Bacillus lugosii and application thereof

Abstract

The invention relates to the technical field of agricultural microorganisms, in particular to bacillus rugosii and application thereof. The bacillus rugosus disclosed by the invention is preserved in the China Center for Type Culture Collection on October 25, 2023, and the preservation number of the bacillus rugosus is CCTCC (China Center for Type Culture Collection) NO: M20232022. The bacillus rugosii and the microbial agent provided by the invention have a very strong antagonistic effect on phytophthora capsici, and have a very good prevention and treatment effect on phytophthora blight of capsici; the ecological adaptability is high; the organic phosphorus decomposing effect is good, and the amylase decomposing activity and the cellulase decomposing activity are remarkable; the fertilizer has strong drought-resistant and saline-alkali-resistant capabilities, and can promote the growth of peppers under saline-alkali conditions, improve the agronomic traits of the peppers and promote the yield increase of the peppers.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural microorganisms, and particularly relates to a Bacillus rugosus and its application. Background Art

[0002] In China, saline-alkali lands are large in area, diverse in type, and widespread in distribution. Unreasonable chemical fertilizer application and irrigation methods have led to soil compaction and reduced fertility in some cultivated lands, and the soil salinization has been aggravated. As an important main production area of protected vegetables (such as peppers and tomatoes) in China, the Hexi area in Gansu Province has long faced difficulties such as arid climate, long low-temperature period, and serious soil salinization. Moreover, due to the extensive use of chemical fertilizers and pesticides, the salt and disease resistance of crops has been weakened, the soil has become compacted, and the quality of crops has declined, hindering the development of sustainable agriculture. Taking salinization as an example, 93% of the cultivated land in the Hexi area is saline-alkali land. It is urgent to screen strains that can not only tolerate drought and adapt to saline-alkali environments, but also improve soil texture and promote the growth of peppers, tomatoes, etc. This is the key to increasing the yield of peppers using saline-alkali land.

[0003] At present, there are three traditional methods for improving saline-alkali lands: physical, chemical, and biological. Physical and chemical improvements are highly operable and have significant short-term effects, but they are costly, have a short time limit, are prone to cause secondary pollution, the regulation effect is affected by various factors, it is difficult to promote on a large scale, and the sustainability is poor, and the improvement effect is limited. Biological improvement mainly relies on planting salt-tolerant plants and culturing salt-tolerant microorganisms, but the timeliness of improving soil quality is poor. Existing salt-tolerant microorganisms have problems such as unclear improvement effect, weak colonization ability, unstable disease prevention, poor stress resistance, and narrow antibacterial spectrum. The stability and long-term effect of their impact on the ecosystem also need to be observed. Biological control, as a green prevention and control method, can utilize biocontrol microorganisms to antagonize pathogenic bacteria. However, the core strains of current microbial inoculants are not "targeted" enough, and it is urgent to explore new strains with excellent crop affinity, regionality, targeting, adaptability, stress resistance, and productivity to achieve stable application effects. Peppers are crops with relatively high economic value and large market demand. Some improved varieties can adapt to a certain degree of arid and saline-alkali environments, but high concentrations of salt will still inhibit the germination of pepper seeds, the development of roots and plants, and are also prone to induce soil-borne diseases caused by the highly destructive Phytophthora capsici, resulting in plants suffering from phytophthora blight, affecting the fruit quality, and causing huge economic losses to agricultural production. At present, most reports on the anti-Phytophthora capsici of microbial secondary metabolites stay at the separation and identification of products and the preliminary screening and evaluation of anti-Phytophthora capsici activity, and there is little research on its antibacterial mechanism and field experiments.

[0004] Therefore, not only the selection and breeding of drought-tolerant and salt-tolerant pepper varieties are crucial for planting in saline-alkali land, but also biological control technology is of great importance for preventing and controlling pepper fungal diseases, promoting growth under salt stress, and improving the soil. China is rich in microbial resources. Exploring and screening biocontrol strains with high efficiency, stress resistance, and broad-spectrum antibacterial properties can provide technical and resource support for disease prevention and control and the sustainable development of agriculture. The growth performance of existing single-strain microorganisms is limited, and their growth-promoting effect on crops in saline-alkali land also has great limitations. Moreover, there is relatively little research on using biocontrol bacteria to prevent and control pepper diseases in China. Some reports have found that the endophytic bacterium Bacillus velezensis XY40-1 can achieve a control effect of 66.13% on pepper blight, and the growth-promoting effect is obvious; the compound microbial agent of Bacillus amyloliquefaciens and Bacillus subtilis has a disease prevention effect of 75% on pepper blight. There are also a small number of reports that salt-tolerant rhizosphere growth-promoting bacterial agents have obvious effects on soil improvement and rice growth promotion. However, the research on microbial agents that can simultaneously repair saline-alkali land and promote the growth and disease prevention of peppers is almost blank, especially for Bacillus rugosus microbial agents.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The purpose of the present invention is to provide a Bacillus rugosus and its application. This Bacillus rugosus has a strong antagonistic effect against Phytophthora capsici, has a good control effect on pepper blight, and has strong drought and salt-alkali resistance capabilities. It can promote the growth of peppers under saline-alkali conditions, improve the agronomic traits of peppers, and promote the yield increase of peppers.

[0007] In the first aspect of the present invention, a Bacillus rugosus is provided, and its preservation number is CCTCC NO: M20232022.

[0008] The Bacillus rugosus of the present invention is named Bacillus rugosus BEG131. It was preserved in the China Center for Type Culture Collection on October 25, 2023, with the preservation number CCTCC NO: M20232022, and the preservation address is Wuhan University, Wuhan, China.

[0009] The Bacillus rugosus BEG131 of the present invention was isolated from the healthy and well-growing plant Alhagi sparsifolia in the desert of Gaotai County, Zhangye City, Gansu Province. After isolation and purification, a monoclonal strain was obtained. Through the comparison of the 16S rRNA gene sequence similarity, its sequence is closest to that of Bacillus rugosus, and the genetic relationship is the closest. This result is the same as the microscopic morphological identification result, indicating that the isolated BEG131 strain is Bacillus rugosus.

[0010] The Bacillus velezensis BEG131 of the present invention has an antagonistic effect against Phytophthora capsici, and the bacteriostatic rate reaches 82.14%; it has the ability to tolerate drought and resist salt and alkali, and grows well when the mass concentration of sodium chloride is 12% and the pH is 12; it has strong ecological adaptability and can grow well at a culture temperature of 15°C to 45°C; it has strong organic phosphorus-decomposing activity, and significant amylase-decomposing activity and cellulase-decomposing activity.

[0011] Specifically, the 16S rRNA sequence of the Bacillus velezensis BEG131 is as shown in SEQ ID No.1.

[0012] In the second aspect of the present invention, a microbial inoculant is provided, and the microbial inoculant contains the above-mentioned Bacillus velezensis BEG131.

[0013] Preferably, the microbial inoculant takes Bacillus velezensis as the main microorganism.

[0014] Preferably, the microbial inoculant is a solid inoculant.

[0015] Preferably, the microbial inoculant contains viable cells of Bacillus velezensis, dried cells of Bacillus velezensis obtained by spray drying, immobilized cells of Bacillus velezensis, or Bacillus velezensis in any other form.

[0016] Preferably, the viable count of the microbial inoculant ≥ 2.5×10 10 CFU / g.

[0017] The microbial inoculant of the Bacillus velezensis BEG131 of the present invention has a strong antagonistic effect against Phytophthora capsici, and the bacteriostatic rate is as high as 82.14%, and has a good control effect on Phytophthora blight of pepper, and the control effect can reach 76.92%; it has strong ecological adaptability and can grow well at a culture temperature of 15°C to 45°C; it has good organic phosphorus-decomposing effect, and significant amylase-decomposing activity and cellulase-decomposing activity; moreover, it has strong drought and salt-alkali resistance ability, grows well when the mass concentration of sodium chloride is 12% and the pH is 12, can promote the growth of pepper under saline-alkali conditions, improve the agronomic traits of pepper, increase the yield by 262.2 kg / mu compared with conventional fertilization, the yield increase rate is 7.5%, and the increase in efficiency per mu is 329.5 yuan.

[0018] In the third aspect of the present invention, a preparation method of a microbial inoculant is provided, including the following steps:

[0019] S1. Screen and purify the microorganisms in the roots of Alhagi sparsifolia, culture and isolate the purified strains to obtain single colonies of Bacillus velezensis, and culture the single colonies again to obtain purified strains of Bacillus velezensis;

[0020] S2. Inoculate the purified single colony of Bacillus subtilis into LB medium for culture to obtain a Bacillus subtilis seed solution.

[0021] S3. Inoculate the Bacillus subtilis seed solution into a fermentation medium for fermentation to obtain a Bacillus subtilis fermentation broth.

[0022] S4. After centrifuging and removing impurities from the Bacillus subtilis fermentation broth, add a carrier and perform spray drying to obtain a Bacillus subtilis microbial inoculant.

[0023] The microbial inoculant obtained by the preparation method of the present invention is a solid powder, which has low cost, is easy to transport and convenient to use.

[0024] Preferably, in step S2, inoculate the purified strain of Bacillus subtilis into LB medium for culture, place it in a shaking culture at 15 - 45 °C and 180 - 200 r / min for 12 - 24 h to obtain a Bacillus subtilis seed solution, and the OD value of the seed solution is 3.0 - 7.0.

[0025] Preferably, in step S3, the inoculation amount of the Bacillus subtilis seed solution is 1% - 3%, the fermentation temperature is 15 - 45 °C, the fermentation time is 20 - 48 h, and the fermentation culture rotation speed is 180 - 200 r / min.

[0026] Preferably, in step S3, the carbon source in the fermentation medium includes any one of glycerol, soluble starch, sucrose, maltose, corn starch, glucose, lactose, and more preferably maltose. The nitrogen source in the fermentation medium includes any one of urea, peptone, glycine, (NH4)2SO4, yeast extract powder, beef extract, and more preferably (NH4)2SO4. Preferably, the addition ratio of the carbon source to the nitrogen source in the fermentation medium is 1:2.

[0027] Preferably, in step S3, the components of the fermentation medium include: maltose, (NH4)2SO4, KH2PO4, K2HPO4, MnSO4 and MgSO4·7H2O; more preferably, the components of the fermentation medium include: 4 - 5 g / L maltose, 8 - 10 g / L (NH4)2SO4, 4 - 5 g / L KH2PO4, 4 - 5 g / L K2HPO4, 0.1 - 0.3 g / L MnSO4 and 0.3 - 0.5 g / L MgSO4·7H2O.

[0028] In the fourth aspect of the present invention, a method for using a microbial inoculant is provided, including the following steps: After the chili peppers grow to 1 - 2 months old, drip-irrigate and apply the Bacillus subtilis microbial inoculant together with water-soluble fertilizer, and the application amount per mu is 0.5 - 2 kg (preferably 1 kg).

[0029] In the fifth aspect of the present invention, there is provided the application of the above-mentioned Bacillus rugosus microbial inoculum in the prevention and control of pepper phytophthora blight. Through field trials and other in-depth studies at multiple levels and in multiple aspects, after application on pepper phytophthora blight, the control effect can reach 76.92%, and the promotion of pepper yield increase reaches 262.2 kg / mu.

[0030] Preferably, the Bacillus rugosus microbial inoculum is applied in the prevention and control of pepper phytophthora blight caused by Phytophthora capsici.

[0031] The present invention has at least the following beneficial effects:

[0032] (1) The Bacillus rugosus and the microbial inoculum of the present invention have a strong antagonistic effect on Phytophthora capsici, with an inhibition rate as high as 82.14%, and have a good control effect on pepper phytophthora blight, and the control effect can reach 76.92%.

[0033] (2) The Bacillus rugosus and the microbial inoculum of the present invention have strong drought and salt-alkali resistance capabilities. When the mass concentration of NaCl is 12% and the pH is 12, the colony grows well, can promote the growth of peppers under saline-alkali conditions, improve the agronomic traits of peppers, increase the yield by 262.2 kg / mu compared with conventional fertilization, with a yield increase rate of 7.5%, and increase the efficiency per mu by 329.5 yuan.

[0034] (3) The Bacillus rugosus and the microbial inoculum of the present invention have strong ecological adaptability and can grow well at a culture temperature of 15°C to 45°C.

[0035] (4) The Bacillus rugosus and the microbial inoculum of the present invention have good effects on decomposing organic phosphorus, and the activities of amylase decomposition and cellulase decomposition are significant.

[0036] (5) The Bacillus rugosus and its microbial inoculum of the present invention have significant drought and salt-alkali resistance characteristics, and perform excellently in the prevention and control of pepper phytophthora blight and yield increase. This achievement breaks through the disadvantages of limited growth performance of single-strain microorganisms and limited growth promotion effect on saline-alkali land crops. Description of the Drawings

[0037] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0038] Figure 1 It is a colony morphology diagram of Bacillus rugosus provided by the present invention after being cultured at 16°C and 37°C for 12 to 24 hours.

[0039] Figure 2 The effect diagram for the activity determination of Bacillus subtilis rugosus provided by the present invention.

[0040] Figure 3 The result diagram of the growth state of Bacillus subtilis rugosus with salt tolerance provided by the present invention.

[0041] Figure 4 The result diagram of the growth state of Bacillus subtilis rugosus with alkali tolerance provided by the present invention.

[0042] Figure 5 The result diagram of the antagonistic test of Bacillus subtilis rugosus against Phytophthora capsici provided by the present invention. Detailed implementation manners

[0043] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present application belongs.

[0044] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0045] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0046] Example 1 Isolation and identification of Bacillus subtilis rugosus BEG131

[0047] 1. Isolation and purification of Bacillus subtilis rugosus BEG131

[0048] (1) Sample collection

[0049] Select healthy and well-growing plants of Alhagi sparsifolia from the desert in Gaotai County, Zhangye City, Gansu Province. After removing the residual branches and withered leaves on the ground surface, insert a sterile shovel under the surface layer of the soil, dig out the roots of Alhagi sparsifolia, gently shake off the floating soil, put the root system and its rhizosphere soil samples into a sterile sampling bag and bring them back to the laboratory, store them in a refrigerator at 4°C, collect the rhizosphere soil of Alhagi sparsifolia with a sterile brush within 48 hours, and isolate the rhizosphere bacteria.

[0050] (2) Isolation of Bacillus rugosus BEG131

[0051] LB medium was used to isolate bacteria from the rhizosphere soil of Alhagi sparsifolia. Weigh 5 g of rhizosphere soil of Alhagi sparsifolia and put it into 45 mL of sterile water, and shake it thoroughly at 37 °C and 150 r / min for 20 min to obtain a soil suspension. Soil suspensions with concentrations of 10 -1 ~10 -6 were obtained by gradient dilution. Pipette 100 μL of suspensions with concentrations of 10 -4 、10 -5 、10 -6 respectively, and spread them on LB medium, then invert it and incubate it overnight in an incubator at 15 - 45 °C.

[0052] (3) Purification of Bacillus rugosus BEG131

[0053] Observe the colony morphology and select colonies with different morphological characteristics for streak purification. Regularly observe the growth of colonies during this period, pick single colonies with consistent morphological characteristics for cultivation, and purify 3 - 4 times until there are no contaminants in the streaks and confirm by microscopic observation after Gram staining.

[0054] 2. Identification of Bacillus rugosus BEG131

[0055] (1) Morphological observation

[0056] Take the isolated and purified strain and streak it on an LB plate, and observe the colony morphology after culturing at 15 - 45 °C for 12 - 24 h. Among them, the colony morphology diagrams after culturing at 16 °C and 37 °C are as Figure 1 shown, indicating that this bacterium has strong ecological adaptability and can grow well under low - temperature and high - temperature environments.

[0057] (2) Molecular biology analysis

[0058]

[0059] Example 2 Optimization of the Fermentation Medium of Bacillus megaterium BEG131

[0060] 1. Optimization of the Carbon and Nitrogen Source Formulations

[0061] (1) Basic Medium Formulations:

[0062] Nutrient Agar Medium (NA): Beef extract 3 g / L, peptone 10 g / L, NaCl 5 g / L, agar 15 g / L, sterilized at 121 °C for 30 min;

[0063] Peptone Yeast Medium (LB): Peptone 10 g / L, yeast extract powder 5 g / L, sodium chloride 10 g / L, agar 15 g / L, sterilized at 121 °C for 30 min;

[0064] Bacterial Basic Medium (CM): Glucose 5 g / L, (NH4)2SO4 2 g / L, sodium citrate 1 g / L, MgSO4·7H2O 0.2 g / L, K2HPO4 4 g / L, KH2PO4 6 g / L, agar 15 g / L, sterilized at 121 °C for 30 min;

[0065] Beef Extract Yeast Glucose Medium (NYBD): Beef extract 8 g / L, yeast extract powder 5 g / L, glucose 10 g / L, agar 15 g / L, sterilized at 118 °C for 30 min;

[0066] Peptone Yeast Sucrose Medium (YSP): Peptone 10 g / L, yeast extract powder 5 g / L, sucrose 20 g / L, agar 15 g / L, sterilized at 118 °C for 30 min.

[0067] (2) Tested Carbon Sources:

[0068] Glycerol, soluble starch, sucrose, maltose, corn starch, glucose, lactose

[0069] (3) Tested Nitrogen Sources:

[0070] Urea, peptone, glycine, (NH4)2SO4, yeast extract powder, beef extract

[0071] 2. Specific Steps:

[0072] (1) Shake Flask Fermentation Culture: Prepare 100 mL of fermentation medium in a 250 mL conical flask. After sterilization, inoculate 1 mL of Bacillus megaterium seed solution and culture at 37 °C and 200 r / min for 48 h. Regularly detect OD 600 and determine the viable cell count by the plate counting method.

[0073] (2) Screening of the optimal basal medium: Fermentation experiments were carried out using NA, LB, CM, NYBD, and YSP basal media respectively. In a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, 1% of the seed solution was inoculated and placed in a constant temperature (37 °C) shaker (200 r / min) for 48 h, and OD was regularly measured. 600 The viable cell count was determined by the plate counting method to determine the optimal medium.

[0074] (3) Screening and optimization of carbon sources: Glycerol, soluble starch, sucrose, maltose, corn starch, glucose, lactose, etc. were used to replace the carbon source in the optimal basal medium with equal mass, and other components remained unchanged. In a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, 1% of the seed solution was inoculated and placed in a constant temperature (37 °C) shaker (200 r / min) for 48 h, and OD was regularly measured. 600 The viable cell count was determined by the plate counting method to determine the optimal carbon source.

[0075] (4) Screening and optimization of nitrogen sources: Urea, peptone, glycine, (NH4)2SO4, yeast extract powder, beef extract were used to replace the nitrogen source in the optimal basal medium with equal mass, and other components remained unchanged. In a 250 mL Erlenmeyer flask containing 100 mL of liquid medium, 1% of the seed solution was inoculated and placed in a constant temperature (37 °C) shaker (200 r / min) for 48 h, and OD was regularly measured. 600 The viable cell count was determined by the plate counting method to determine the optimal nitrogen source.

[0076] 3. Test results

[0077] (1) Screening of the optimal basal medium:

[0078] Bacillus lugosii was fermented for 48 h using different basal fermentation media, and OD and viable cell count were measured at 16 h, 20 h, 24 h, 40 h, and 48 h. The results are shown in Table 1. Considering various factors comprehensively, the NYBD medium with a stable growth rate and a long stationary phase was determined as the optimal basal fermentation medium. 600 Table 1 Screening of the optimal fermentation medium

[0079] Table 1 Screening of the optimal fermentation medium

[0080]

[0081]

[0082] (2) Screening of the optimal carbon source:

[0083] Based on the NYBD medium, glycerol, soluble starch, sucrose, maltose, corn starch, glucose, and lactose were used to replace the carbon source in the optimal basal medium in equal mass, with other components remaining unchanged. Fermentation was carried out for 48 h, and OD was measured at 16 h, 20 h, 24 h, 40 h, and 48 h. 600 And the viable cell count. The results are shown in Table 2. After comprehensive consideration in multiple aspects, maltose was determined as the optimal carbon source.

[0084] Table 2 Screening of the Optimal Carbon Source

[0085]

[0086] (3) Screening of the Optimal Nitrogen Source:

[0087] Based on 8 g / L beef extract, 5 g / L yeast extract powder, and 10 g / L maltose, urea, peptone, glycine, (NH4)2SO4, yeast extract powder, and beef extract were used to replace the yeast extract powder in the basal medium in equal mass, with other components remaining unchanged. Fermentation was carried out for 48 h, and OD was measured at 16 h, 20 h, 24 h, 40 h, and 48 h. 600 And the viable cell count. The results are shown in Table 3. After comprehensive consideration in multiple aspects, it was determined that the replacement effect of yeast extract powder was the best.

[0088] Table 3 Screening of the Optimal Nitrogen Source

[0089]

[0090] Based on 8 g / L beef extract, 5 g / L yeast extract powder, and 10 g / L maltose, urea, peptone, glycine, (NH4)2SO4, yeast extract powder, and beef extract were used to replace the beef extract in the basal medium in equal mass, with other components remaining unchanged. Fermentation was carried out for 48 h, and OD was measured at 16 h, 20 h, 24 h, 40 h, and 48 h. 600 And the viable cell count. The results are shown in Table 4. After comprehensive consideration in multiple aspects, it was determined that the replacement effects of (NH4)2SO4 and peptone were the best.

[0091] Table 4 Screening of the Optimal Nitrogen Source

[0092]

[0093] (4) Conclusion:

[0094] In this experiment, by screening different basal fermentation media, carbon sources, and nitrogen sources, and using the OD of the fermentation broth during the fermentation process 600 And the viable cell count as references, the optimal carbon source was determined to be maltose, and the optimal nitrogen sources were yeast extract powder, (NH4)2SO4, and peptone. Considering the cost, (NH4)2SO4 was finally selected as the optimal nitrogen source.

[0095] 4. Optimal addition amounts of carbon source and nitrogen source: maltose : (NH4)2SO4 = 1 : 2.

[0096] 5. Fermentation medium formula: 4 - 5 g / L maltose, 8 - 10 g / L (NH4)2SO4, 4 - 5 g / L KH2PO4, 4 - 5 g / L K2HPO4, 0.1 - 0.3 g / L MnSO4, and 0.3 - 0.5 g / L MgSO4·7H2O.

[0097] Example 3 Activity determination of Bacillus megaterium BEG131

[0098] 1. Activity determination

[0099] (1) Determination of phosphate - solubilizing effect on plate

[0100] Inoculate the isolated strain onto the pre - prepared organic phosphorus medium plate, with four inoculation points per petri dish, and repeat three times. Incubate at 37°C for 5 days, observe and record the growth of the strain and the size of the decomposition zone. Determine the phosphate - solubilizing activity of the bacteria according to the size of the decomposition zone and the value of decomposition zone diameter / colony diameter (D / d). The larger the decomposition zone and the larger the D / d value, the stronger the phosphate - solubilizing activity.

[0101] (2) Amylase activity

[0102] Activate the preserved strain by the method of three - zone streaking, then pick single colonies with a sterile toothpick and inoculate them onto the amylase plate screening medium, with four inoculation points per petri dish, and repeat three times. Then place them in a 37°C biochemical incubator for static incubation in an inverted position. After 36 h of incubation, take out the amylase screening plate, pour Lugol's iodine solution onto the plate until the iodine solution covers the whole plate, stain for 15 min, then pour out Lugol's iodine solution, observe the formation of the transparent zone around the colonies, and determine the amylase activity of the bacteria according to the size of the decomposition zone and the value of decomposition zone diameter / colony diameter (D / d).

[0103] (3) Cellulase activity

[0104] Activate the preserved strain by the method of three - zone streaking, then pick single colonies with a sterile toothpick and inoculate them onto the cellulase plate screening medium, with four inoculation points per petri dish, and repeat three times. Then place them in a 37°C biochemical incubator for static incubation in an inverted position. After 36 h of incubation, take out the cellulase screening plate, pour Congo red staining solution onto the plate until the staining solution covers the whole plate, stain for 20 min, then discard the Congo red staining solution, then pour in sodium chloride washing solution for decolorization for 20 min, and finally discard the sodium chloride washing solution, observe the transparent zone around the colonies, and determine the cellulase activity of the bacteria according to the size of the decomposition zone and the value of decomposition zone diameter / colony diameter (D / d).

[0105] 2. Medium

[0106] (1) Organophosphorus plate culture medium

[0107] Glucose 10g, (NH4)2SO4 0.5g, NaCl 0.3g, KCl 0.3g, MnSO4 0.03g, FeSO4 0.03g, lecithin 0.2g, CaCO3 5.0g, yeast extract 0.4g, agar 20g, distilled water 1000mL, pH 7.0-7.2.

[0108] (2) Amylase plate screening medium

[0109] Sodium chloride 10g, peptone 10g, beef extract 5g, soluble starch 2g, distilled water 1000mL, heat the above ingredients to fully dissolve, cool and adjust the pH to 7.0-7.2, then add 18g agar, dissolve and dispense, and sterilize at 121℃ for 30min. The plate making method is the same as LB solid medium.

[0110] (3) Cellulase plate screening medium

[0111] 15g sodium carboxymethyl cellulose, 10g peptone, 5g yeast extract powder, 5g sodium chloride, 1g potassium dihydrogen phosphate, 0.2g magnesium sulfate, 1000mL distilled water. Heat the above ingredients to fully dissolve them, cool and adjust the pH to 7.0-7.2, then add 18g agar, dissolve and dispense, and sterilize at 121℃ for 30min. The method of making plates is the same as that of LB solid medium.

[0112] 3. Staining Reagents

[0113] (1) Lugol's iodine solution

[0114] Weigh 2.0 g of potassium iodide and dissolve it in 100 mL of distilled water. Then add 1.0 g of iodine. After it is fully dissolved, add more distilled water to make the total volume of the solution 300 mL.

[0115] (2) Congo red staining solution

[0116] Weigh 0.1 g of Congo red reagent and dilute it to 100 mL of distilled water to prepare a Congo red staining solution with a mass concentration of 1 g / L.

[0117] (3) Sodium chloride rinse solution

[0118] Accurately weigh 5.844 g of sodium chloride, dilute it to 100 mL of distilled water, and prepare a sodium chloride rinse solution with a molar concentration of 1 mol / L.

[0119] 4. Results

[0120] like Figure 2As shown, the strain Bacillus subtilis BEG131 has strong organophosphorus-decomposing activity. The diameter of the decomposition zone is 2.2 cm, and the ratio of the diameter of the decomposition zone to the colony diameter (D / d) is 2.2 / 1.6, that is, 1.375, indicating that the phosphorus-solubilizing activity of this bacterium is very strong. The amylase activity and cellulase activity of the strain Bacillus subtilis BEG131 are also obvious. The sizes of the decomposition zones are 5.0 cm and 4.5 cm respectively, and the ratios of the diameter of the decomposition zone to the colony diameter (D / d) are 5.0 / 2.5 and 4.5 / 1.5 respectively, that is, 2 and 3, which is sufficient to show that the activities of Bacillus subtilis BEG131 in decomposing amylase and cellulase are very strong.

[0121] Example 4 Drought Tolerance and Salt-alkali Resistance Experiment

[0122] 1. Experimental Design and Research Methods

[0123] 1.1 Medium Type

[0124] LB medium: Tryptone 2 g / L, Yeast Extract 1 g / L, NaCl 2 g / L, Agar 18 g / L, pH 7.0 - 7.4.

[0125] 1.2 Experimental Methods

[0126] 1.2.1 Drought Tolerance Experiment of Bacillus subtilis BEG131

[0127] PEG6000 was used to increase the osmotic pressure to artificially simulate a drought environment to verify the drought tolerance of Bacillus subtilis BEG131. The single colony of Bacillus subtilis obtained by isolation and purification was inoculated into LB medium and cultured at 37°C and 180 r / min for 16 h as the seed solution. It was inoculated into 100 mL of drought stress medium (LB medium containing 5%, 12%, 20%, 40%, 60% PEG6000 respectively) at an inoculation amount of 2%, and cultured at 37°C and 150 r / min. Samples were taken every 4 hours to measure the OD value at a wavelength of 600 nm.

[0128] 1.2.2 Salt Tolerance Experiment of Bacillus subtilis BEG131

[0129] The single colony of Bacillus subtilis activated on LB was inoculated into LB medium and cultured at 37°C and 180 r / min for 16 h as the seed solution. 100 μL was taken and spread on the salt stress medium (LB medium containing 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14% NaCl respectively), and cultured overnight in an incubator at 37°C to observe the colony growth situation.

[0130] 1.2.3 Alkaline Tolerance Experiment of Bacillus subtilis BEG131

[0131] Inoculate a single colony of Bacillus subtilis activated on LB medium into LB medium, and culture it at 37°C with shaking at 180 r / min for 16 h as the seed liquid. Pipette 100 μL and spread it on the alkali stress-resistant medium (LB medium with pH values of 7, 8, 9, 10, 11, 12, and 13 respectively), and culture it overnight in an incubator at 37°C, and observe the colony growth situation.

[0132] 2. Result statistics

[0133] 2.1 Drought tolerance experiment of Bacillus subtilis BEG131

[0134] The growth characteristics of Bacillus subtilis BEG131 cultured for 48 h under different drought stress levels are shown in Table 5. With the increase of drought stress level, the growth rate of strain BEG131 shows a decreasing trend. Among them, it can grow well in the culture medium with 5% PEG6000 concentration. When the PEG6000 concentration increases to 20%, the growth of strain BEG131 is affected to a certain extent. However, the growth of the strain under 40% PEG6000 drought stress is more vigorous than that under 20% PEG6000 drought stress. The growth of the strain is significantly inhibited under 60% PEG6000 drought stress but relatively stable, indicating that this bacterium has a certain drought-loving property.

[0135] Table 5 Growth characteristics of culture for 48 h under different drought stress levels

[0136]

[0137] 2.2 Salt tolerance experiment of Bacillus subtilis BEG131

[0138] The growth situation of Bacillus subtilis BEG131 on the salt stress-resistant medium is shown in Figure 3 , and its colonies can grow normally in LB medium with salt concentrations of 1%, 2%, 4%, 6%, 8%, 10%, and 12% NaCl. When the mass concentration of NaCl is 14%, the growth is inhibited. It can be seen that Bacillus subtilis BEG131 has extremely strong salt tolerance and can grow on LB medium with 12% NaCl.

[0139] 2.3 Alkali tolerance experiment of Bacillus subtilis BEG131

[0140] The growth situation of Bacillus subtilis BEG131 on the alkali stress-resistant medium is shown in Figure 4 , and its colonies can grow in LB medium with pH values of 7, 8, 9, 10, 11, and 12 respectively. When the pH is 13, the colony growth is inhibited. It can be seen that Bacillus subtilis BEG131 has extremely strong alkali tolerance and can grow on LB medium with a maximum pH of 12.

[0141] Example 5 Evaluation of the antagonistic effect of Bacillus velezensis BEG131 against Phytophthora capsici

[0142] The test fungus was Phytophthora capsici: After the test fungus was activated and cultured on rye medium, a mycelial cake with a diameter of 0.6 cm was punched at the edge of the colony where the mycelium grew vigorously, inoculated in the center of the rye medium, and then the mycelial cake of the antagonistic bacterium Bacillus velezensis BEG131 was inoculated 2.5 cm away from the mycelial cake of Phytophthora capsici. There were 4 points per plate and 3 replicates. It was cultured in a biochemical incubator at 28 °C for about 7 days. The pathogenic bacteria without the antagonistic bacterium were used as the control. The data were statistically analyzed and the inhibition rate was calculated.

[0143] Inhibition rate = (diameter of the control fungal colony - diameter of the inhibited fungal colony) / (diameter of the control fungal colony - 0.6) × 100%

[0144] The results of the antagonistic experiment are as Figure 5 shown, and the inhibition rate is as high as 82.14%.

[0145] Example 6 Preparation of a microbial inoculum of Bacillus velezensis BEG131

[0146] This example provides a method for preparing a microbial inoculum of Bacillus velezensis BEG131, which specifically includes the following steps:

[0147] 1. Inoculate the purified single colony of Bacillus velezensis BEG131 into LB medium, and shake culture at 15 - 45 °C and 180 - 200 r / min for 12 - 24 h as the seed liquid. The OD value of the seed liquid is 3.0 - 7.0;

[0148] 2. Inoculate the seed liquid of Bacillus velezensis BEG131 into the fermentation medium to obtain the fermentation broth of Bacillus velezensis BEG131. The inoculation amount is 1% - 3%, the fermentation temperature is 15 - 45 °C, the fermentation time is 20 - 48 h, and the fermentation rotation speed is 180 - 200 r / min;

[0149] 3. Centrifuge the fermentation broth of Bacillus velezensis BEG131 to remove impurities, add a carrier, and spray dry to obtain the microbial inoculum of Bacillus velezensis BEG131;

[0150] 4. The viable count of the microbial inoculum of Bacillus velezensis BEG131 ≥ 2.5×10 10 CFU / g.

[0151] Example 7 A microbial inoculum of Bacillus velezensis BEG131 for controlling Phytophthora capsici and its application

[0152] The test site was set at the vegetable planting base of Gaoqiao Saline-alkali Land Farm, Xuanhua Town, Gaotai County, Zhangye City, with an east longitude of 99.38° and a north latitude of 39.24°, and an altitude of 1298 m. The terrain is flat, with convenient irrigation, uniform fertility, and the previous crop was corn. The tested soil was tidal irrigation desert soil, and the measured values of soil nutrients before the test were: pH value 10.17, available nitrogen content 16.5 mg / kg, available phosphorus 3.68 mg / kg, available potassium 142.4 mg / kg, and soil organic matter content 2.32 g / kg.

[0153] The pepper seedlings of the variety Jufeng No. 8 were raised on April 5, 2024. The land was prepared, plots were arranged, and base fertilizers were applied on April 18; the seedlings were transplanted and planted on May 9. The ridge width was 0.7 m, the row spacing was 60 cm × 40 cm, and the number of seedlings per mu was 2,600. Starting from the second drip irrigation, the crop began to top-dress with the integration of water and fertilizer, and a total of 6 top-dressings were carried out in combination with the drip irrigation water. Except for the fertilizers in each test plot being strictly implemented according to the test design, other farming operation measures were kept consistent.

[0154] The test had 4 treatments, 3 replicates, and was arranged in a randomized block design. The plot area was 76.8 m2. The specific treatments were as follows:

[0155] Treatment 1: Conventional fertilization + Bacillus rugosus BEG131 1 kg / mu;

[0156] Treatment 2: Conventional fertilization (i.e., basal application of compound fertilizer (17-17-17) 40 kg / mu, and a total of 30 kg of top-dressing was carried out 6 times in total with the integration of water and fertilizer along with the drip irrigation water during the whole growth period; 5 kg / mu of water-soluble fertilizer (30-13-13) was dripped in the early stage, 5 kg / mu of water-soluble fertilizer (18-18-18) was dripped in the middle stage, and 5 kg / mu of water-soluble fertilizer (13-13-30) was dripped during the fruiting period);

[0157] Treatment 3: Conventional fertilization + inactivated matrix;

[0158] Treatment 4: Blank control (no fertilization).

[0159] 1. Effects of different treatments on the growth period of pepper

[0160] Table 6 Record table of pepper growth period Unit: day / month

[0161]

[0162] Through the investigation of each growth period of pepper (Table 6), it can be seen that there was no obvious difference in the growth periods of Treatment 1, Treatment 2, and Treatment 3; in Treatment 4, due to no fertilizer application, the initial harvest period was postponed and the harvest ended earlier.

[0163] 2. Effects of different treatments on the main agronomic traits of pepper

[0164] Table 7 Survey table of main agronomic traits of pepper Unit: cm, cm, mm, mm, piece, g

[0165]

[0166] Thirty pepper plants were randomly selected from each plot to measure their main agronomic traits. From the results (Table 7), it can be seen that the agronomic traits of peppers in treatment 1 are better than those in other treatments. The single fruit weight in treatment 1 is 38.2 g, which is 2.6 g more than that in treatment 2; 2.4 g more than that in treatment 3; and 7.1 g more than that in treatment 4.

[0167] 3. Effects of different treatments on pepper yield

[0168] By randomly selecting 30 pepper plants in each plot in S-type and measuring yields on the spot, the yield results (Table 8) show that treatment 1 has the highest yield of 3773.3 kg / mu, which is 262.2 kg / mu higher than treatment 2, with an increase rate of 7.5%, and 233.3 kg / mu higher than treatment 3, with an increase rate of 6.6%. There is little difference in the increase in yield between treatment 2 and treatment 3.

[0169] Table 8 Yield measurement results Unit: kg, %

[0170]

[0171] Table 9 Variance analysis table

[0172]

[0173] Table 10 Multiple analysis table

[0174]

[0175] Note: Lowercase letters (a, b, c) indicate a significance level of α = 0.05, and uppercase letters (A, B, C) indicate a significance level of α = 0.01.

[0176] The results of variance analysis of yield measurement showed (Table 9): there was no significant difference between replicates, and the difference between treatments reached an extremely significant level (F=242.36>F0.01). The multiple comparison method (Table 10) showed that treatment 1 was significantly different from other treatments; there was no significant difference between treatments 2 and 3.

[0177] 4. Comparison of economic benefit analysis

[0178] From the economic benefit analysis of pepper treatments (Table 11), it can be seen that treatment 1 has the highest benefit of 5514.3 yuan per mu, which is 329.5 yuan higher than treatment 2 and 283.4 yuan higher than treatment 3. There is little difference between treatment 2 and treatment 3. The input-output ratios of treatment 1, treatment 2 and treatment 3 are 11.54:1, 12.97:1 and 13.08:1 respectively.

[0179] Table 11 Economic Benefit Analysis Table Unit: kg, yuan

[0180]

[0181] Example 8 Evaluation of the Control Effect of Bacillus velezensis BEG131 on Phytophthora capsici on Living Pepper Plants

[0182] The pepper plants were treated by the root irrigation method. In the field continuously cultivated with Phytophthora capsici, healthy pepper seedlings with basically the same size were respectively irrigated with 500 g, 1000 g, and 1500 g per mu of Bacillus velezensis BEG131 microbial inoculum with water. The root irrigation with clear water was used as the negative control. The disease incidence of the peppers was observed daily. At harvest, 100 pepper plants treated with Bacillus velezensis BEG131 microbial inoculum and 100 pepper plants not treated with Bacillus velezensis BEG131 microbial inoculum were selected, and the disease incidence, disease index, and control effect were calculated. The whole-plant disease grading standard is as follows:

[0183] Grade 0: The whole plant is disease-free;

[0184] Grade 1: The lesion on the stem does not exceed 1 / 2 or less than 1 / 2 of the stem circumference, the leaves are slightly wilted, or there are lesions on a few lower leaves;

[0185] Grade 2: The lesion on the stem exceeds 1 / 2 or more than 1 / 2 of the stem circumference, and the leaves are wilted;

[0186] Grade 3: The lesion on the stem surrounds the stem circumference or more than 2 / 3, and the leaves are wilted;

[0187] Grade 4: The whole leaves of the diseased plant are wilted or dead.

[0188] Disease incidence = (number of diseased plants / total number of investigated plants) × 100%

[0189] Disease index = Σ (number of plants at each level × value of that disease level) / (total number of investigated plants × highest disease level value) × 100

[0190] Control effect = (control disease index - treatment disease index) / control disease index × 100%

[0191] Bacillus velezensis BEG131 microbial inoculum has a good control effect on Phytophthora capsici on living pepper plants. The results are shown in Table 12. When 500 g / acre, 1000 g / acre, and 1500 g / acre of Bacillus velezensis BEG131 microbial inoculum are respectively applied with water to pepper seedlings with basically the same size, the control effects can reach 51.92%, 76.92%, and 79.49% respectively. Considering the comprehensive effect and cost of the inoculum, when 1000 g / acre of Bacillus velezensis BEG131 microbial inoculum is applied with water, the best control effect on living pepper plants can reach 76.92%.

[0192] Table 12 Control effect of Bacillus rugosus BEG131 microbial inoculum on Phytophthora blight of living pepper plants

[0193]

[0194] In summary, Bacillus rugosus BEG131 of the present invention and the microbial inoculum have a strong antagonistic effect on Phytophthora capsici, with an inhibition rate as high as 82.14%, and have a good control effect on Phytophthora blight of pepper, with a control efficacy of up to 76.92%; Bacillus rugosus BEG131 of the present invention and the microbial inoculum have strong drought and salt-alkali resistance capabilities. The colony grows well when the mass concentration of NaCl is 12% and the pH is 12, which can promote the growth of pepper under saline-alkali conditions, improve the agronomic traits of pepper, increase the yield by 262.2 kg / mu, with a yield increase rate of 7.5%, and increase the efficiency by 329.5 yuan per mu; Bacillus rugosus BEG131 of the present invention and the microbial inoculum have strong ecological adaptability and can grow well at a culture temperature of 15°C to 45°C; Bacillus rugosus BEG131 of the present invention and the microbial inoculum have good effects on decomposing organic phosphorus, and the activities of amylase decomposition and cellulase decomposition are significant; Bacillus rugosus BEG131 of the present invention and its microbial inoculum have significant drought and salt-alkali resistance characteristics and perform excellently in the control of Phytophthora blight of pepper and yield increase. This achievement breaks through the disadvantages of limited growth performance of single-strain microorganisms and limited growth promotion effect on saline-alkali land crops.

[0195] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Bacillus rugosus, characterized in that, Its preservation number is CCTCC NO: M20232022.

2. The Bacillus rugosus according to claim 1, wherein The 16S rRNA sequence of the Bacillus velezensis is shown in SEQ ID No.

1.

3. The Bacillus rugosus according to claim 1, characterized in that, The Bacillus velezensis is isolated from Alhagi sparsifolia.

4. The Bacillus rugosus according to claim 1, characterized in that, The Bacillus velezensis has an antagonistic effect against Phytophthora capsici, with an inhibition rate as high as 82.14%. It has strong ecological adaptability and can grow well at a culture temperature of 15°C to 45°C. It has drought and salt-alkali resistance capabilities, and can grow well with a NaCl mass concentration of 12% and a pH of 12. It has strong organic phosphorus-degrading activity, and significant amylase-degrading and cellulase-degrading activities.

5. A microbial inoculant, characterized in that, The microbial inoculant contains the Bacillus velezensis described in any one of claims 1-4.

6. The microbial inoculant according to claim 5, wherein, The viable count of the microbial inoculum ≥ 2.5×10 10 CFU / g.

7. The preparation method of the microbial inoculum according to claim 5, characterized in that, It includes the following steps: S1. Screen and purify the microorganisms in the roots of Alhagi sparsifolia, culture and isolate the purified strains to obtain single colonies of Bacillus velezensis, and culture the single colonies again to obtain purified strains of Bacillus velezensis; S2. Inoculate the purified single colonies of Bacillus velezensis into LB medium for culture to obtain a Bacillus velezensis seed solution; S3. Inoculate the Bacillus velezensis seed solution into a fermentation medium for fermentation to obtain a Bacillus velezensis fermentation broth; S4. After centrifuging and removing impurities from the Bacillus velezensis fermentation broth, add a carrier and spray-dry to obtain a Bacillus velezensis microbial inoculant.

8. The preparation method of the microbial inoculum according to claim 7, wherein, In step S3, the inoculation amount of the Bacillus velezensis seed solution is 1% to 3%, the fermentation temperature is 15 to 45°C, the fermentation time is 20 to 48 h, and the fermentation rotation speed is 180 to 200 r / min.

9. The preparation method of the microbial inoculum according to claim 7, wherein, In step S3, the components of the fermentation medium include: maltose, (NH4)2SO4, KH2PO4, K2HPO4, MnSO4, and MgSO4·7H2O.

10. Use of Bacillus rugosus according to any one of claims 1-4 or microbial inoculum according to any one of claims 5-6 in controlling Phytophthora blight of pepper, characterized in that, After application in pepper blight, the control effect is significant, with a control efficacy of up to 76.92%, and it can promote the growth and yield increase of peppers under saline-alkali conditions.

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