An antagonistic complex Bacillus spore bacterium agent for Gastrodia elata continuous cropping obstacle and its application

Abstract

The present invention belongs to the field of microbial technology, and specifically relates to an antagonistic composite bacillus agent for Gastrodia elata continuous cropping obstacle and its application. Specifically, the agent provided by the present invention 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 , deposit number: CGMCC NO.33089; the Bacillus velezensis SWFU000440, classified and named as Bacillus velezensis Bacillus velezensis , deposit number: CGMCC NO.33088. Applying the microbial agent of the present invention can not only 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.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to an antagonistic complex Bacillus spore bacterium 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 the most famous, with extremely high economic value and medicinal and edible value. With the continuous expansion of the cultivation area of Gastrodia elata, the problem of continuous cropping obstacle has gradually emerged. Continuous cropping of 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 Gastrodia elata diseases as the starting point to alleviate and prevent Gastrodia elata continuous cropping obstacle is a key problem that urgently needs to be solved in the Gastrodia elata industrial chain. In recent years, biological control technology has shown significant 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 bacterium 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 20% reduction in yield every year. The most important problem in Gastrodia elata planting 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 continuous cropping has become increasingly common, and the diseases of Gastrodia elata have become increasingly 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 pathogenic bacteria species causing diseases of Gastrodia elata, find out the widely distributed pathogenic bacteria, and screen antagonistic bacteria from the isolated pathogenic 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 complex Bacillus spore bacterium 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:

[0005] On the one hand, the present invention provides a special composite microbial inoculant for Gastrodia elata. The inoculant is a microbial fermentation broth, and the microorganisms are Bacillus amyloliquefaciens SWFU000435 and Bacillus velezensis SWFU000440;

[0006] The Bacillus amyloliquefaciens SWFU000435 is classified and named as Bacillus amyloliquefaciens Bacillus amyloliquefaciens , and the preservation number is: CGMCC NO.33089;

[0007] The Bacillus velezensis SWFU000440 is classified and named as Bacillus velezensis Bacillus velezensis , and the preservation number is: CGMCC NO.33088;

[0008] The special composite 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.

[0009] The effective viable count of the fermentation broth of Bacillus amyloliquefaciens SWFU000435 is ≥6.9 × 10 8 CFU / g; the effective viable count of the fermentation broth of Bacillus velezensis SWFU000440 is ≥7.1 × 10 8 CFU / g.

[0010] Furthermore, the fermentation broth medium of Bacillus amyloliquefaciens SWFU000435 is: 20.59 g / L of glucose, 5.55 g / L of yeast extract powder, 10.26 g / L of peptone, and 2 g / L of potassium chloride; the culture temperature is 31°C, the initial pH of the medium is 6.0, and the shaker speed is 180 r / min;

[0011] The fermentation broth medium of Bacillus velezensis 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 medium is 7.0, and the shaker speed is 180 r / min.

[0012] On the other hand, the present invention provides a special composite microbial fertilizer for Gastrodia elata, which includes microorganisms, carriers or additives. It is characterized in that the microorganisms are the composite microbial inoculant.

[0013] Furthermore, for the composite microbial fertilizer, the additives include one or more of wetting agents, dispersants, disintegrants, binders, synergists or protectants.

[0014] Furthermore, for the compound microbial fertilizer, the carrier includes talcum 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.

[0015] Furthermore, for the compound microbial fertilizer, the dosage form of the fertilizer includes liquid, powder, granule, wettable powder, suspending agent or water dispersible granule.

[0016] 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.

[0017] Furthermore, for the compound microbial fertilizer, when the fertilizer is in water dispersible granule form, the water dispersible granule, by mass percentage, is 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%. The mixture is formulated into a preparation, and then based on the preparation, 18% - 20% of distilled water is added, mixed evenly and granulated, and then dried to obtain the water dispersible granule.

[0018] Furthermore, for the compound microbial fertilizer, when the fertilizer is in water dispersible granule form, the water dispersible granule, by mass percentage, is 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%. The mixture is formulated into a preparation, and then based on the preparation, 18% - 20% of distilled water is added, mixed evenly and granulated, and then dried to obtain the water dispersible granule.

[0019] Furthermore, the present invention provides the application of the inoculant, or any of the compound microbial fertilizers, in the 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.

[0020] Furthermore, the present invention provides a method for preventing and treating continuous cropping obstacles of Gastrodia elata, which comprises applying the inoculant, or any of the compound microbial fertilizers, to the land with continuous cropping obstacles of Gastrodia elata.

[0021] The technical effects achieved by the present invention are as follows:

[0022] 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 complex 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 complex bacteria water dispersible granule can be well colonized in the soil, and have a good control effect on Gastrodia elata diseases. The application of the complex water dispersible granule in the Gastrodia elata planting obstacle area can play a very 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 obstacle of Gastrodia elata. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Partial symptom diagram of Gastrodia elata tuber in field diseases;

[0024] Figure 2 Determination result diagram of the affinity of the complex strain (a: B. amyloliquefaciens SWFU000435,b: B. velezensis SWFU000440 );

[0025] Figure 3 Effect diagram of the complex bacteria fermentation broth on the growth of Gastrodia elata pathogenic bacteria hyphae;

[0026] Figure 4 Product diagrams of different dosage forms of Xinanlin 0001 complex bacteria fertilizer. DETAILED DESCRIPTION OF THE INVENTION

[0027] 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, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained by purchase. The present invention will be further described in detail below with reference to the examples.

[0028] The Bacillus amyloliquefaciens SWFU000435 provided in the present invention is classified and named as Bacillus amyloliquefaciens Bacillus amyloliquefaciens (abbreviated as B. amyloliquefaciens) , Preservation number: CGMCC NO.33089, Preservation institution: 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 tested by the preservation center on December 16, 2024, and the result was that it was viable.

[0029] Bacillus velezensis SWFU000440, taxonomically named Bacillus velezensis Bacillus velezensis (Abbreviated as B. velezensis ) , Preservation number: CGMCC NO.33088, Preservation institution: 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 tested by the preservation center on December 16, 2024, and the result was that it was viable.

[0030] Example 1

[0031] Bacillus amyloliquefaciens ( B. amyloliquefaciens ) SWFU000435 and Bacillus velezensis ( B. velezensis ) SWFU000440's antibacterial effect on Gastrodia elata pathogenic bacteria

[0032] 1. Isolation, purification, identification and verification of Gastrodia elata disease pathogens

[0033] Collect disease samples ( Figure 1 ), 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 onto healthy Gastrodia elata for testing. After analysis and screening, 13 pathogenic bacteria all caused varying degrees of rot in Gastrodia elata, which were respectively 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 , Xylariasp began to develop symptoms 3 days after inoculation. E. scabrosa , P. epimiltina , F. oxysporum、T. koningii , N. diffusa , F. solani , T. atroviride The disease started to occur 5 days after inoculation. The lesion site of Gastrodia elata was re-isolated and purified after inoculation, and compared with the inoculation site. Through molecular biological identification, it was determined that the 13 isolated strains were all pathogenic bacteria of Gastrodia elata.

[0034] The present invention uses high-throughput sequencing technology to analyze the changes in the fungal community structure of the rhizosphere soil of Gastrodia elata with different continuous cropping numbers. The results show that the relative abundance of Mucor in the soil gradually decreases with the increase in the number of planting crops, which reveals that the soil itself contains pathogenic bacteria that cause Gastrodia elata to rot, and Gastrodia elata planting is more conducive to the pathogenic bacteria. Mucor circinelloides The growth and reproduction of Gastrodia elata led to an increase in the incidence of the pathogen, which in turn caused the problem of continuous cropping of Gastrodia elata. With the increase in the number of continuous cropping, the relative abundance of Ascomycota and Mucorales increased significantly, among which the relative abundance of Agaricomycetes and Sordariomycetes, Polyporales and Hypocreales increased with the increase in the number of continuous cropping; the macro-micro-molecular corresponding fitting results revealed that the trend of the change in the fungal community structure and the group of the rhizosphere soil fungi of Gastrodia elata with different continuous cropping numbers based on high-throughput sequencing technology were consistent with the pathogenic group of the pathogenic test disease; the continuous cropping of Gastrodia elata led 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, resulting in a decrease in the yield and quality of Gastrodia elata, which directly led to the formation of continuous cropping of Gastrodia elata.

[0035] 2. Screening of antagonistic bacteria

[0036] The plate confrontation method was used to initially screen strains with antagonistic effects on the pathogens of soil-borne diseases of Gastrodia elata, and the strains with an inhibition rate greater than 50% were rescreened; after multiple screenings, fungi with antagonistic activity against the pathogens of Gastrodia elata were obtained. The rescreening results are shown in Table 1 B. velezensis SWFU000440 The inhibition rates of 13 pathogenic bacteria were all above 50%. B. amyloliquefaciens SWFU000435 right Trichoderma. atroviride The antibacterial rate was below 50%, and the others were all above 50%; B. subtilis(Bacillus subtilis) right Fusarium. oxysporum and Trichoderma. atroviride The inhibition rate is below 50%.

[0037] Table 1 Antibacterial rate of antagonistic bacteria rescreening

[0038]

[0039] 3. Optimization of the culture fermentation conditions of the antagonistic bacteria

[0040] Two strains of bacteria with stable antagonistic effects screened by the present invention are B. velezensis SWFU000440 and B. amyloliquefaciens SWFU000435 , and through single-factor experiments, it is determined that B. velezensis SWFU000440 the optimal culture medium is the 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 culture medium is the 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 initial pH of the optimal culture medium is 7.0, and the optimal shaker speed is 180 r / min; after culturing for 18 h, the average value of the OD 600 of the fermentation broth measured three times is 0.711 ± 0.015.

[0041] B. amyloliquefaciens SWFU000435 the optimal fermentation medium of B. B. 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. Under the condition of controlling a single fermentation culture condition; B. B. amyloliquefaciens SWFU000435 the optimal culture temperature is 31 °C, the initial pH of the optimal culture medium is 6.0, and the optimal shaker speed is 180 r / min. After culturing for 18 h, the average value of the OD 600 value of the fermentation broth measured three times is 0.699 ± 0.018.

[0042] 4. Determination of the affinity between composite strains

[0043] The antagonistic bacteria are streaked crosswise in pairs on the 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 is an inhibition zone between the strains. If there is an inhibition zone, it indicates that the two strains are incompatible and there is antagonism. If there is no inhibition zone, it indicates that the two strains are compatible and can be co-cultured. The determination results of the affinity between the composite strains show that: B. velezensis SWFU000440 and B. B. amyloliquefaciens SWFU000435 the two strains are streaked and cultured in pairs on the surface of the NA medium, and the results are as Figure 2 shown B.B. velezensis SWFU000440 and B. amyloliquefaciens SWFU000435 Both of the two antagonistic bacteria can grow normally on the culture medium, and no inhibition zone is produced between the two strains, indicating that B. velezensis SWFU000440 and B. B. amyloliquefaciens SWFU000435 there is an affinity between the two strains and no mutual antagonism, meeting the prerequisite for developing a compound microbial agent.

[0044] 5. Determination of the antibacterial effect of the compound strain in the petri dish

[0045] 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 compound bacterium, numbered Xinanlin 0001. The antibacterial effect of the fermentation broth of the compound 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, pathogenic fungal cakes of Gastrodia elata (diameter d = 5 mm) were respectively inoculated with the mycelium side facing down. Centered on the pathogenic fungus, circular filter papers (diameter d = 5 mm) soaked with the compound bacterium Xinanlin 0001 were respectively inoculated at the three vertex positions in an equilateral "△" shape. The petri dish with a circular filter paper without inoculating the compound bacterium Xinanlin 0001 was used as the control group. Each treatment had 3 replicate 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 by the cross method, and the area of the experimental group was measured by the irregular figure area calculation method. Then, the growth inhibition rate was calculated accordingly, and the antibacterial abilities of the single strain and the compound microbial strain were compared. The calculation formula is as follows:

[0046]

[0047] The antibacterial effect of the compound bacterium Xinanlin 0001 against the pathogenic bacteria of Gastrodia elata is shown in Table 2. The compound 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 compound bacterium Xinanlin 0001 has an antibacterial effect on Mucor circinelloides, Geotrichum sp., Bionectria ochroleuca, Trichoderma hamatum, Earliella scabrosa and Fusarium solaniThe bacteriostatic rates were between 80% and 90%, being 84.31%, 83.24%, 89.33%, 80.22%, 81.38% and 85.43% respectively, showing good bacteriostatic effects; The compound bacterium Xinanlin 0001 had good bacteriostatic effects on Porogramme epimiltina, Fusarium oxysporum and Xylaria sp., three kinds of pathogenic bacteria. The bacteriostatic rates were between 70% and 80%, being 72.78%, 79.56% and 79.87% respectively. Compared with the single B. velezensis SWFU000440 and B. amyloliquefaciens SWFU000435 bacteriostatic rates, the bacteriostatic effects of the compound bacterium Xinanlin 0001 were all significantly improved. Therefore, the bacteriostatic effect of the compound bacterium Xinanlin 0001 formed by the combination of the antagonistic bacteria B. velezensis SWFU000440 and B. amyloliquefaciens SWFU000435 showed an increasing trend compared with that of the single strain.

[0048] Table 2

[0049]

[0050] Use a pipette to suck 100 μL of the fermentation broth of the compound bacterium 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 ) bacterial cake (d = 5 mm) at the center position of the plate, with the hyphal surface facing down. Use the medium coated with sterile water as a control, with 3 replicates for each treatment. After culturing in a constant temperature incubator at 28°C for 5 days, observe the hyphal morphology of the pathogenic bacteria.

[0051] The results were as Figure 3 , on the PDA plate coated with the fermentation broth of the compound bacterium Xinanlin 0001, the hyphal growth of the Gastrodia elata pathogenic bacterium was abnormal, mainly manifested as: the hyphae of the pathogenic bacterium showed swelling, increased hyphal branching, deformity, and apical swelling. In addition, under the action of the fermentation broth of the compound bacterium Xinanlin 0001, it was observed under the microscope that the number of spores in the pathogenic bacteria decreased significantly, while the hyphae of the pathogenic bacteria in the control group could produce spores normally and form a dense spore group. It can be seen that the fermentation broth of the compound bacterium has a certain inhibitory effect on the hyphal growth and spore production of the Gastrodia elata pathogenic bacterium. Example 2

[0052] Development of a compound microbial inoculant

[0053] 1 Test inoculants

[0054] Xinanlin 0001 Compound Water Dispersible Granules, Xinanlin 0001 Compound Bacterial Powder, Xinanlin 0001 Compound Bacterial Stock Solution (such as Figure 4 ), microbial inoculant (brand: Zhongke Chemical Industry; dosage form: granular; effective viable bacteria count ≥ 100 million / g, effective bacterial species names: Bacillus licheniformis, Paenibacillus mucilaginosus, organic matter content ≥ 50%), Trichoderma harzianum inoculant (brand: Muyu Stone Biology; dosage form: wettable powder; effective viable bacteria count ≥ 5 billion / g), biological control war zone inoculant (brand: Microcore Biology; dosage form: powder; effective bacterial species names: Beauveria bassiana, Bacillus subtilis; effective viable bacteria count ≥ 10 billion / g), Bacillus amyloliquefaciens inoculant (brand: Nongbao Biology; dosage form: powder; effective viable bacteria quantity ≥ 100 billion / g).

[0055] 1.1 Preparation of Xinanlin 0001 Compound Bacterial Stock Solution:

[0056] B. amyloliquefaciens SWFU000435 Liquid optimized medium: 20.59 g of glucose, 5.55 g of yeast extract powder, 10.26 g of peptone, 2 g of potassium chloride, 1000 mL of distilled water, pH adjusted to 6.0, culture temperature at 31°C, shaker speed at 180 r / min,

[0057] B. velezensis SWFU000440 Liquid optimized medium: 11.3 g of glucose, 5.43 g of beef extract powder, 10.28 g of peptone, 5.87 g of yeast extract powder, 5.05 g of potassium chloride, 1000 mL of distilled water, pH adjusted to 7.0, culture temperature at 28°C, shaker speed at 180 r / min;

[0058] B. amyloliquefaciens SWFU000435 and B. velezensis SWFU000440 were respectively inoculated in the liquid medium for culture, and the fermentation broths of the two bacteria were mixed in a ratio of 1:1 to form Xinanlin 0001 Compound Bacterial Stock Solution. The effective viable bacteria count of the fermentation broth of Bacillus amyloliquefaciens SWFU000435 was 6.9 × 10 8 CFU / g; the effective viable bacteria count of the fermentation broth of Bacillus velezensis SWFU000440 was 7.1 × 10 8 CFU / g.

[0059] 1.2 Preparation of Xinanlin 0001 Compound Bacterial Powder

[0060] Take 5 equal masses of Xinanlin 0001 Compound Bacterial Stock Solution and 5 parts of carrier talc powder. After fully wetting and mixing, place the mixture in an oven and dry it at 50°C, then crush it into powder form to obtain Xinanlin 0001 Compound Bacterial Powder.

[0061] 1.3 Preparation of Xinanlin 0001 composite water dispersible granules

[0062] 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.

[0063] 2. Targets of microbial application

[0064] 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).

[0065] 3. Application and treatment of fungicides

[0066] 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.

[0067] Table 3

[0068]

[0069] 4. Prevention effectiveness investigation methods

[0070] 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.

[0071] Table 4 Classification standards for Gastrodia elata tuber diseases

[0072]

[0073] 5 Results and Analysis

[0074] An efficacy test of different bacterial 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 types of bacterial fertilizers for first-crop and continuous-crop Gastrodia elata. After applying microbial bacterial agents, the incidence rate of continuous-crop Gastrodia elata diseases can be reduced, indicating that applying microbial bacterial agents has a good preventive effect on reducing the incidence rate of continuous-crop Gastrodia elata diseases. Among them, after applying the dilution solution of 100-fold Xinanlin 0001 compound water dispersible granule, the incidence rate of first-crop Gastrodia elata decreased by 100%, the preventive effect reached 100%, the average weight of Gastrodia elata increased by 5.58%, the incidence rate of continuous-crop Gastrodia elata decreased by 73.34%, the preventive effect reached 52.02%, and the average weight of Gastrodia elata increased by 69.52%; after applying the dilution solution of 300-fold Xinanlin0001 compound water dispersible granule, the incidence rate of first-crop Gastrodia elata decreased by 100%, the preventive effect reached 100%, the average weight of Gastrodia elata increased by 9.31%, the incidence rate of continuous-crop Gastrodia elata decreased by 100%, the preventive effect reached 100%, and the average weight of Gastrodia elata increased by 77.47%; after applying the dilution solution of 500-fold Xinanlin 0001 compound water dispersible granule, the incidence rate of first-crop Gastrodia elata decreased by 66.67%, the preventive effect reached 50%, the average weight of Gastrodia elata increased by 2.42%, the incidence rate of continuous-crop Gastrodia elata decreased by 69.24%, the preventive effect reached 58.45%, and the average weight of Gastrodia elata increased by 72.52%.

[0075] After applying the dilution solution of 100-fold Xinanlin 0001 compound bacterial powder, the incidence rate of first-crop Gastrodia elata decreased by 100%, the preventive effect reached 100%, the average weight of Gastrodia elata increased by 1.16%, the incidence rate of continuous-crop Gastrodia elata decreased by 73.34%, the preventive effect reached 64%, and the average weight of Gastrodia elata increased by 68.15%; after applying the dilution solution of 300-fold Xinanlin 0001 compound bacterial powder, the incidence rate of first-crop Gastrodia elata decreased by 100%, the preventive effect reached 100%, the average weight of Gastrodia elata increased by 3.09%, the incidence rate of continuous-crop Gastrodia elata decreased by 86.66%, the preventive effect reached 75.98%, and the average weight of Gastrodia elata increased by 69.41%; after applying the dilution solution of 500-fold Xinanlin 0001 compound bacterial powder, the incidence rate of first-crop Gastrodia elata decreased by 68.89%, the preventive effect reached 53.29%, the average weight of Gastrodia elata increased by 3.03%, the incidence rate of continuous-crop Gastrodia elata decreased by 69.24%, the preventive effect reached 58.49%, and the average weight of Gastrodia elata increased by 66.8%.

[0076] After applying the 100-fold dilution of Xinanlin 0001 complex bacterial solution, 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 second crop of 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 Xinanlin 0001 complex bacterial solution, 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 second crop of 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 Xinanlin 0001 complex bacterial solution, 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 second crop of Gastrodia elata decreased by 57.16%, the prevention effect reached 35.7%, and the average weight of Gastrodia elata increased by 65.49%.

[0077] After applying the 200-fold dilution of microbial inoculum, 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 second crop of 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 second crop of 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 biocontrol war zone solution, 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 second crop of 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 second crop of Gastrodia elata decreased by 71.42%, the prevention effect reached 48.56%, and the average weight of Gastrodia elata increased by 65.73%.

[0078] Table 5 Control effect of compound water dispersible granule on Gastrodia elata diseases

[0079] The results showed that: Applying different kinds of microbial inoculants had good inhibitory effects 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 bacteria stock solution, 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 preventive effects of applying 500-fold compound bacteria stock solution diluted solution and 200-fold microbial inoculant diluted solution were below 50%, and the rest were above 50%. Among them, the preventive effects of applying 100-fold and 300-fold Xinanlin 0001 compound water dispersible granule diluted solution, 100-fold and 300-fold Xinanlin 0001 compound bacteria powder diluted solution, and 300-fold Bacillus amyloliquefaciens diluted solution reached 100%. In the continuous-cropping treatment, the preventive effects of applying 100-fold and 500-fold Xinanlin 0001 compound bacteria stock solution diluted solution, 300-fold Trichoderma harzianum diluted solution, 300-fold biocontrol zone diluted solution and 300-fold Bacillus amyloliquefaciens diluted solution 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 the stress resistance ability of Gastrodia elata plants was different after use. After applying Xinanlin 0001 compound water dispersible granule diluted solution, Xinanlin 0001 compound bacteria powder diluted solution and Xinanlin 0001 compound bacteria stock solution diluted solution, 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 preventive 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 kinds of microbial inoculants from good to bad were: Xinanlin 0001 compound water dispersible granule diluted solution > Xinanlin 0001 compound bacteria powder diluted solution > Xinanlin 0001 compound bacteria stock solution 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 preventive 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 good control effects 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.

[0080] The present invention is based on a variety of 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 . Through screening the carrier, wetting agent, dispersant, disintegrant and binder of the compound bacteria Xinanlin 0001 water dispersible granule and their optimal usage amounts, and screening the concentrations of the wetting agent and dispersant by using single factor test and orthogonal test, the results show that: B. 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 dodecylbenzenesulfonate, 6% of the dispersant sodium hexametaphosphate, 4% of the disintegrant calcium chloride, 4% of the binder polyethylene glycol, and the carrier diatomite 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 field trials, the biocontrol effect of the 300-fold dilution of the Xinanlin 0001 compound bacteria water dispersible granule is the most ideal. B. amyloliquefaciens SWFU000435 and B. velezensis SWFU000440 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 preventive effects of applying

[0081] and B. amyloliquefaciens SWFU000435 and B. velezensis SWFU000440 the 300-fold dilution of the Xinanlin 0001 compound bacteria water dispersible granule are significant. The incidence of Gastrodia elata diseases is reduced, and the yield of Gastrodia elata is significantly increased, indicating that the two Bacillus strains in the Xinanlin 0001 compound bacteria water dispersible granule can colonize well in the soil and have good control effects on Gastrodia elata diseases. Applying the compound water dispersible granule in the Gastrodia elata field can play a very 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 achieves a breakthrough in the aspect of biological control to alleviate the continuous cropping obstacles of Gastrodia elata.

Claims

1. Use of a special compound microbial inoculant for Gastrodia elata in the preparation of a control agent for continuous cropping obstacle of Gastrodia elata, characterized in that, The microbial 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 , preservation number: CGMCC NO.33089; The Bacillus velezensis SWFU000440 is classified and named as Bacillus velezensis Bacillus velezensis , with the preservation number: CGMCC NO.33088; The special compound microbial agent 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.

2. The application according to claim 1, wherein 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.

3. A special compound microbial fertilizer for Gastrodia elata continuous cropping obstacle, comprising microorganisms, carriers or auxiliaries, wherein the auxiliaries include one or more of wetting agents, dispersants, disintegrants, binders, synergists or protectants, and is characterized in that, The microorganism is the compound microbial agent described in claim 1.

4. The compound microbial fertilizer according to claim 3, wherein 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 mineral source humate, chitosan or amino acids; and / or The protective agent includes ascorbic acid VC, humic acid or cyclodextrin.

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

6. The compound microbial fertilizer according to claim 5, wherein The microbial fertilizer is in powder form, which is obtained by fully mixing the compound microbial agent and the carrier in equal mass, drying the mixture, and then crushing it.

7. The compound microbial fertilizer according to claim 5, characterized in that The microbial fertilizer is in water dispersible granule form. The water dispersible granule, by mass percentage, is 10%-80% of the compound microbial agent, 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%. The preparation is mixed and formulated, and then 18%-20% distilled water is added based on the preparation, mixed evenly and granulated, and dried to obtain the water dispersible granule.

8. The compound microbial fertilizer according to claim 7, characterized in that, The water dispersible granule, by mass percentage, comprises 10%-80% of compound microbial inoculum, 5% of wetting agent sodium dodecyl benzene sulfonate, 6% of dispersant sodium hexametaphosphate, 4% of disintegrant calcium chloride, 4% of binder polyethylene glycol, and the carrier diatomaceous earth is added up to 100%. They are mixed to prepare the preparation, and then 18% - 20% of distilled water is added based on the preparation, mixed evenly and granulated, and the water dispersible granule is obtained after drying.

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

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