata diseases is reduced, and the yield of Gastrodia elata increases significantly, indicating that the two Bacillus strains in the Xinanlin 0001 compound bacteria water dispersible granule can be well colonized in the soil and have a good control effect on Gastrodia elata diseases. Applying the compound water dispersible granule in the planting area with obstacles of Gastrodia elata can play a good role in preventing and controlling the occurrence of Gastrodia elata diseases. On the one hand, this research lays a foundation for the development of microbial inoculants for Gastrodia elata diseases, and on the other hand, it has made a breakthrough in the aspect of biological control to alleviate the continuous cropping obstacles of Gastrodia elata. Description of the Drawings

[0016] Figure 1 Partial symptom diagram of Gastrodia elata tuber in field diseases; Figure 2 Determination result diagram of the affinity of the compound strain (a: B. amyloliquefaciens SWFU000435,b: B. velezensis SWFU000440 ); Figure 3 Diagram of the effect of the compound bacteria fermentation broth on the growth of the mycelia of Gastrodia elata pathogens; Figure 4 Product diagrams of different dosage forms of Xinanlin 0001 compound bacteria fertilizer. Detailed Embodiments

[0017] Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those not specified in the examples regarding specific techniques or conditions, the techniques or conditions described in the literature in this field or according to the product specifications shall be followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through purchase. The present invention will be further described in detail below with reference to the examples.

[0018] Bacillus amyloliquefaciens SWFU000435 provided in the present invention is classified and named as Bacillus amyloliquefaciens Bacillus amyloliquefaciens (abbreviated as B. amyloliquefaciens ), deposit number: CGMCC NO.33089, depositary institution: China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, deposit date: December 16, 2024. The viability of this biological material was detected by the depositary institution on December 16, 2024, and the result was that it was viable.

[0019] Bacillus velezensis SWFU000440, classified and named as Bacillus velezensis Bacillus velezensis (abbreviated as B. velezensis ), preservation number: CGMCC NO.33088, preservation unit: China General Microbiological Culture Collection Center (CGMCC), address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, preservation date: December 16, 2024. The viability of this biological material was detected by the preservation center on December 16, 2024, and the result was that it survived.

[0020] Example 1 Bacillus amyloliquefaciens ( B. amyloliquefaciens ) SWFU000435 and Bacillus velezensis ( B. velezensis ) SWFU000440's antibacterial effect on Gastrodia elata pathogenic bacteria 1. Isolation, purification, identification and verification of Gastrodia elata disease pathogens Collect disease samples ( Figure 1 ), and a total of multiple strains were isolated from all diseased Gastrodia elata by tissue isolation method. Genomic DNA was extracted from all isolates, and the diseased Gastrodia elata samples were identified. Then the pathogenic bacteria were inoculated back to healthy Gastrodia elata for inspection. After analysis and screening, 13 pathogens all caused varying degrees of rot in Gastrodia elata, which were Mucor circinelloides、Geotrichum sp., Bionectria ochroleuca , Trichoderma hamatum , Earliella scabrosa , Clonostachys rosea , Porogramme epimiltina , Fusarium oxysporum , Trichoderma koningii , Nemania diffusa , Fusarium solani , Trichoderma atroviride , Xylaria sp. Among them M. circinelloides , Geotrichum sp., B. ochroleuca , T. hamatum , C. rosea , Xylaria sp started to get sick 3 days after inoculation, E. scabrosa , P. epimiltina , F. oxysporum、T. koningii , N. diffusa , F. solani , T. atrovirideThe disease onset began 5 days after inoculation. The diseased parts of the Gastrodia elata after re-inoculation were subjected to re-isolation and purification experiments, and compared with the inoculation. Through molecular biological identification, it was determined that these 13 isolates were all pathogenic bacteria of Gastrodia elata.

[0021] Based on the results of analyzing the changes in the fungal community structure of the rhizosphere soil of Gastrodia elata with different continuous cropping cycles by high-throughput sequencing technology, this invention reveals that: the relative abundance of Mucoromycota in the soil gradually decreases with the increase in the number of planting cycles, which reveals that there are pathogenic bacteria in the soil itself that cause the rot of Gastrodia elata, and the planting of Gastrodia elata is more conducive to the Mucor circinelloides growth and reproduction of this pathogenic bacterium, resulting in an increase in the incidence of this pathogen, and further leading to the problem of continuous cropping obstacles of Gastrodia elata. With the increase in the number of continuous cropping times, the relative abundances of Ascomycota and Mucoromycota increase significantly. Among them, the relative abundances of Agaricomycetes and Sordariomycetes, Polyporales and Hypocreales all increase with the increase in the number of continuous cropping times; the results of this macroscopic-microscopic-molecular correspondence fitting reveal that: based on the analysis of the changes in the fungal community structure of the rhizosphere soil of Gastrodia elata with different continuous cropping cycles by high-throughput sequencing technology, the trends and taxa are fitted with the pathogenic taxa of pathogenicity tests; the continuous cropping of Gastrodia elata leads to an increase in the relative abundance of some pathogenic bacteria in the soil, resulting in an increase in the incidence of Gastrodia elata diseases, leading to a decrease in the yield and quality of Gastrodia elata, thus directly leading to the formation of continuous cropping obstacles of Gastrodia elata.

[0022] 2. Screening of antagonistic bacteria The plate confrontation method was used to preliminarily screen the strains with antagonistic effects against the pathogens of soil-borne diseases of Gastrodia elata, and the strains with an initial screening inhibition rate greater than 50% were re-screened; after multiple screenings, fungi with antagonistic activity against the pathogenic bacteria of Gastrodia elata were obtained, and the re-screening results are shown in Table 1 B. velezensis SWFU000440 The inhibition rates against 13 kinds of pathogenic bacteria were all above 50%; B. amyloliquefaciens SWFU000435 For Trichoderma. atroviride the inhibition rate was below 50%, and the rest were all above 50%; B. subtilis(Bacillus subtilis) For Fusarium. oxysporum and Trichoderma. atroviride the inhibition rate was below 50%.

[0023] Table 1 Re-screening inhibition rates of antagonistic bacteria

[0024] 3. Optimization of fermentation culture conditions for antagonistic bacteria Two strains with stable antagonistic effects screened in this invention are B. velezensis SWFU000440 and B. amyloliquefaciens SWFU000435 . Through single-factor experiments, it was determined that B. velezensis SWFU000440The optimal medium is BPY medium, and the best carbon source, nitrogen source, and inorganic salt are glucose, beef extract powder, peptone, yeast extract powder, and potassium chloride; B. amyloliquefaciens SWFU000435 The optimal medium is YSP medium, and the best carbon source, nitrogen source, and inorganic salt are glucose, yeast extract powder, peptone, and potassium chloride; The analysis results show that: B. velezensis SWFU000440 The optimal fermentation medium is: glucose 11.3 g / L, beef extract powder 5.43 g / L, peptone 10.28 g / L, yeast extract powder 5.87 g / L, potassium chloride 5.05 g / L. Under the condition of controlling a single fermentation culture condition, B. velezensis SWFU000440 The optimal culture temperature is 28 °C, the optimal initial pH of the medium is 7.0, and the optimal shaker speed is 180 r / min; After culturing for 18 h, the OD of the fermentation broth was actually measured three times 600 The average value is 0.711 ± 0.015.

[0025] B. amyloliquefaciens SWFU000435 The optimal fermentation medium of is: glucose 20.59 g / L, yeast extract powder 5.55 g / L, peptone 10.26 g / L, and potassium chloride 2 g / L. Under the condition of controlling a single fermentation culture condition; B. amyloliquefaciens SWFU000435 The optimal culture temperature is 31 °C, the optimal initial pH of the medium is 6.0, and the optimal shaker speed is 180 r / min. After culturing for 18 h, the OD of the fermentation broth was actually measured three times 600 The average value of the value is 0.699 ± 0.018.

[0026] 4. Determination of the affinity between composite strains The antagonistic strains were cross-streaked pairwise in NA medium (NA: peptone 10 g, beef extract 5 g, NaCl 1.5 g, agar 20 g, distilled water 1000 ml, natural pH), and cultured in an incubator at 37 °C for 2 d to observe whether there was an inhibition zone between the strains. If there was an inhibition zone, it indicated that the two strains were incompatible and there was antagonism. If there was no inhibition zone, it indicated that the two strains were compatible and could be co-cultured. The determination results of the affinity between the composite strains show that: B. velezensis SWFU000440 and B. amyloliquefaciens SWFU000435 The two strains were streaked pairwise on the surface of NA medium, and the results were as Figure 2 shown, B. velezensis SWFU000440 and B. amyloliquefaciens SWFU000435 Both of the two antagonistic strains could grow normally on the medium, and no inhibition zone was produced between the two strains, indicating that B. velezensis SWFU000440 and B. amyloliquefaciens SWFU000435 There was an affinity between the two strains, and there was no mutual antagonism, meeting the prerequisite conditions for developing a composite microbial agent.

[0027] 5. Determination of the antibacterial effect of the composite strain in the petri dish The B. velezensis SWFU000440 and B. amyloliquefaciens SWFU000435 two strains were respectively inoculated into two Erlenmeyer flasks containing 50 mL of liquid medium under their respective optimal culture media and fermentation culture conditions. After culturing for 18 h, the fermentation broths of the two strains were mixed at a ratio of 1:1 to form a composite bacterium, numbered Xinanlin 0001. The antibacterial effect of the fermentation broth of the composite bacterium Xinanlin 0001 against the pathogenic bacteria of Gastrodia elata was determined by the plate confrontation method. At the central position of the PDA medium plate, a mycelial cake of the pathogenic fungus of Gastrodia elata (diameter d = 5 mm) was inoculated with the mycelial surface facing down. Centered on the pathogenic fungus, circular filter papers (diameter d = 5 mm) soaked with the composite bacterium Xinanlin 0001 were inoculated at the three vertex positions in an equilateral "△" shape. The petri dish with circular filter papers without inoculating the composite bacterium Xinanlin 0001 was used as the control group. Each treatment had 3 repeated experiments. In a constant temperature incubator at 28 °C, when the diameter of the pathogenic bacteria in the control group grew to 3 / 4 of the diameter of the petri dish, the diameter and area of the pathogenic fungal colony in the control group were measured using the cross method, and the area of the experimental group was measured using the irregular figure area calculation method. Then, the growth inhibition rate was calculated based on this, and the antibacterial abilities of the single strain and the composite microbial strain were compared. The calculation formula is:

[0028] The results of the antibacterial effect of the composite bacterium Xinanlin 0001 against the pathogenic bacteria of Gastrodia elata are shown in Table 2: The composite bacterium Xinanlin 0001 has a good antibacterial effect in the PDA medium. Among them, the antibacterial effects on Clonostachys rosea , Trichoderma koningii and Nemania diffusa are the best, and the antibacterial rates are 90%, 90.99%, 93.37% and 95.12% respectively; the antibacterial rates of the composite bacterium Xinanlin 0001 against Mucor circinelloides, Geotrichum sp., Bionectria ochroleuca, Trichoderma hamatum, Earliella scabrosa and Fusarium solani are between 80% and 90%, which are 84.31%, 83.24%, 89.33%, 80.22%, 81.38% and 85.43% respectively, and the antibacterial effect is better; the composite bacterium Xinanlin 0001 has a good antibacterial effect on Porogramme epimiltina, Fusarium oxysporum and Xylaria sp. The three pathogenic bacteria all have good antibacterial effects, and the antibacterial rates are between 70% and 80%, which are 72.78%, 79.56% and 79.87% respectively. Compared with the single B. velezensis SWFU000440 and B. amyloliquefaciens SWFU000435Compared with the antibacterial rate, the antibacterial effect of the compound bacteria Xinanlin 0001 is significantly improved. Therefore, the antagonistic bacteria B. velezensis SWFU000440 and B. amyloliquefaciens SWFU000435 The combined compound bacteria Xinanlin 0001 shows an increasing trend in antibacterial rate compared with single strains in terms of antibacterial effect.

[0029] Table 2

[0030] Use a pipette to suck 100 μL of the fermentation broth of compound bacteria Xinanlin 0001 onto a PDA medium (200 g of potato, 10.0 g of glucose, 20 g of agar, 1000 ml of distilled water, natural pH), spread it evenly with a spreader, and inoculate a vigorously growing Gastrodia elata pathogenic bacterium ( Mucor circinelloides, Bionectria ochroleuca, Trichoderma hamatum, Clonostachys rosea, Fusarium solani ) agar disc (d = 5 mm) at the center of the plate, with the mycelium side facing down. Use the medium coated with sterile water as a control. Each treatment has 3 replicates. After culturing in a constant temperature incubator at 28°C for 5 days, observe the mycelial morphology of the pathogenic bacterium.

[0031] The results are as Figure 3 shown. On the PDA plate coated with the fermentation broth of compound bacteria Xinanlin 0001, the mycelial growth of the Gastrodia elata pathogenic bacterium is abnormal, mainly manifested as: the mycelium of the pathogenic bacterium shows swelling, increased mycelial branching, deformity, and apical swelling. In addition, under the action of the fermentation broth of compound bacteria Xinanlin 0001, the number of spores in the pathogenic bacterium is significantly reduced under the microscope, while the mycelia of the pathogenic bacterium in the control group can produce spores normally and form a dense spore cluster. It can be seen that the fermentation broth of compound bacteria has a certain inhibitory effect on the mycelial growth and spore production of the Gastrodia elata pathogenic bacterium. Example 2

[0032] Development of compound microbial inoculants

[0033] 1 Tested inoculants Xinanlin 0001 compound water dispersible granule, Xinanlin 0001 compound bacterial powder, Xinanlin 0001 compound bacterial stock solution (as Figure 4) Microbial inoculant (Brand: Zhongke Chemical; Dosage form: Granular; Effective viable bacteria count ≥ 100 million / g, Effective strain names: Bacillus licheniformis, Paenibacillus mucilaginosus, Organic matter content ≥ 50%), Trichoderma harzianum inoculant (Brand: Muyushi Biology; Dosage form: Wettable powder; Effective viable bacteria count ≥ 5 billion / g), Biocontrol war zone inoculant (Brand: Wehe Biology; Dosage form: Powder; Effective strain names: Beauveria bassiana, Bacillus subtilis; Effective viable bacteria count ≥ 10 billion / g), Bacillus amyloliquefaciens inoculant (Brand: Nongbao Biology; Dosage form: Powder; Effective viable bacteria count ≥ 100 billion / g).

[0034] 1.1 Preparation of Xinanlin 0001 compound bacteria stock solution: B. amyloliquefaciens SWFU000435 Liquid optimized medium: Glucose 20.59 g, Yeast extract powder 5.55 g, Peptone 10.26 g, Potassium chloride 2 g, Distilled water 1000 mL, pH adjusted to 6.0, Culture temperature 31°C, Shaker speed 180 r / min, B. velezensis SWFU000440 Liquid optimized medium: Glucose 11.3 g, Beef extract powder 5.43 g, Peptone 10.28 g, Yeast extract powder 5.87 g, Potassium chloride 5.05 g, Distilled water 1000 mL, pH adjusted to 7.0, Culture temperature 28°C, Shaker speed 180 r / min; B. amyloliquefaciens SWFU000435 and B. velezensis SWFU000440 Inoculate them into the liquid medium for culture respectively, and mix the fermentation broths of the two bacteria in a ratio of 1:1 to form Xinanlin 0001 compound bacteria stock solution, in which the effective viable bacteria count of the fermentation broth of Bacillus amyloliquefaciens SWFU000435 is 6.9 × 10 8 CFU / g; The effective viable bacteria count of the fermentation broth of Bacillus velezensis SWFU000440 is 7.1 × 10 8 CFU / g.

[0035] 1.2 Preparation of Xinanlin 0001 compound bacteria powder Take 5 equal masses of Xinanlin 0001 compound bacteria stock solution and 5 masses of carrier talc powder, fully wet and mix them evenly, then place the mixture in an oven, dry at 50°C, and crush it into powder to obtain Xinanlin 0001 compound bacteria powder.

[0036] 1.3 Preparation of Xinanlin 0001 compound water dispersible granule Calculated by mass percentage, 50% of Xinanlin 0001 composite bacteria stock solution, 5% of wetting agent sodium dodecylbenzene sulfonate, 6% of dispersant sodium hexametaphosphate, 4% of disintegrant calcium chloride, 4% of binder polyethylene glycol, and carrier diatomaceous earth are supplemented to 100%, mixed to form a preparation, and then 18% to 20%, preferably 19% of distilled water is added based on the preparation, mixed evenly and granulated, and the particle size range of 30 to 60 mesh is judged as granulated, and after drying, Xinanlin 0001 composite water-dispersible granules are obtained.

[0037] 2. Targets of microbial application The continuous cropping site of Gastrodia elata in Haiba Village, Qiaoshan Town, Yiliang County, Zhaotong City, Yunnan Province (1650 m above sea level, 27°39'33'' N, 104°16'14'' E).

[0038] 3. Application and treatment of fungicides The experiment mainly used three dosage forms of stock solution, granules and powder to apply Xinanlin 0001 compound microbial agent. The microbial agent was applied to the first and second crops of Gastrodia elata in a cycle of 30 days, for a total of 3 times. The treatments are shown in Table 3. In January 2021, a plot of land with a crop of Gastrodia elata was selected, the land was prepared, and each microbial agent was applied to the fungus pond according to the treatment design, once a month, for a total of 3 times, with 5 replicates for each treatment, and one month apart. The fungus material with honey fungus grown in advance was buried in the soil for growth. In January 2022, the surface soil layer of the fungus pond was removed, and the healthy, disease-free, and uniform size of honey fungus was evenly spread on the fungus material and covered with soil. After Gastrodia elata has grown for two months, 8 different microbial agents were applied once a month for a total of 3 times.

[0039] Table 3

[0040]

[0041] 4. Prevention effectiveness investigation methods In November 2022, when the farmers harvested Gastrodia elata, the incidence of Gastrodia elata diseases was investigated, and the collected Gastrodia elata samples were put into ziplock bags and brought back to the laboratory, and the number of healthy Gastrodia elata, the number of diseased Gastrodia elata, the type of disease, the weight of Gastrodia elata, the size of Gastrodia elata, and the incidence of disease were recorded in each treatment. The classification standards for Gastrodia elata tuber diseases are shown in Table 4.

[0042] Table 4 Classification standards for Gastrodia elata tuber diseases

[0043] 5 Results and Analysis An efficacy test of different microbial agents was carried out in Qiaoshan Town, Yiliang County, Zhaotong City. The results are shown in Table 5. It can be seen that there are significant differences in the incidence rate of Gastrodia elata and the weight of Gastrodia elata after applying different kinds of microbial fertilizers for the first crop and continuous cropping of Gastrodia elata. After applying microbial agents, the incidence rate of continuous cropping Gastrodia elata diseases can be reduced, indicating that applying microbial agents has a good preventive effect on reducing the incidence rate of continuous cropping Gastrodia elata diseases. Among them, after applying the dilution solution of 100-fold Xinanlin 0001 compound water dispersible granule, the incidence rate of the first crop Gastrodia elata decreased by 100%, the preventive effect reached 100%, the weight of Gastrodia elata increased by 5.58% on average, the incidence rate of continuous cropping Gastrodia elata decreased by 73.34%, the preventive effect reached 52.02%, and the weight of Gastrodia elata increased by 69.52% on average; after applying the dilution solution of 300-fold Xinanlin0001 compound water dispersible granule, the incidence rate of the first crop Gastrodia elata decreased by 100%, the preventive effect reached 100%, the weight of Gastrodia elata increased by 9.31% on average, the incidence rate of continuous cropping Gastrodia elata decreased by 100%, the preventive effect reached 100%, and the weight of Gastrodia elata increased by 77.47% on average; after applying the dilution solution of 500-fold Xinanlin 0001 compound water dispersible granule, the incidence rate of the first crop Gastrodia elata decreased by 66.67%, the preventive effect reached 50%, the weight of Gastrodia elata increased by 2.42% on average, the incidence rate of continuous cropping Gastrodia elata decreased by 69.24%, the preventive effect reached 58.45%, and the weight of Gastrodia elata increased by 72.52% on average.

[0044] After applying the dilution solution of 100-fold Xinanlin 0001 compound microbial powder, the incidence rate of the first crop Gastrodia elata decreased by 100%, the preventive effect reached 100%, the weight of Gastrodia elata increased by 1.16% on average, the incidence rate of continuous cropping Gastrodia elata decreased by 73.34%, the preventive effect reached 64%, and the weight of Gastrodia elata increased by 68.15% on average; after applying the dilution solution of 300-fold Xinanlin 0001 compound microbial powder, the incidence rate of the first crop Gastrodia elata decreased by 100%, the preventive effect reached 100%, the weight of Gastrodia elata increased by 3.09% on average, the incidence rate of continuous cropping Gastrodia elata decreased by 86.66%, the preventive effect reached 75.98%, and the weight of Gastrodia elata increased by 69.41% on average; after applying the dilution solution of 500-fold Xinanlin 0001 compound microbial powder, the incidence rate of the first crop Gastrodia elata decreased by 68.89%, the preventive effect reached 53.29%, the weight of Gastrodia elata increased by 3.03% on average, the incidence rate of continuous cropping Gastrodia elata decreased by 69.24%, the preventive effect reached 58.49%, and the weight of Gastrodia elata increased by 66.8% on average.

[0045] After applying the 100-fold dilution of the Xinanlin 0001 complex bacterial liquid, the incidence rate of the first crop of Gastrodia elata decreased by 68.86%, the prevention effect reached 53.29%, the average weight of Gastrodia elata increased by 3.35%, the incidence rate of the replanted Gastrodia elata decreased by 69.24%, the prevention effect reached 44.63%, and the average weight of Gastrodia elata increased by 63.03%; after applying the 300-fold dilution of the Xinanlin 0001 complex bacterial liquid, the incidence rate of the first crop of Gastrodia elata decreased by 66.67%, the prevention effect reached 50%, the average weight of Gastrodia elata increased by 8.16%, the incidence rate of the replanted Gastrodia elata decreased by 71.42%, the prevention effect reached 61.44%, and the average weight of Gastrodia elata increased by 67.87%; after applying the 500-fold dilution of the Xinanlin 0001 complex bacterial liquid, the incidence rate of the first crop of Gastrodia elata decreased by 37.77%, the prevention effect reached 6.65%, the average weight of Gastrodia elata increased by 2.96%, the incidence rate of the replanted Gastrodia elata decreased by 57.16%, the prevention effect reached 35.7%, and the average weight of Gastrodia elata increased by 65.49%.

[0046] After applying the 200-fold dilution of the microbial inoculant, the incidence rate of the first crop of Gastrodia elata decreased by 37.77%, the prevention effect reached 29.97%, the average weight of Gastrodia elata increased by 1.14%, the incidence rate of the replanted Gastrodia elata decreased by 73.34%, the prevention effect reached 52.02%, and the average weight of Gastrodia elata increased by 66.5%; after applying the 300-fold dilution of Trichoderma harzianum, the incidence rate of the first crop of Gastrodia elata decreased by 66.67%, the prevention effect reached 59.59%, the average weight of Gastrodia elata decreased by 2.19%, the incidence rate of the replanted Gastrodia elata decreased by 33.34%, the prevention effect reached 39.99%, and the average weight of Gastrodia elata increased by 39.6%; after applying the 300-fold dilution of the biocontrol war zone, the incidence rate of the first crop of Gastrodia elata decreased by 66.67%, the prevention effect reached 50%, the average weight of Gastrodia elata increased by 0.37%, the incidence rate of the replanted Gastrodia elata decreased by 38.46%, the prevention effect reached 16.91%, and the average weight of Gastrodia elata increased by 48.63%; after applying the 300-fold dilution of Bacillus amyloliquefaciens, the incidence rate of the first crop of Gastrodia elata decreased by 100%, the prevention effect reached 100%, the average weight of Gastrodia elata increased by 4.11%, the incidence rate of the replanted Gastrodia elata decreased by 71.42%, the prevention effect reached 48.56%, and the average weight of Gastrodia elata increased by 65.73%.

[0047] Table 5 Control effects of compound water-dispersible granules on Gastrodia elata diseases

[0048] The results showed that: Applying different microbial inoculants had a good inhibitory effect on the diseases of the first-crop and continuous-cropping Gastrodia elata, among which the control effect on continuous-cropping Gastrodia elata was significantly higher than that on the first-crop Gastrodia elata, and the weight of continuous-cropping Gastrodia elata increased significantly. In the continuous-cropping treatment, after applying the diluted solution of 500-fold Xinanlin 0001 compound bacterial liquid, 300-fold Trichoderma harzianum diluted solution and 300-fold Trichoderma harzianum diluted solution, the incidence rate of Gastrodia elata was above 20%, and the rest were below 20%. In the first-crop treatment, the prevention effects of applying the diluted solution of 500-fold compound bacterial liquid and 200-fold microbial inoculant were below 50%, and the rest were above 50%. Among them, the prevention effects of applying the diluted solutions of 100-fold and 300-fold Xinanlin 0001 compound water dispersible granule, 100-fold and 300-fold Xinanlin 0001 compound bacterial powder, and 300-fold Bacillus amyloliquefaciens reached 100%. In the continuous-cropping treatment, the prevention effects of applying the diluted solutions of 100-fold and 500-fold Xinanlin 0001 compound bacterial liquid, 300-fold Trichoderma harzianum diluted solution, 300-fold biocontrol zone diluted solution and 300-fold Bacillus amyloliquefaciens were below 50%, and the rest were above 50%. Among them, the control effect of applying 300-fold Xinanlin 0001 compound water dispersible granule diluted solution was 100%, indicating that the microbial strains in different types of bio-fertilizers were different, and their resistance abilities to Gastrodia elata plants were different after application. After applying the diluted solutions of Xinanlin 0001 compound water dispersible granule, Xinanlin 0001 compound bacterial powder and Xinanlin 0001 compound bacterial liquid, the incidence rate of the first-crop Gastrodia elata showed an increasing trend with the increase of the dilution multiple of the inoculant diluted solution, and the incidence rate of continuous-cropping Gastrodia elata showed a trend of first decreasing and then increasing. The prevention effect and the weight of Gastrodia elata showed a trend of first increasing and then decreasing with the increase of the dilution multiple of the inoculant diluted solution in the first-crop and continuous-cropping treatments. Generally speaking, the control effects of the 7 microbial inoculants from good to bad were: Xinanlin 0001 compound water dispersible granule diluted solution > Xinanlin 0001 compound bacterial powder diluted solution > Xinanlin 0001 compound bacterial liquid diluted solution > Bacillus amyloliquefaciens diluted solution > microbial inoculant diluted solution > biocontrol zone diluted solution > Trichoderma harzianum diluted solution. When applying 300-fold Xinanlin 0001 compound water dispersible granule diluted solution, the incidence rates of the first-crop and continuous-cropping Gastrodia elata were the lowest, and the prevention effect and the weight of Gastrodia elata were the highest. Therefore, when the most suitable dilution multiple of Xinanlin 0001 compound water dispersible granule was 300, the control effect was the best and the yield of Gastrodia elata was the highest. It can be seen from this that applying microbial inoculants not only had a good control effect on reducing the incidence rate of continuous-cropping Gastrodia elata diseases, but also significantly increased the yield of Gastrodia elata, thus indirectly alleviating the problem of continuous cropping obstacle of Gastrodia elata.

[0049] The present invention is based on various Bacillus strains isolated from the rhizosphere soil of Gastrodia elata, and two microbial strains with significant antagonistic activity against the main pathogens of Gastrodia elata are screened out, which are Bacillus amyloliquefaciens B. amyloliquefaciens SWFU000435 and Bacillus velezensis B. velezensis SWFU000440 。The present invention screens the carrier, wetting agent, dispersant, disintegrant and binder of the compound bacteria Xinanlin 0001 water dispersible granule and their optimal usage amounts, and screens the concentrations of the wetting agent and dispersant by using single factor test and orthogonal test. The results show that: B. amyloliquefaciens SWFU000435 and B. velezensis SWFU000440 The final formula of the Xinanlin 0001 compound bacteria water dispersible granule is: 10% - 80% of the compound bacteria technical material, 5% of the wetting agent sodium dodecyl benzene sulfonate, 6% of the dispersant sodium hexametaphosphate, 4% of the disintegrant calcium chloride, 4% of the binder polyethylene glycol, and the carrier diatomaceous earth is supplemented to 100%, and the water addition amount is controlled between 18% and 20%. Under laboratory conditions, the inhibitory activities of these two Bacillus strains against the pathogens are both above 50%. In the field experiment, the biocontrol effect of the 300-fold dilution of the Xinanlin 0001 compound water dispersible granule is the most ideal. B. amyloliquefaciens SWFU000435 and B. velezensis SWFU000440 The test results of the present invention show that the application of microbial inoculants can not only promote the reconstruction of the soil microbial community structure, reduce the incidence of Gastrodia elata diseases, but also effectively increase the yield of Gastrodia elata and alleviate the economic losses caused by continuous cropping obstacles. Compared with the single Bacillus amyloliquefaciens inoculant, the application of

[0050] and B. amyloliquefaciens SWFU000435 and B. velezensis SWFU000440 The 300-fold dilution of the Xinanlin 0001 compound bacteria water dispersible granule has a significant preventive effect, the incidence of Gastrodia elata diseases is reduced, and the yield of Gastrodia elata increases significantly, indicating that the two Bacillus strains in the Xinanlin 0001 compound bacteria water dispersible granule can colonize well in the soil and have a good control effect on Gastrodia elata diseases. Applying the compound water dispersible granule in the Gastrodia elata field can play a good role in preventing and controlling the occurrence of Gastrodia elata diseases. On the one hand, this research lays a foundation for the development of microbial inoculants for Gastrodia elata diseases, and on the other hand, it has made a breakthrough achievement in the aspect of biological control to alleviate the continuous cropping obstacles of Gastrodia elata.

Claims

1. A composite microbial agent for Gastrodia elata, characterized in that: The bacterial agent is a microbial fermentation liquid, and the microorganisms are Bacillus amyloliquefaciens SWFU000435 and Bacillus velez SWFU000440; The Bacillus amyloliquefaciens SWFU000435 is classified and named as Bacillus amyloliquefaciens Bacillus amyloliquefaciens , Deposit Number: CGMCC NO.33089; The Velez Bacillus SWFU000440 is classified as Velez Bacillus Bacillus velezensis , Deposit Number: CGMCC NO.33088; The fermentation liquid of Bacillus amyloliquefaciens SWFU000435 and the fermentation liquid of Bacillus velez SWFU000440 were mixed in a volume ratio of 1:1 to prepare a composite microbial agent for Gastrodia elata. The effective viable count of the fermentation broth of Bacillus amyloliquefaciens SWFU000435 is ≥ 6.9 × 10 8 / g; effective viable count of Bacillus Velezii SWFU000440 fermentation broth ≥7.1 × 10 8 Pieces / g.

2. The composite microbial agent according to claim 1, characterized in that: The fermentation medium of the Bacillus amyloliquefaciens SWFU000435 is: 20.59 g / L glucose, 5.55 g / L yeast extract powder, 10.26 g / L peptone and 2 g / L potassium chloride; the culture temperature is 31° C., the initial pH of the culture medium is 6.0, and the shaking speed is 180 r / min; The fermentation medium of the Velez Bacillus SWFU000440 is 11.3 g / L of glucose, 5.43 g / L of beef extract powder, 10.28 g / L of peptone, 5.87 g / L of yeast extract powder and 5.05 g / L of potassium chloride; the culture temperature is 28°C, the initial pH of the culture medium is 7.0, and the shaking speed is 180 r / min.

3. A composite microbial fertilizer for Gastrodia elata, comprising microorganisms, carriers or adjuvants, wherein the adjuvants include one or more of a wetting agent, a dispersant, a disintegrating agent, a binder, a synergist or a protective agent, characterized in that: The microorganism is the composite microbial agent according to claim 1.

4. The composite microbial fertilizer according to claim 3, characterized in that: The carrier includes talc, kaolin, bentonite, diatomaceous earth or light calcium carbonate; and / or The wetting agent includes sodium dodecylbenzene sulfonate (SDBS), sodium dodecyl sulfate (SDS), polyvinyl alcohol (PVA) or sodium carboxymethyl cellulose (CMC-Na); and / or The dispersant includes sodium hydroxymethylcellulose, sodium hexametaphosphate, polyvinyl pyrrolidone or sodium polyphosphate, and / or The disintegrant comprises sodium chloride, calcium chloride, ammonium sulfate or sodium alginate; and / or The binder includes adding dextrin, polyethylene glycol (PEG) or soluble starch; and / or The synergist includes mineral-derived potassium humate, chitosan or amino acid; and / or The protective agent includes ascorbic acid VC, humic acid or cyclodextrin.

5. The composite microbial fertilizer according to claim 4, characterized in that: The dosage form of the bacterial fertilizer includes liquid, powder, granule, wettable powder, suspension or water dispersible granule.

6. The composite microbial fertilizer according to claim 5, characterized in that: The bacterial fertilizer is a powder, which is obtained by fully mixing equal masses of composite microbial agents and carriers, drying the mixture, and crushing it.

7. The composite microbial fertilizer according to claim 5, characterized in that: The bacterial fertilizer is a water-dispersible granule, which is composed of 10%-80% of a composite microbial agent, 5% of a wetting agent, 6% of a dispersant, 4% of a disintegrant, and 4% of a binder, and the carrier is supplemented to 100%, mixed to form a preparation, and then 18% to 20% of distilled water is added to the preparation, mixed evenly and granulated, and dried to obtain water-dispersible granules.

8. The composite microbial fertilizer according to claim 7, characterized in that: The water dispersible granules are composed of 10%-80% of a composite microbial agent, 5% of a wetting agent sodium dodecylbenzene sulfonate, 6% of a dispersant sodium hexametaphosphate, 4% of a disintegrant calcium chloride, 4% of a binder polyethylene glycol, and a carrier diatomaceous earth to make up to 100%, which are mixed to form a preparation, and then 18%-20% of distilled water is added to the preparation as a basis, mixed evenly, granulated, and dried to obtain water dispersible granules.

9. Use of the microbial agent according to claim 1 or the composite microbial fertilizer according to any one of claims 3 to 8 in biological control of Gastrodia elata diseases or continuous cropping disorders, or in the preparation of a control agent for Gastrodia elata diseases or continuous cropping disorders.

10. A method for preventing and controlling the continuous cropping of Gastrodia elata, characterized in that: Apply the microbial agent described in claim 1 or the composite microbial fertilizer described in any one of claims 3-8 to land with continuous cropping of Gastrodia elata.

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