Bacillus Velez and its application in agricultural production

Abstract

The present invention discloses Bacillus Velez and its application in agricultural production. The present invention isolates strains from soil samples rich in corn stalks, and finally screens out a strain LF-9 that can efficiently produce cellulase, promote plant growth and prevent and treat plant diseases. Through molecular biology combined with morphological identification, it is determined that strain LF-9 is Bacillus Velez, and its microbial preservation number is CGMCC NO.29001. Experiments have shown that the LF-9 strain can not only efficiently produce cellulase, but also promote plant growth and prevent and treat potato black mole and potato black leg disease; using the Bacillus Velez LF-9 for agricultural waste treatment can significantly shorten the composting fermentation cycle and improve the composting fermentation quality. The Bacillus Velez LF-9 strain provided by the present invention has application prospects in producing cellulase, preparing agricultural waste compost, promoting plant growth and preventing and treating plant diseases.

Description

Technical Field

[0001] The present invention relates to a Bacillus strain isolated from soil, and in particular to a Bacillus Velezii strain isolated from soil rich in corn stalks. Bacillus velezensis ) and its application in agricultural production, belonging to the field of Bacillus Velez and its application. Background Art

[0002] Crop straw is the main by-product of grain production, and it contains a large amount of cellulose, which is an important biological resource in agricultural production. In addition, among the returned straw, the returned straw that has not been harmlessly treated carries a large number of harmful substances such as pathogens and insect eggs, which will have a serious impact on the growth and yield of later crops. In addition, the carbon-nitrogen ratio in the straw is too high, and the straw degradation rate after returning to the field is too slow, which is easy to compete with crop seedlings for nitrogen sources, which will also lead to serious agricultural production problems and trigger a series of ecological and environmental problems. At the same time, with the development of animal husbandry, the problem of livestock and poultry manure pollution under modern breeding production environment conditions has gradually become prominent. If it is not scientifically and effectively treated, it will bring a series of environmental problems. The annual output of sheep manure is huge. As one of the agricultural wastes, untreated sheep manure contains a large number of harmful substances such as pathogens and parasite eggs. If it is directly applied to the field without being decomposed, it will not only pollute the environment, but also cause serious crop diseases, insect pests and weeds. At present, composting fermentation is one of the effective ways to realize the recycling of agricultural waste resources. However, in actual production, there are problems such as long composting fermentation time and poor fermentation quality. Therefore, screening microorganisms that can shorten the composting production cycle and improve the composting fermentation quality for agricultural waste composting is an effective method.

[0003] With the rapid development of agriculture and animal husbandry, related environmental pollution problems have become increasingly prominent, especially the treatment of livestock manure and agricultural straw waste has become an urgent problem of environmental pollution and ecological protection. Therefore, an efficient, convenient and environmentally friendly way to treat agricultural waste is of great significance to promoting the green and sustainable development of agriculture. Summary of the invention

[0004] One of the objects of the present invention is to provide a strain of Bacillus Velezii ( Bacillus velezensis ) strain;

[0005] The second object of the present invention is to provide a fermentation liquid, a microbial preparation or a compost prepared by the Bacillus Velezii strain;

[0006] The third purpose of the present invention is to apply the Bacillus Velezii strain to agricultural production.

[0007] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0008] One aspect of the present invention is to provide a strain of Bacillus Velezii ( Bacillus velezensis ) LF-9, its microbial preservation number is CGMCC No.29001; its classification name is: Bacillus Velez Bacillus velezensis ; The preservation time is: November 14, 2023; The preservation unit is: General Microbiology Center of China Culture Collection Administration; The preservation address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0009] The colony and bacterial morphology of the Bacillus Velezii described in the present invention are as follows: after being cultured on LB medium, the colony is observed to be nearly circular in shape, with protruding wrinkles in the middle, sticky when picked up, milky white in color at the initial growth stage, and light yellow after maturity.

[0010] The isolated Bacillus Velez LF-9 can produce high-yield cellulase, shorten the compost fermentation cycle, and improve the compost fermentation quality; the Bacillus Velez LF-9 can produce IAA and effectively promote the increase of plant height, root length or fresh weight; the Bacillus Velez LF-9 has a significant inhibitory effect on bacteria or fungi including Rhizoctonia solani or Erwinia carotovora subspecies potato black shank, and therefore has a definite preventive and therapeutic effect on plant diseases including potato black mole disease or potato black shank disease.

[0011] Another aspect of the present invention is to provide a fermentation liquid prepared by the Bacillus Velez subtilis LF-9, or a microbial preparation or compost containing the Bacillus Velez subtilis LF-9; wherein the effective viable count of Bacillus Velez subtilis LF-9 in the microbial preparation or compost is 1×10 6~8 cfu / mL.

[0012] In a preferred specific embodiment, the present invention provides a method for preparing the fermentation broth, comprising: activating the strain of Bacillus Velez LF-9 and inoculating it into a liquid culture medium for fermentation to obtain a fermentation broth; wherein the composition of the liquid culture medium is: by mass percentage, 1.5% tryptone, 0.58% yeast extract, 0.54% NaCl, and the remainder is distilled water, and the pH value is 6.8; the fermentation culture conditions include: the fermentation culture temperature is 27°C, the fermentation culture is carried out in a shaker, and the shaker speed is 150 r•min -1 , the fermentation time is 3 days.

[0013] Those skilled in the art can prepare the Bacillus Velez subtilis LF-9 provided by the present invention into various conventional microbial preparations or composts according to conventional preparation methods of microbial preparations, which are all technical means well known to those skilled in the art.

[0014] For reference, the present invention provides a method for preparing compost using Bacillus Velez subtilis LF-9, comprising: mixing corn stalks and sheep manure in a mass ratio of 3:1, inoculating activated bacterial liquid of Bacillus Velez subtilis LF-9, composting and fermenting; the fermentation conditions may be constant temperature cultivation at 25°C for 30 days; and turning the compost every 10 days during the fermentation process.

[0015] Another aspect of the present invention is to apply the Bacillus Velez LF-9, the fermentation liquid prepared by the Bacillus Velez LF-9, or the microbial preparation or compost containing the Bacillus Velez LF-9 to agricultural production.

[0016] In a preferred embodiment of the present invention, the Bacillus Velez subtilis LF-9, the fermentation broth prepared by the Bacillus Velez subtilis LF-9, or the microbial preparation containing the Bacillus Velez subtilis LF-9 is used to produce cellulase; wherein the activity of the produced cellulase (CL) can reach up to 235.897 U•mL -1 .

[0017] In a preferred specific embodiment of the present invention, the Bacillus Velez LF-9, the fermentation liquid prepared by the Bacillus Velez LF-9, or the microbial preparation containing the Bacillus Velez LF-9 is used to promote plant growth; wherein promoting plant growth includes increasing the plant height, root length or fresh weight of the plant; wherein the fresh weight refers to the weight of the whole plant; more preferably, the plant includes potato or wheat.

[0018] In a preferred specific embodiment of the present invention, the Bacillus Velez subsp. LF-9, the fermentation liquid prepared by the Bacillus Velez subsp. LF-9, or the microbial preparation containing the Bacillus Velez subsp. LF-9 is applied to plant disease prevention and control; wherein the pathogens of the plant diseases include Rhizoctonia solani or Erwinia carotovora subsp. potato black shank; wherein the plant diseases include potato black mole disease or potato black shank disease.

[0019] The present invention isolates strains from soil and finally screens out a strain LF-9 that can efficiently produce cellulase, be used to prepare agricultural waste compost, promote plant growth, and prevent and treat plant fungal diseases or bacterial diseases. The classification status of strain LF-9 is determined to be Bacillus velezinus ( Bacillus velezensis), experiments have shown that the Bacillus Velez LF-9 can produce high levels of cellulase, shorten the compost fermentation cycle, and improve the compost fermentation quality; the Bacillus Velez LF-9 produces IAA, which effectively promotes the increase of plant height, root length or fresh weight; the Bacillus Velez LF-9 has a significant preventive and therapeutic effect on fungal or bacterial plant diseases including potato black mole or potato black shank. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the colony morphology of Bacillus Velez LF-9;

[0021] Figure 2 This is the Congo red staining result of Bacillus Velez LF-9;

[0022] Figure 3 This is the time curve of cellulase production by Bacillus Velez LF-9;

[0023] Figure 4 This is a comparison of the apparent results of agricultural waste compost after inoculation with Bacillus Velez LF-9 27 days later;

[0024] Figure 5 This is a graph showing the temporal variation of the fermentation center temperature during the composting process of agricultural waste;

[0025] Figure 6 This is a graph showing the temporal changes in organic matter and total nitrogen during the composting process of agricultural waste; Figure 6 -a is the time variation pattern of organic matter in the process of agricultural waste composting, Figure 6 -b is the time variation pattern of total nitrogen in the process of agricultural waste composting;

[0026] Figure 7 This is the result diagram of IAA production ability of Bacillus Velez LF-9;

[0027] Figure 8 This is a graph showing the results of promoting potato plant growth by Bacillus Velez LF-9;

[0028] Fig. 9 This is a diagram showing the effect of agricultural waste compost on the growth of wheat plants;

[0029] Fig.10 This is a diagram showing the plate inhibition effect of Bacillus Velez LF-9 on Rhizoctonia solani. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, it should be understood that the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0031] Example 1 Isolation and identification of strain LF-9

[0032] 1. Strain Isolation

[0033] The formula of CMC-Na culture medium is: CMC-Na 10 g, KH2PO4 2.5 g, Na2HPO4 1.5 g, peptone 2.5 g, NH4NO3 1.0 g, MgSO4•7H2O 0.5 g, yeast extract 1.0 g, distilled water 1 L, pH natural.

[0034] The dilution plate method was used. Weigh 2 g of the collected soil sample rich in corn stalks, add 20 mL of sterile water, shake on a shaker for 30 min, and then let it stand for 20 min to make a soil suspension. Take 1 mL of the supernatant and perform gradient dilution to 10 -3 ~10 -5 There were 3 gradients in total, and 0.2 mL of each was applied to CMC-Na medium and inverted at 24°C for 2-3 days until a single colony appeared. The selected microorganism was named LF-9 and was purified by continuous streaking with LB medium. The obtained strain LF-9 was stored in a -81°C ultra-low temperature refrigerator with 70% glycerol.

[0035] 2 Identification of strain LF-9

[0036] After culturing the strain LF-9 on the LB solid medium plate at 25°C for 48 h, the colony morphology of the strain LF-9 on the LB solid medium plate was observed. Figure 1 As shown in the figure, the colony is nearly round in shape, with a protruding wrinkle in the middle, sticky when picked up, and milky white in color. Pick a single colony of the strain to be identified in a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 1 min to collect the bacteria. Bacterial DNA was extracted using a bacterial genomic DNA extraction kit purchased from Tiangen Biochemical Technology Co., Ltd. For detailed steps, please refer to the product manual. Using 16S rDNA universal primers and yrA The primers gyrA-F and gyrA-R were used to amplify the 16S rDNA and yrAThe gene fragment was amplified by PCR using conventional methods. The PCR product was sequenced after being tested by 1% agarose gel electrophoresis. The obtained sequence was analyzed by NCBI online BLAST. Combined with the morphological characteristics, the strain LF-9 was identified as Bacillus velezsima ( Bacillus velezensis ).

[0037] Example 2 Preparation of Bacillus Velez LF-9 Fermentation Broth

[0038] 1. Plate activation

[0039] Bacillus Velezii LF-9 was inoculated on LB solid medium plates and cultured at 28°C for 3 days.

[0040] 2 Liquid fermentation

[0041] The culture medium formula is calculated by weight percentage, specifically: 1.5% tryptone, 0.58% yeast extract, 0.54% NaCl, and the rest is distilled water, and the pH value is 6.8.

[0042] The activated bacteria were inoculated into 250 mL Erlenmeyer flasks (100 mL per flask) and incubated at 27°C with a shaking speed of 150 r•min. -1 After 3 days of fermentation, the fermentation liquid of Bacillus Velez LF-9 was obtained. The effective viable cell count of Bacillus Velez LF-9 was 1×10 8 cfu / mL.

[0043] Test Example 1 Test on Cellulase Production by Bacillus Velezii LF-9

[0044] 1 Test method

[0045] The formula of enzyme production culture medium is: 3.0 g peptone, 3.0 g ammonium sulfate, 1.0 g potassium dihydrogen phosphate, 0.2 g magnesium sulfate, 5.0 g sodium chloride, and 1 L distilled water.

[0046] Bacillus Velez LF-9 was inoculated on CMC-Na medium by the spot inoculation method, with three replicates for each strain. After constant temperature culture at 27℃ for 72 h, 1 mg / mL Congo red solution was poured in for staining for 30 min. The Congo red solution was poured out and the culture was decolorized by soaking in 1 mol / L NaCl solution for 20 min. The ratio (D / d) of the transparent hydrolysis zone diameter (D) and the colony diameter (d) was calculated to preliminarily determine the cellulase production ability of Bacillus Velez LF-9.

[0047] A single colony of Bacillus Velezii LF-9 was inoculated into LB liquid medium at 35 ℃ and 160 r•min. -1After culturing for 24 h, 1 mL of bacterial solution was inoculated into 100 mL of enzyme production medium and incubated at 35 °C and 160 r•min. -1 After 24 h of shaking culture in a constant temperature shaker, samples were taken regularly to determine the enzyme activity of the fermentation broth, and the fermentation broth enzyme activity determination cycle was 168 h. The cellulase activity of Bacillus Velezii LF-9 was determined using a cellulase (CL) activity detection kit purchased from Beijing Box Biotechnology Co., Ltd. For detailed steps, please refer to the product manual.

[0048] 2 Test results

[0049] The preliminary results are as follows Figure 2 As shown, the D / d of Bacillus Velez LF-9 was 7.94, which had a strong ability to produce cellulase.

[0050] The results of cellulase activity assay were as follows Figure 3 As shown in the figure, with the extension of culture time, the cellulase activity of Bacillus Velez LF-9 showed a trend of first increasing and then approaching stability. The cellulase activity of Bacillus Velez LF-9 was the highest at 3 days of fermentation, reaching 235.897 U•mL -1 , and then began to decline and stabilize.

[0051] Test Example 2 Test of Bacillus Velez LF-9 in Composting Agricultural Waste

[0052] 1 Test method

[0053] Corn stalks and sheep manure were mixed in a mass ratio of 3:1, and then the effective viable bacteria count was 1×10 8The bacterial solution of Bacillus Velez LF-9 with a concentration of 320 mL / Kg was inoculated into the pile and mixed again. After being placed in a 25×25 cm flower pot, a 20 cm high compost fermentation pot was made. The initial moisture content of each group was adjusted to about 50% with sterile water, covered with plastic film, and cultured at 25°C for 30 days. Each treatment was repeated three times, namely N1, N2, and N3 treatments. The same amount of sterile water was added as a blank control (CK). The treatment group and the control group were placed in a 23°C constant temperature artificial climate room for fermentation. The pile was turned manually every 10 days during the fermentation process. When collecting samples, 10 g of compost samples were collected from the upper, middle, and lower parts of the pile 5 cm away from the surface of the pile, mixed evenly as one sample, placed in a sterile self-sealing bag, and quickly brought back to the laboratory for testing. The first sampling was carried out 3 hours after the pile was made as the 0 day sample. Thereafter, the central temperature of the compost fermentation was measured once every 5 days. Samples were taken on the first day (Incubation period, IP), the third day (Medium temperature period, MP), the ninth day (High temperature period, HP), and the 27th day (Rotten period, RP) of compost fermentation to test the maturity, central temperature, moisture content, organic matter, total nitrogen and carbon-nitrogen ratio (C / N) of the compost.

[0054] 2 Test results

[0055] The characterization test results during composting are as follows Figure 4 As shown in the figure, with the extension of fermentation time, the color of compost gradually deepened. After 30 days of composting, the color of the compost inoculated with Bacillus Velez LF-9 turned dark brown compared with the control, the straw was crushed to a high degree, the hand felt soft and delicate, and the volume of compost was significantly reduced. This shows that inoculation with Bacillus Velez LF-9 can accelerate the progress of compost fermentation and improve the quality of compost.

[0056] The change of the center temperature of the fermentation during the composting process can reflect the change of the activity of microorganisms in the compost. It can also reduce the survival rate of plant pathogens and pest eggs, and reduce the occurrence of plant diseases and insect pests. It is an important indicator for evaluating the quality of compost fermentation. Figure 5 As shown in the figure, after inoculation of Bacillus Velez LF-9, the temperature of the pile gradually increased, and the central temperature of the fermentation pile reached a maximum of 44.6℃ on the 5th day, indicating that the pile was in the high temperature period of fermentation. The central temperature of the fermentation fluctuated around 40℃ during the composting period of 1-10 days, and the cumulative high temperature period reached more than 8 days, reaching the harmless treatment standard of composting. After 10 days, the central temperature of the fermentation pile gradually decreased, and the pile entered the mature stage.

[0057] The results of the change of water content during composting are shown in Table 1. As the composting time increases, the water content of the piles in the treatment group inoculated with the fermentation liquid of Bacillus Velez LF-9 and the control group shows a downward trend overall, but the water content of the treatment group decreases more dramatically overall. The water content of the pile after the maturity period decreases by 19.38% compared with the water content of the pile during the latent period. This indicates that the microbial life activities in the pile are more intense during the composting process inoculated with Bacillus Velez LF-9, which consumes part of the water in the pile.

[0058] Table 1 Changes in water content in the compost pile at different composting periods

[0059]

[0060] Note: Data are expressed as mean ± standard deviation. Different lowercase letters after the data in the same column indicate significant differences (P < 0.05).

[0061] The results of organic matter content during composting are as follows Figure 6 -a shows that the trend of organic matter content in the treatment group inoculated with Velez Bacillus LF-9 and the control group is basically the same. The organic matter in the pile is rapidly decomposed during the latent period to the mesophilic period of composting, resulting in an increase in the content of water-soluble organic carbon, and thus an increase in the content of organic matter in the pile. After the high temperature period, the content of organic matter in the pile shows a downward trend. The increase in the degradation rate of organic matter degrades the organic matter in the pile into humus, and the downward trend of the content of organic matter in the pile of the treatment group is higher than that of the control group. Compared with the initial treatment period, the content of organic matter in the pile of the treatment group and the control group decreased by 20.88% and 12.02%, respectively.

[0062] The total nitrogen content during composting is as follows Figure 6 -b shows that the total nitrogen content in the treatment group inoculated with Bacillus Velez LF-9 and the control group showed opposite trends. The total nitrogen content in the pile of the treatment group showed an increasing trend with the extension of composting time. During the composting process, the total nitrogen content in the pile reached its peak around the high temperature period, and there was no significant difference in the total nitrogen content in the pile until the end of the maturity period (p>0.05), which was 40.36% higher than that in the initial period of composting; while the total nitrogen content in the pile of the control group showed a downward trend with the extension of composting time. At the end of the maturity period, the total nitrogen content in the pile of the control group reached the lowest, which was significantly lower than that in the initial period of composting, and decreased by 39.25%.

[0063] C / N is an important indicator to measure the progress of composting. The changes in C / N during composting are shown in Table 2. In the early stage of composting, the C / N in the pile showed an upward trend. When the C / N reached a suitable C / N for microbial growth, the conversion efficiency of ammonia in the pile gradually accelerated, which was higher than the decomposition efficiency of carbon. In the mature stage of composting, the C / N of the pile inoculated with Bacillus Velez LF-9 was 19.27, which had reached the standard of mature composting, while the C / N of the control pile was still at a high point, indicating that the inoculation of Bacillus Velez LF-9 can significantly accelerate the composting process.

[0064] Table 2 Changes of C / N in the compost pile at different composting periods

[0065]

[0066] Note: Data are expressed as mean ± standard deviation. Different lowercase letters after the data in the same column indicate significant differences (P < 0.05).

[0067] Test Example 3 Test of Bacillus Velezii LF-9 in promoting plant growth

[0068] 1 Test method

[0069] Salkowski colorimetric solution: Weigh 12 g of ferric sulfate and dissolve it in distilled water. Then slowly add 429.7 mL of concentrated sulfuric acid. After the solution cools, dilute to 1 L with distilled water. The measurement range is 0.3~20 mg / mL.

[0070] The formula of King'B medium is: 20 g peptone, 1.5 g magnesium sulfate heptahydrate, 1.5 g potassium dihydrogen phosphate, distilled water to 1000 mL, pH 7.0~7.2.

[0071] The classic Salkowski reagent colorimetric method was used to determine the IAA production capacity of Bacillus Velezii LF-9. A single colony was picked from the pure bacterial plate and inoculated into LB medium for activation. After a single colony grew out, a single colony was picked and inoculated into liquid LB medium to make (OD 660 =1) Bacterial suspension. Inoculate the bacterial suspension with 1% inoculation volume into King'B medium without tryptophan, and use sterile water as a control. Place the above-mentioned conical flask in a shaking incubator at 28°C and 160 rpm for 7 days. Take 1 mL of the bacterial suspension and drop it into the test tube. Add IAA to one of the test tubes as a control. Add equal amounts of Salkowski colorimetric solution. Place the test tubes at room temperature for 15 minutes to develop color. Observe the color change. The colorimetric results are compared with IAA and King'B medium without tryptophan with sterile water. The redder the solution color, the stronger the ability to produce IAA. If it does not change color, it is negative and does not produce IAA.

[0072] Punch 5 bacterial cakes of Bacillus Velezii LF-9 with a 5 mm diameter puncher and transfer them to a conical flask containing 200 mL LB liquid medium. Incubate at 27 °C and 180 r•min. -1 After culturing for 3 days under the same conditions, the excess bacterial cake and agar blocks were removed by filtering through double-layer gauze, and the fermentation liquid was diluted 100 times with sterile water for later use.

[0073] The virus-free potato tissue culture seedlings (Zhongjia No. 7) were transplanted into sterilized nutrient soil pots with a size of 11 cm × 9 cm. The sterile soil and sterilized vermiculite were mixed in a volume ratio of 1:1. When the seedlings grew to the 3-5 leaf stage, they were treated. The prepared Bacillus Velez LF-9 fermentation liquid was inoculated into the roots of the plants by the root injury inoculation method, 10 mL per plant, and the root irrigation treatment with an equal amount of blank LB liquid culture medium was used as a control. There were 12 pots per treatment. The temperature was maintained at 23°C and the relative humidity was 60%. The LED light source in the climate chamber was used for supplementary lighting. The light and dark conditions were alternating for 12 hours, and watering was performed every 5 days. After 30 days, the survey was conducted, the growth indicators were recorded, and the growth promotion effect was calculated.

[0074] After the surface of wheat seeds was disinfected, they were plated for germination. The specific steps of the surface disinfection were to select full and uniform seeds, place them in a 5% sodium hypochlorite solution for surface disinfection for 2 min, and then wash them with sterile water 3 times to remove residual sodium hypochlorite. After mixing nutrient-free sand and sterilized vermiculite in a volume ratio of 1:1, they were sterilized at 121°C for 4 h, and the soil moisture content was adjusted to about 60% with sterile water. After filling to two-thirds of the volume of a 11 cm×9 cm flower pot, 15 g of compost sample inoculated with Bacillus Velez LF-9 after fermentation for 27 days was added and evenly spread, and then 15 g of sterile sand was spread, and the sterile water compost sample after adding the same amount of compost for 27 days was used as the control. Different treatments were set up with 12 pots for repetition. The temperature was maintained at 25°C and the relative humidity was 65%. The LED light source in the climate chamber was used for supplementary lighting. The light and dark conditions were alternating for 12 h, and watering was done every 3 days. An investigation was conducted after 14 days, the growth indicators were recorded, and the growth-promoting effect was calculated.

[0075] 2 Test results

[0076] The results of IAA production test are as follows Figure 7 As shown: after adding Salkowski colorimetric solution to the bacterial solution of Bacillus Velez LF-9, the color of the fermentation solution turned light red, indicating that Bacillus Velez LF-9 has a certain ability to produce IAA.

[0077] The results of the growth promotion test on potato plants are shown in Table 3 and Figure 8As shown in the figure, the plant height, root length and fresh weight of potato plants increased by 23.36%, 18.07% and 70.31% respectively after inoculation with Bacillus Velez LF-9, indicating that Bacillus Velez LF-9 has a significant growth-promoting effect on potato plants.

[0078] Table 3 Growth-promoting effect of Bacillus Velezii LF-9 strain on potato plants

[0079]

[0080] The results of the indoor compound bacterial fertilizer growth promotion test are as follows Fig. 9 As shown in the figure, compared with the control group of sterile water compost sample added with compost 27 days later, the plant height and fresh weight of wheat plants in the treatment group of compost sample inoculated with Bacillus Velez LF-9 solution added with compost 27 days later increased significantly, and the wheat plants increased by 63.54%, 50.64% and 81.21% respectively. Compared with the treatment group, the leaves of the control group were yellowish. This shows that inoculation with Bacillus Velez LF-9 can shorten the compost fermentation cycle, improve the nutritional quality of compost, and significantly promote the growth of wheat plants.

[0081] Test Example 4 Test of Bacillus Velezii LF-9 in plant disease control

[0082] 1 Test method

[0083] Bacillus velez LF-9 was cultured in the plate activation step of Example 2 for later use; Rhizoctonia solani was inoculated on a potato dextrose medium (PDA) plate and cultured at 25° C. for 7 days; Erwinia carotovora subsp. luteola was cultured on an LB plate for later use.

[0084] The plate confrontation method was used. The activated Rhizoctonia solani was placed in the clean bench, and the pathogen cakes with a diameter of 5 mm were punched out with a hole puncher. They were placed in the center of the PDA plate, and Bacillus Velezii LF-9 was inoculated with an inoculation loop at 2.5 cm around the Rhizoctonia solani cakes. This was repeated 3 times. A PDA plate inoculated with only Rhizoctonia solani was set as a control (CK). The activated Erwinia carotovora subsp. potato blackleg was placed in the clean bench, 60 μL of the Erwinia carotovora subsp. potato blackleg was aspirated and inoculated in the LB plate. It was evenly spread with a spreader. Bacillus Velezii LF-9 was inoculated with an inoculation loop at 2.5 cm around the four sides. This was repeated 3 times. An LB plate inoculated with only Erwinia carotovora subsp. potato blackleg was set as a control (CK). After inoculation, the plate was sealed with a petri dish sealing film, placed in a 28℃ incubator and inverted for culture, and the colony growth was observed daily. The inhibition rate was investigated after 5 days.

[0085] The inhibition rate calculation formula is: inhibition rate (%) = [(control colony radius - treated colony radius) / control colony radius] × 100%.

[0086] The virus-free potato seedlings (Xisen No. 6) were transplanted into sterilized nutrient soil pots. The nutrient-free sandy soil and sterilized vermiculite were mixed in a volume ratio of 1:1 and sterilized at 121℃ for 4 h. The soil moisture content was adjusted to about 60% with sterile water. After filling to two-thirds of the volume of a 11 cm×9 cm pot, 15 g of the compost sample inoculated with Bacillus Velez LF-9 after fermentation for 27 days was added and evenly spread, and then 15 g of sterile sandy soil was spread. The sterile water compost sample after adding the same amount of compost for 27 days was used as the control. Different treatments were set up with 12 pots of replication. After 6 days, the spore suspension of Rhizoctonia solani was inoculated by the root injury inoculation method, 10 mL per plant. The temperature was maintained at 25℃, the relative humidity was 65%, the LED light source in the climate chamber was used for supplementary light, and the light and dark were alternating for 12 hours. Water was poured every 3 days. After 28 days, the disease was investigated, the disease grade and fresh weight were recorded, and the disease index and control effect were calculated.

[0087] The disease classification standard and the calculation of the disease index are shown in Table 4:

[0088] Disease index = [Σ (number of diseased plants at each level × representative value of relative level) / (total number of plants surveyed × 5)] × 100.

[0089] Table 4 Indoor grading standards for potato black mole

[0090]

[0091] 2 Test results

[0092] The results of the plate confrontation test are as follows: Bacillus Velezii LF-9 has a good inhibitory effect on Rhizoctonia solani and Erwinia carotovora subsp. Fig.10 As shown, the inhibition rate was 79.77%; the inhibition rate of Bacillus Velez LF-9 against Erwinia carotovora subsp. solanacearum was 80.78%.

[0093] Table 5 Control effect of agricultural waste compost on potato black mole disease after 27 days of composting

[0094]

[0095] The results of the control test of potato black mole disease by compost containing Bacillus Velez LF-9 bacterial solution are shown in Table 5. The compost of agricultural waste inoculated with Bacillus Velez LF-9 bacterial solution 27 days later had a good control effect on potato black mole disease, with a control effect of 79.37%. At the same time, it also had a certain promoting effect on the growth of potato plants.

Claims

1. A strain of Bacillus velez Bacillus velezensis ) LF-9, characterized in that, Its microbial preservation number is: CGMCC NO.29001.

2. A fermentation broth, characterized in that: The fermentation broth is prepared by fermenting the Bacillus Velez subtilis LF-9 described in claim 1.

3. A method for preparing the fermentation broth according to claim 2, characterized in that: include: The strain of Bacillus Velez LF-9 described in claim 1 is activated and inoculated into a liquid culture medium for fermentation to obtain a fermentation liquid; wherein the liquid culture medium is composed of: in mass percentage, 1.5% tryptone, 0.58% yeast extract, 0.54% NaCl, and the remainder is distilled water, and the pH value is 6.

8.

4. Use of the Bacillus Velez LF-9 according to claim 1 in the production of cellulase or IAA, or use of the fermentation broth according to claim 2 in the production of cellulase or IAA.

5. Use of the Bacillus Velez LF-9 of claim 1 in preparing agricultural waste compost, or use of the fermentation liquid of claim 2 in preparing agricultural waste compost; wherein, The agricultural waste includes corn stalks or sheep dung.

6. Use of the Bacillus Velez LF-9 of claim 1 in promoting plant growth, or use of the fermentation liquid of claim 2 in promoting plant growth; wherein, The plant is potato or wheat.

7. The use according to claim 6, characterized in that: The promoting plant growth includes increasing the plant height, root length or fresh weight of the plant; wherein the fresh weight refers to the weight of the entire plant.

8. Use of the Bacillus Velez LF-9 of claim 1 in preventing and controlling plant diseases, or use of the fermentation liquid of claim 2 in preventing and controlling plant diseases; wherein: The plant disease is potato black spot disease caused by Rhizoctonia solani or potato black shank disease caused by Erwinia carotovora subsp.

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