CN111100820B - Bacillus belgii HMB28023 and application thereof - Google Patents
Bacillus belgii HMB28023 and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological control, and particularly discloses a Bacillus velezensis strain HMB28023 with the preservation number of CGMCC No.19002 and a microbial agent containing the strain, wherein the HMB28023 strain has the prevention effect on botrytis cinerea of more than 84.39 percent, has a wide prevention spectrum, has high prevention effect on botrytis cinerea, also has good prevention effect on Fusarium oxysporum, Phyllostachys malorum, Poplar rot, walnut ulcer and Fusarium Juglandis, has strong specificity on botrytis cinerea and is difficult to generate drug resistance, and is safe to people and livestock and free of environmental pollution.
Description
Technical Field
The invention belongs to the field of biological control, and particularly relates to Bacillus belgii, a microbial agent produced by using the Bacillus belgii, and application of the Bacillus belgii and the microbial agent.
Background
Grape gray mold is a fungal disease caused by botrytis cinerea (borttytis cinerea), and multiple parts of the grape, such as inflorescences, crowns, fruits, young stems and leaves, can develop diseases, so that the grape loss caused by the grape gray mold is 20-30% each year, and is seriously more than 50%, and therefore, the grape gray mold has serious harm to grape production.
The prevention and control of grape gray mold mainly adopts chemical agents, cultivation measures, biological prevention and control methods and the like. The chemical prevention and control has good prevention and control effect, but has the problems of environmental pollution, easy generation of drug resistance and the like. The prevention and control effect of adopting cultivation measures is poor. Biological control is increasingly favored because of the advantages of high control efficiency, strong specificity, good drug effect durability, environmental friendliness, difficult generation of drug resistance and the like. At present, the microorganisms used for preventing and treating grape gray mold are mainly: bacillus subtilis (CN102876605A, CN106818881A, CN101870959A), Lactobacillus plantarum (CN108220212A, CN108522647A), Bacillus amyloliquefaciens (CN103243044A), Paenibacillus polymyxa (CN104694446A), actinomycetes (CN106434499A), Saccharomyces cerevisiae (CN107904181A), Streptomyces corchorusii (CN102899356A), Hansenula botryis cinerea (CN107868759A), Pichia pastoris (CN107988088A, CN107937289A), Metschnikowia (CN107904180A) and Saccharomyces cerevisiae (CN 107881122A).
Bacillus velezensis belongs to a new species of Bacillus, and is currently used for preventing and treating dozens of diseases such as wheat sharp eyespot, wheat scab, sclerotinia rot of colza, root rot of hot pepper, fusarium wilt of kidney bean, alternaria alternata, banana fusarium wilt, rice blast and the like, but no report for preventing and treating grape gray mold is found.
Disclosure of Invention
Aiming at the damage of grape gray mold to grapes and the like, the invention provides a Bacillus velezensis (Bacillus velezensis) strain HMB28023 which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 19002.
The invention also provides application of the Bacillus velezensis HMB28023 in prevention and treatment of Bortrytis cinerea, Blakeslea tabescens (Sphaeropsis sapienea), Verticillium malorum (Botryosphaeria bereniana), Populus aspergillum (Valsa sordida), Juglans canker (Dothiorella gregaria) or Juglans regia (Cercospora insulana).
The invention also provides a microbial agent which contains the bacillus belgii HMB28023 bacteria and spores.
The microbial agent is a liquid preparation.
The invention also provides a preparation method of the microbial agent, which comprises the following steps:
(1) activating strains: activating the HMB28023 strain stored at low temperature on an LB plate culture medium, selecting a single colony on an LB slant culture medium, and culturing at 25-35 ℃ for 10-16 hours to obtain an activated strain;
(2) preparing a seed solution: scraping a ring of the activated strain obtained in the step (1) by using a sterile inoculating ring, inoculating the ring into 100mL of LB liquid culture medium, and culturing for 10-16 hours at the temperature of 25-35 ℃ and the rotating speed of a shaking table of 150-220 rpm to obtain seed liquid;
(3) fermentation culture: inoculating the seed solution obtained in the step (2) into a corn flour and soybean meal culture medium with the pH value of 7.0 according to the volume ratio of 1-3%, and performing fermentation culture for 35-40 h at the temperature of 25-35 ℃ and the rotating speed of a shaking table of 150-220 rpm to obtain a fermentation liquid;
(4) detecting the quantity of bacteria and spores in the fermentation liquid, and stopping fermentation culture when mature spores in the fermentation liquid account for 95 percent of the total quantity of the spores and the bacteria; the obtained product is a liquid preparation of HMB 28023.
The LB plate culture medium or LB inclined plane culture medium in the step (1) of the preparation method comprises the following components in percentage by weight: 8-12 g of tryptone, 4-6 g of yeast extract, 4-6 g of sodium chloride, 12-18 g of agar powder and 1000mL of water.
The LB liquid culture medium in the step (2) of the preparation method comprises the following components in parts by weight: 8-12 g of tryptone, 4-6 g of yeast extract, 4-6 g of sodium chloride and 1000mL of water.
The LB plate culture medium, the LB slant culture medium and the LB liquid culture medium are all prepared according to a conventional method.
The corn meal and soybean meal culture medium in the step (3) of the preparation method comprises the following components in percentage by weight: 1.0-3.0% of corn flour, 1.0-3.0% of soybean flour, 0.1-0.8% of NaCl, and MnSO4·H20.5-1.0% of O and the balance of water.
The preparation method of the corn flour and soybean meal culture medium comprises the steps of mixing corn flour, soybean meal, NaCl and MnSO in percentage by weight4·H2And O, mixing, adding water, adjusting the pH value, and uniformly stirring.
The invention also provides application of the microbial agent in preventing and treating botrytis cinerea (Bortrytis cinerea), sphacelotheca tabaci (Sphaeropsis sapienea), apple ring spot bacteria (Botryospora bergeriana), poplar rot bacteria (Valsa sordida), walnut canker bacteria (Dothiorella gregaria) or walnut brown spot bacteria (Cercospora insulana).
The use method of the microbial agent comprises the following steps: diluting the obtained microbial agent with water until viable cell number is 107cfu/mL, and the purpose of preventing the grape gray mold can be achieved by spraying the leaf surfaces 2 weeks before the grape gray mold occurs.
The invention also provides a molecular marker for identifying the strain HMB28023, which consists of a nucleotide sequence shown in SEQ ID No.1 or SEQ ID No. 3.
The invention also provides a genetically engineered bacterium, which is obtained by taking HMB28023 as a spawn, and has the function of preventing and treating grape gray mold.
The invention has the advantages and beneficial effects that: (1) the bacterial strain HMB28023 has high control effect on botrytis cinerea, and the average control effect is more than 84.39%; (2) the strain HMB28023 has strong specificity to botrytis cinerea and is not easy to generate drug resistance; (3) the strain HMB28023 has a wide control spectrum, has a high control effect on grape gray mold, and also has a good control effect on Chinese pine blight, apple ring rot, poplar canker, walnut canker and walnut brown spot; (4) the microbial agent is safe to people and livestock, and has no problem of environmental pollution; (5) the preparation method is simple, low in cost and simple to use.
And (3) biological preservation: the Bacillus velezensis (Bacillus velezensis) strain HMB28023 is preserved in the China general microbiological culture Collection center in 2019 in 11 and 25 months, and the preservation addresses are as follows: the microbial research institute of China academy of sciences No.3, Xilu No.1, Beijing, Chaoyang, with the collection number of CGMCC No. 19002.
Drawings
FIG. 1 is a phylogenetic tree of the HMB28023 strain obtained from the 16S rDNA sequence.
FIG. 2 is a phylogenetic tree of HMB28023 strain obtained based on the gyrB gene sequence.
FIG. 3 is a phylogenetic tree of the HMB28023 strain obtained from the ropB gene sequence.
Detailed Description
The invention is further illustrated by the following specific examples, which are not to be construed as limiting in any way. The experimental methods in the following examples are all conventional methods unless otherwise specified; the percentages in the following examples are by weight unless otherwise specified.
Example 1 screening isolation procedure for HMB28023 Strain
In 2013, 16 days to 20 days in 1 month, 22 parts of glacier lowland, grassland, lakeside, valley gorge and mountain estuary are collected by plant protection research institute of agriculture and forestry academy of sciences in Hebei province from 9 counties such as Rizhi city Milin county, Gongbu county, Gongjiang county, Rituzi county, Ringbu county, Razi county, Shannan city Liangka county, Lasa city Qushui county, Mozhucai county and the likeTaking various types of soil samples back to a laboratory, respectively weighing 1g of soil samples, putting the soil samples into a 250mL sterilized triangular flask, adding 100mL of sterile water, putting the sterilized triangular flask on a shaker, oscillating the sterilized triangular flask at 170rpm for 30min, standing the sterilized triangular flask for 2h, taking 10mL of supernate, adding the supernate into a 50mL sterilized centrifuge tube, carrying out constant-temperature water bath at 80 ℃ for 30min, then taking 1mL of soil samples, adding 9mL of sterile water, and obtaining 10mL10-3Multiplying the soil microorganism suspension, and then diluting the soil suspension to 10-4、10-5、10-6Taking 200 mu L of microbial suspension with each concentration of the dilution liquid, coating the microbial suspension on an LB culture medium plate, repeating each concentration for 3 times, culturing for 1 d-3 d at the constant temperature of 30 ℃, and separating and purifying bacteria. And screening biocontrol bacteria by using grape gray mold as a target through a flat plate confronting method and a pot experiment method. As a result, a strain with good control effect on grape gray mold was selected from 1805 strains of soil bacteria and named as HMB 28023.
Example 2 taxonomic identification of HMB28023 strain:
(1) morphological characterization
The thallus is in a rod shape and cultured on an LB culture medium for 10 hours to generate spores which are Zhongsheng, oval, no cyst expansion, negative acid-fast staining, no parasporal crystal, motion and flagellum perigenesis. On a nutrient agar plate, the colony is light milky white, purulent, round and neat in edge at the initial culture stage, the colony is raised into a steamed bun shape, and the surface is moist; the bacterial colony in the later culture period is light yellow, the edge is irregular, and the surface is dry and has wrinkles; carrying out streak culture on a nutrient agar inclined plane to form a straight line shape; the white mycoderm is formed on the surface of the culture medium by static culture in the liquid culture medium. These morphological characteristics were substantially identical to those of Bacillus described in the handbook of identification of common bacterial systems (east elegans bead et al, science publishers, 2001), and it was preliminarily judged that the HMB28023 strain belongs to Bacillus.
(2) Identification and classification using 16S rDNA sequences
Performing PCR amplification by using genome DNA of HMB28023 as a template and universal primers F27 and R1492 as primers to obtain a PCR amplification product; wherein the primer sequence is as follows:
27F：5’-AGAGTTTGATCATGGCTCAG-3’
R1492：5’-GGCTACCTTGTTACGACTT-3’
the 16S rDNA PCR reaction system (50. mu.L) was 10 XPCR Buffer (Mg)2+)5 mu L of the solution; dNTP mix (2.5mM) 5. mu.L; 1 μ L of Taq (5U/. mu.L), 1 μ L of 27F (10 μmol/L), and 1 μ L of R1492(10 μmol/L); HMB28023 genomic DNA 50 ng; ddH2O make up to 50. mu.L. The reaction condition of PCR is 95 ℃ for 5 min; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1.5 min; 10min at 72 ℃. And carrying out gel electrophoresis on the obtained PCR amplification product, and sending the product to Shanghai biological engineering Co., Ltd for sequencing to obtain a 16S rDNA sequence (shown as SEQ ID No.1) of the HMB 28023. The 16S rDNA sequence of the obtained HMB28023 is subjected to homology comparison in Genbank, and as a result, the homology of the strain HMB28023 and the 16S rDNA of the bacillus reaches 99.91 percent; meanwhile, a phylogenetic tree was constructed using MEGA software (Molecular evolution Genetics Analysis), and as a result (see FIG. 1), HMB28023 was aggregated with Bacillus, indicating that HMB28023 belongs to Bacillus.
(3) Identification and classification using gyrB gene sequences
Carrying out PCR amplification by using HMB28023 genome DNA as a template and degenerate primers gyrB-F and gyrB-R of a gyrB gene of bacillus as primers to obtain a PCR amplification product; wherein the sequences of the gyrB-F primers and the gyrB-R primers are as follows: gyrB-F: 5 '-TTGRCGGHRGYGGHTATAAAGT-3', gyrB-R: 5 '-TCCDCCSTCAGARTCWCCCTC-3'.
The PCR amplification reaction system (50. mu.L) of gyrB was 10 XPCR Buffer (Mg)2+)5 mu L of the solution; dNTP mix (2.5mM) 5. mu.L; 1 μ L of Taq (5U/. mu.L); 1 μ L of gyrB-F (10 μmol/L), 1 μ L of gyrB-R (10 μmol/L); HMB28023 genomic DNA 50 ng; ddH2O make up to 50. mu.L. The reaction condition of PCR is 95 ℃ for 5 min; 30 cycles of 95 ℃ for 30s, 55 ℃ for 45s, and 72 ℃ for 1 min; 10min at 72 ℃. And (3) delivering the amplified product to Shanghai biological engineering Co., Ltd for sequencing to obtain the gyrB gene sequence (shown as SEQ ID No.2) of the HMB28023 strain. The gyrB gene sequences of the obtained HMB28023 strain were subjected to homology comparison in Genbank. The result shows that the sequence homology of HMB28023 and gyrB genes of Bacillus belgii strains UCMB5007, UCMB5044 and VCC-2003 is the highest and reaches 99.89%; at the same time, MEGA software (Molecular evolution Genetics Analysis, Molecular Analysis) was usedEvolutionary genetic analysis) and the results (see fig. 2) that the HMB28023 strain was aggregated with Bacillus belgii, indicating that HMB28023 is Bacillus belgii (Bacillus velezensis) and is a new strain, unlike known Bacillus belgii strains.
(3) Identification of classes Using rpoB Gene sequences
Carrying out PCR amplification by using HMB28023 genome DNA as a template and primers rpoB1206-F and rpoB3202-R of a bacillus rpoB gene as primers to obtain a PCR amplification product; wherein the sequence of the primer is as follows: rpoB1206-F: 5'-ATCGAAACGCCTGAAGGTCCAAACAT-3', rpoB3202-R: 5'-ACACCCTTGTTACCGTGACGACC-3'.
The PCR amplification reaction system (50. mu.L) for rpoB was 10 XPCR Buffer (Mg)2+)5 mu L of the solution; dNTP mix (2.5mM) 5. mu.L; 1 μ L of Taq (5U/. mu.L); rpoB1206-F (10. mu. mol/L) 1. mu.L, rpoB3202-R (10. mu. mol/L) 1. mu.L; HMB28023 genomic DNA 50 ng; ddH2O make up to 50. mu.L. The reaction condition of PCR is 95 ℃ for 5 min; 30 cycles of 95 ℃ for 30s, 60 ℃ for 45s, and 72 ℃ for 2 min; 10min at 72 ℃. And (3) delivering the amplified product to Shanghai biological engineering Co., Ltd for sequencing to obtain the rpoB gene sequence of the HMB28023 strain (see SEQ ID No. 3). The sequence of rpoB gene of the obtained HMB28023 strain is subjected to homology comparison in Genbank, and the result shows that the sequence homology of the obtained HMB28023 with rpoB gene of B.beilai strains BH21, K2 and LC1 is the highest and reaches 99.63 percent; meanwhile, a phylogenetic tree is constructed by using MEGA software (Molecular evolution Genetics Analysis), and the result (shown in figure 3) shows that the HMB28023 strain is polymerized with Bacillus belgii, which indicates that the HMB28023 is Bacillus belgii (Bacillus velezensis) and is a new strain.
By combining the morphological characteristics, the results of sequence homology comparison analysis of 16S rDNA, gyrB and rpoB genes, it is known that HMB28023 belongs to Bacillus belgii (Bacillus velezensis), and is a new Bacillus belgii strain different from the existing Bacillus belgii strain.
Example 3 preparation of HMB28023 microbial Agents
The method comprises the following steps:
(1) activating strains: activating (30 ℃) a Belgium strain HMB28023 (the strain HMB28023 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.19002) stored at minus 80 ℃ on an LB plate culture medium (the components and the weight ratio of the components are 1g of tryptone, 0.5g of yeast extract, 1g of sodium chloride, 15g of agar powder and 1000mL of water), and picking a single bacterium to fall on an LB slant culture medium (the components and the weight ratio of the components are 1g of tryptone, 0.5g of yeast extract, 1g of sodium chloride, 15g of agar powder and 1000mL of water) to culture at 30 ℃ for 30 hours to obtain an activated strain;
(2) preparing a seed solution: preparing an LB liquid culture medium (the components and the weight ratio thereof are 1g of tryptone, 0.5g of yeast extract, 1g of sodium chloride and 1000mL of water) according to a conventional method, filling 100mL of LB culture solution into a 250mL triangular flask, carrying out high-pressure damp-heat sterilization, inoculating a strain activated in the inoculating loop step (1) into each flask after the temperature is reduced to room temperature, and carrying out shaking culture for 24 hours at the temperature of 30 ℃ and the rotation speed of a shaking table of 190rpm to obtain a seed solution;
(3) preparing a corn flour and soybean flour culture medium: according to the weight percentage, 1.5 percent of corn flour, 2.0 percent of soybean meal, 0.5 percent of NaCl and MnSO4·H2Adding 0.6% of O into water, and stirring and mixing uniformly to obtain a corn flour and soybean meal culture medium; subpackaging in 500mL triangular bottles, 200mL each; sterilizing the corn flour and soybean meal culture medium at 121 ℃ for 30 minutes, and then cooling to 30 ℃ for later use;
(4) fermentation culture: inoculating 2mL of the seed solution obtained in the step (2) into 200mL of the culture medium of each bottle of corn flour and soybean meal obtained in the step (3); carrying out fermentation culture at 30 ℃ and the rotation speed of a shaking table of 180rpm for 36h, sampling from a triangular flask at intervals of 30 minutes, carrying out microscopic examination, counting spores and total thallus in a visual field, and calculating the spore rate (%) -mature spore number/(mature spore number + thallus number) × 100); stopping fermentation culture when the spore rate reaches 90%; and performing co-fermentation culture for about 48 hours to obtain the liquid preparation of the Bacillus belgii HMB 28023.
Example 4 antagonism of HMB28023 Strain against Botrytis cinerea
(I) the sources of botrytis cinerea to be tested: the botrytis cinerea GBC-1 strain is collected from botrytis cinerea leaves in greenhouse of Manchu villages in North of Shanghai county of Hebei province, is separated and purified by a plant protection research institute of the academy of agriculture and forestry of Hebei province, and is identified as botrytis cinerea (Bortrytis cinerea) as a plant pathological system of the plant protection academy of Hebei agriculture university, and the pathogenicity is determined to be strong pathogenicity.
(II) test method:
plate confrontation experiment: firstly, the botrytis cinerea GBC-1 strain is activated and cultured for 5 days on a PDA (personal digital assistant) plate, and then a hole puncher is used for
The edge area of the colony was punched to make a plate, the Botrytis cinerea plate was transferred to the center of another PDA plate, and the activated Bacillus belgii HMB28023 of step (1) of example 3 was spotted at a distance of 2.0 cm from the indicator plate, and a blank control (no spot of HMB28023 strain) was set. Culturing at constant temperature of 25 deg.C, measuring control growth amount (colony radius) and treatment growth amount (growth inhibiting radius after inoculating HMB 28023) of Botrytis cinerea when blank control is about to grow on the whole culture dish, and expressing antagonistic action by antibacterial rate.
The bacteriostatic rate (%) (control growth amount-treated growth amount)/control growth amount × 100.
Results (see table 1) the inhibition rate of bacillus belgii HMB28023 against botrytis cinerea reached 73.7%; the transparent bacteriostatic bandwidth is 8.7 mm; the bacillus beilesensis HMB28023 has obvious inhibition effect on botrytis cinerea and has biocontrol potential for preventing and treating botrytis cinerea.
TABLE 1 results of experiments on the antagonistic action of the HMB28023 strain on Botrytis viticola
Example 5 comparative experiment of in vitro control effect of HMB28023 strain and its microbial inoculum on grape gray mold
(I) test treatment:
(1) a 100-fold aqueous dilution of the HMB28023 liquid formulation prepared in example 3.
(2) Blank control: and (4) clear water.
(II) test method:
the test is carried out in a plant disease biocontrol laboratory of plant protection research institute of academy of agriculture and forestry in Hebei province.
(1) Cultivating grape plants: and (3) filling garden soil in a plastic flowerpot with the diameter of 30cm and the depth of 20cm, planting the Victoria grape seedlings, and normally managing the greenhouse. And (3) supporting and fixing grape seedlings for culture by using bamboo poles, and pinching off young buds to ensure the vegetative growth of the plants.
(2) Preparing a grape botrytis cinerea bacterial disc: activating and culturing Botrytis cinerea GBC-1 strain on PDA plate for 5 days, and performing cell-punching with a puncher
And punching holes on the edge area of the bacterial colony to prepare bacterial sheets for later use.
(3) The in vitro leaf method is used for determining the prevention and treatment effect: healthy grape leaves with consistent growth are selected, washed clean with sterile water, soaked in 100-fold aqueous dilution of the HMB28023 liquid preparation prepared in example 3 for 30 minutes, then the petioles are wrapped with absorbent cotton with the leaf back facing up, and placed in a petri dish with sterile wet gauze pads
In the culture dish, one leaf is placed in each culture dish, sealed by a preservative film, and cultured for 1d at 25 ℃. And (3) inoculating the botrytis cinerea slices cultured in the step (2) at the main veins of the leaf back, sealing again, and culturing at 25 ℃. Observing every day, measuring the diameter of the lesion spots by adopting cross phase multiplication after the contrast is fully developed, and calculating the area of the lesion spots. Blank controls were run and 6 replicates of each treatment were run.
Area of lesion (mm)2) Length of lesion (mm) × width of lesion (mm) × 3.14/4
Control effect (%) - (control lesion area-treatment lesion area)/control lesion area x 100
Results (see Table 2) HMB28023 liquid formulation handlingThe area of the lesion is 83.18mm2Significantly lower lesion area than the blank (408.20 mm)2) The prevention effect of the HMB28023 liquid preparation on grape gray mold is 79.62%. The Bacillus belgii strain HMB28023 and the HMB28023 liquid preparation have an outstanding prevention and treatment effect on grape gray mold.
TABLE 2 comparative test results of the prevention of botrytis of the HMB28023 strain of the invention on grape
| Treatment of | Area of lesion (mm)2) | Control effect (%) |
| HMB28023 liquid formulations | 83.18b | 79.62 |
| Blank control | 408.20a | -- |
Example 6 comparative test of prevention and control effects of HMB28023 strain and its microbial inoculum on grape gray mold potted plant
(I) test treatment:
(1) a 100-fold aqueous dilution of the HMB28023 liquid formulation prepared in example 3.
(2) Chemical agents: 42.8% Fluopyram-trifloxystrobin suspension (Lunason) 1000-fold aqueous dilution.
(3) Blank control: and (4) clear water.
(II) test method:
the test is carried out in a plant disease biocontrol laboratory of plant protection research institute of academy of agriculture and forestry in Hebei province.
(1) Cultivating grape plants: and (3) filling garden soil in a plastic flowerpot with the diameter of 30cm and the depth of 20cm, planting two grape seedlings of summer black and the vine root, and normally managing in a greenhouse. And (3) supporting and fixing grape seedlings for culture by using bamboo poles, and pinching off young buds to ensure the vegetative growth of the plants.
(2) Preparing a grape botrytis cinerea bacterial disc: activating and culturing Botrytis cinerea GBC-1 strain on PDA plate for 5 days, and performing cell-punching with a puncher
And punching holes on the edge area of the bacterial colony to prepare bacterial sheets for later use.
(3) The pot culture method is used for determining the prevention and treatment effect: selecting healthy grape leaves growing consistently on grape plants, spraying sterile water to wash the surfaces of the leaves, spraying 100 times of water diluent of the HMB28023 liquid preparation prepared in the embodiment 3 after drying, inoculating the botrytis cinerea bacterial tablets cultured in the step (2) on the grape leaves after 24 hours, controlling the temperature to be 20-25 ℃ and the humidity to be about 90%. Chemical control and blank control were set up with 6 replicates of each treatment. Observing every day, measuring the diameter of the lesion spots by adopting cross phase multiplication after the contrast is fully developed, and calculating the area of the lesion spots.
Area of lesion (mm)2) Length of lesion (mm) × width of lesion (mm) × 3.14/4.
Control effect (%) - (control lesion area-treatment lesion area)/control lesion area x 100
Results (see Table 3) on summer black grapes, the HMB28023 liquid formulation treated the lesion area to 19.49mm2Significantly lower lesion area than chemical control (88.61 mm)2) The lesion area is extremely significantly lower than that of the blank control (246.40 mm)2) The prevention effect of the HMB28023 liquid preparation on gray mold of summer black grapes is 92.09%; the area of lesion of HMB28023 liquid preparation on the Sida grape is 61.09mm2Lesion area (64.94 mm) in contrast to chemical agents2) The difference is not significant and is significantly lower than the lesion area of the blank control (264.87 mm)2) HMB28023 liquid formulation on the ash of Sida grapeThe control effect of the mildew is 76.94 percent. The Bacillus belgii strain HMB28023 and the HMB28388 liquid preparation thereof have an outstanding prevention and treatment effect on grape gray mold.
TABLE 3 comparative test results of Bacillus belgii HMB28023 on control of grape gray mold
Example 7 comparative experiment of the HMB28023 Strain and its inoculum on the control of Botrytis cinerea
(I) test treatment:
(1) a 100-fold aqueous dilution of the HMB28023 liquid formulation prepared in example 3.
(2) Chemical agents: 42.8% Fluopyram-trifloxystrobin suspension (Lunason) 1000-fold aqueous dilution.
(3) Blank control: and (4) clear water.
(II) test method:
(1) the test is carried out in a facility cultivation grape garden in the north of the Hebei province Heyushui city, Shanyang county, north of the Hehou province, the Heyushui county, the Heyuanhui county and the village. The grape variety is Victoria, and is normally managed in the field. In 2019, month 4, this test was performed at the initial onset of grape gray mold.
(2) Preparing a grape botrytis cinerea bacterial disc: activating and culturing Botrytis cinerea GBC-1 strain on PDA plate for 5 days, and performing cell-punching with a puncher
And punching holes on the edge area of the bacterial colony to prepare bacterial sheets for later use.
(3) And (3) field test: the grape gray mold fungus plate prepared in the step (2) is inoculated after 100 times of water dilution of the HMB28023 liquid preparation prepared in the example 3 is sprayed, and then the grape gray mold fungus plate prepared in the step (2) is inoculated after 24 hours, and then the plastic packaging bag of 220mm multiplied by 280mm is used for sealing and moisturizing. Chemical control and blank control were set for 6 replicates per treatment. Observing every day, measuring the diameter of the lesion spots by adopting cross phase multiplication after the contrast is fully developed, and calculating the area of the lesion spots.
Area of lesion (mm)2) Length of lesion (mm) × width of lesion (mm) × 3.14/4.
Control effect (%) - (control lesion area-treatment lesion area)/control lesion area x 100
Results (see Table 4) the HMB28023 liquid formulation treated the lesion area at 26.82mm2Lesion area (20.80 mm) in contrast to chemical agents2) The difference is not significant and is significantly lower than the lesion area of the blank control (241.49 mm)2) The prevention effect of the HMB28023 liquid preparation on the grape gray mold is 88.89%. The Bacillus belgii strain HMB28023 and the HMB28023 liquid preparation have an outstanding prevention and treatment effect on grape gray mold.
TABLE 4 comparative test results of field control effect of HMB28023 strain and its microbial inoculum on grape gray mold
| Treatment of | Area of lesion (mm)2) | Control effect (%) |
| HMB28023 liquid formulations | 26.82b | 88.89 |
| Chemical agent | 20.80b | 91.39 |
| Blank control | 241.49a | -- |
Example 8 test of the inhibitory Effect of HMB28023 Strain on five pathogenic strains of fruit Tree
The test is carried out in a plant disease biological control laboratory of plant protection research institute of agriculture and forestry academy of sciences in Hebei province in 2018 in 5 months.
(II) test method:
(1) pathogenic fungi and sources of diseases to be tested: the Sclerotinia tabescens (Sphaeropsis sapienea), Phyllostachys malus (Botryospora berengiana), Populus canker (Valsa sordida), Juglans canker (Dothiorella gregaria) and Juglans regia (Cercospora insulana) tested in this example were provided by the woodpathology laboratory of the university of agriculture in Hebei.
(2) Plate determination test:
firstly, the pathogenic fungi to be tested are activated on a PDA plate, cultured for 3-7 days and then arranged in the edge area of a colony, and a hole puncher is used
A plate was punched out, and the test pathogenic fungi plate was transferred to the center of another PDA plate, and then the activated Bacillus belgii HMB28023 in step (1) of example 3 was spotted at a distance of 2.0 cm from the indicator plate, and a blank control (without spotting the HMB28023 strain) was set. Culturing at constant temperature of 25 deg.C for 3-7 days, observing growth conditions of HMB28023 strain and pathogenic fungi to be tested day by day, observing and recording growth radius and width of antibacterial band of control pathogenic bacteria when the control pathogenic bacteria grow to the edge of the culture dish. The "-" indicates that the HMB28023 strain has no bacteriostatic effect; "+" indicates that the HMB28023 strain has 1.00-5.00mm of antibacterial zone and has weak antibacterial effect; "+ +" indicates that the HMB28023 strain has a moderate bacteriostatic action of 6.00-10.00 mm; the "+++" shows that the HMB28023 strain has 11.00-15.00mm of antibacterial zone and strong antibacterial effect; the "+++" shows that the HMB28023 strain has 16.00-20.00mm of antibacterial zone and has super antibacterial effect.
TABLE 5 test results of the inhibition of five pathogenic fungi for fruit tree diseases by HMB28023 Strain
Results (see table 5) the bacillus beleisi HMB28023 has more than moderate bacteriostatic action on five tested important forest fruit disease pathogenic bacteria, has strong bacteriostatic action on apple ring rot and has super strong bacteriostatic action on poplar rot. It is shown that HMB28023 has strong bacteriostatic ability on Ralstonia tabescens (Sphaeropsis apinea), Verticillium pomonella (Botryospuaera berengiana), Populus asperatus (Valsa sordida), Juglans regia (Dothiorella gregaria) and Juglans regia (Cercospora insulana), i.e. has good biocontrol potential for the above 5 kinds of forest and fruit diseases.
Sequence listing
<110> institute of plant protection of academy of agriculture, forestry and science of Hebei province
<120> Bacillus belgii HMB28023 and application thereof
<130> 2020S1700IHCY
<141> 2020-01-12
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1069
<212> DNA
<213> Bacillus velezensis
<400> 1
ggaactggcg ggcgtgctat acatgcaagt cgagcggaca gatgggagct tgctccctga 60
tgttagcggc ggacgggtga gtaacacgtg ggtaacctgc ctgtaagact gggataactc 120
cgggaaaccg gggctaatac cggatggttg tttgaaccgc atggttcaga cataaaaggt 180
ggcttcggct accacttaca gatggacccg cggcgcatta gctagttggt gaggtaacgg 240
ctcaccaagg cgacgatgcg tagccgacct gagagggtga tcggccacac tgggactgag 300
acacggccca gactcctacg ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc 360
tgacggagca acgccgcgtg agtgatgaag gttttcggat cgtaaagctc tgttgttagg 420
gaagaacaag tgccgttcaa atagggcggc accttgacgg tacctaacca gaaagccacg 480
gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttgtc cggaattatt 540
gggcgtaaag ggctcgcagg cggtttctta agtctgatgt gaaagccccc ggctcaaccg 600
gggagggtca ttggaaactg gggaacttga gtgcagaaga ggagagtgga attccacgtg 660
tagcggtgaa atgcgtagag atgtggagga acaccagtgg cgaaggcgac tctctggtct 720
gtaactgacg ctgaggagcg aaagcgtggg gagcgaacag gattagatac cctggtagtc 780
cacgccgtaa acgatgagtg ctaagtgtta gggggtttcc gccccttagt gctgcagcta 840
acgcattaag cactccgcct ggggagtacg gtcgcaagac tgaaactcaa aggaattgac 900
gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga agaaccttac 960
caggtcttga catcctctga caatcctaga gataggacgt ccccttcggg ggcagagtga 1020
caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg ttgggttaa 1069
<210> 2
<211> 876
<212> DNA
<213> Bacillus velezensis
<400> 2
ggcgtctgtc gtaacgcctt gtcgaccact cttgacgtta cggttcatcg tgacggaaaa 60
atccactatc aggcgtacga gcgcggtgta cctgtggccg atcttgaagt gatcggtgat 120
actgataaga ccggaacgat tacgcacttc gttccggatc cggaaatttt caaagaaaca 180
accgaatacg actatgacct gctttcaaac cgtgtccggg aattggcctt cctgacaaaa 240
ggtgtaaaca tcacgattga agacaaacgt gaaggacaag aacggaaaaa cgagtaccac 300
tacgaaggcg gaatcaaaag ctatgttgag tacttaaacc gttccaaaga agtcgttcat 360
gaagagccga tttatatcga aggcgagaaa gacggcataa cggttgaagt tgcattgcaa 420
tacaacgaca gctatacaag caatatttat tctttcacaa ataatatcaa cacatacgaa 480
ggcggcacgc acgaagccgg atttaaaacc ggtctgaccc gtgttataaa cgactatgca 540
agaagaaaag ggattttcaa agaaaatgat ccgaatttaa gcggggatga tgtgagggaa 600
gggctgactg ccattatttc aattaagcac cctgatccgc aattcgaagg gcagacgaaa 660
acgaagctcg gcaactccga agcgagaacg atcactgata cgctgttttc ttctgcgctg 720
gaaacattcc ttcttgaaaa tccggactca gcccgcaaaa tcgttgaaaa aggtttaatg 780
gccgcaagag cgcggatggc agcgaaaaaa gcgcgggaat tgacccgccg caaaagtgcg 840
cttgagattt ccaatctgcc gggcaaactg gcggac 876
<210> 3
<211> 1076
<212> DNA
<213> Bacillus velezensis
<400> 3
acaggaaaag taacgcctag aatcgactac ctgactgctg atgaagagga taactatgtc 60
gtagcccaag cgaatgctaa gctgagcgat gacggttctt tcttggatga cagcatcgta 120
gcgcgtttca gaggggaaaa caccgttgta gcccgcaacc gcgtggatta catggacgtt 180
tctcctaaac aggttgtttc tgctgcgaca gcatgtattc cgttcttgga aaacgatgac 240
tctaaccgtg ctctaatggg agcgaacatg cagcgtcagg ctgtgccttt gatgcagccg 300
gaagctccga tcgtcggaac gggtatggaa tacgtatccg gtaaagactc cggtgcagcc 360
gttatttgta aacaccctgg tatcgttgaa cgggtggaag cgaaaaacgt atgggtgcgc 420
cgctatgaag aaattgacgg ccaaaaagta aaaggcaacc tggataagta cagcttgctg 480
aaatttgtcc gctccaacca aggtacgtgc tacaaccagc gtccgatcgt cagtgtcggc 540
gatgaagtag tcaaaggaga aatccttgct gacggacctt caatggagct tggcgaactt 600
gctctcggcc gcaacgtaat ggtcggcttc atgacatggg atggttacaa ctatgaggat 660
gccatcatca tgagtgaacg ccttgtgaaa gatgatgtat acacatctat tcacattgaa 720
gaatatgaat cagaagcacg tgatacaaag cttgggcctg aagagatcac ccgcgatatt 780
ccaaacgtag gggaagacgc gcttcgcaat cttgatgacc gcggaattat ccgtatcggt 840
gcggaagtca acgacggaga ccttctcgta ggtaaagtaa cgcctaaagg tgtaactgag 900
cttacggctg aagaacgcct tctgcatgcg atctttggag aaaaagcgcg tgaagtccgt 960
gatacttctc tccgtgtgcc tcacggcggc ggcggaatta tccacgacgt aaaagtcttc 1020
aaccgtgaag acggcgacga acttcctccg ggagtgaacc agcttgtacg cgtata 1076
Claims (5)
1. Bacillus belgii (B.), (B.), (B.beijerinckii)Bacillus velezensis) The strain HMB28023 is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 19002.
2. Use of the bacillus beilesiensis strain HMB28023 of claim 1 for the control of botrytis cinerea, fusarium oxysporum, verticillium malorum, populus canker, canker walnut, and phaeosphaera Juglandis.
3. A microbial agent comprising the Bacillus belgii strain HMB28023 according to claim 1 as a fungus and a spore.
4. The microbial agent according to claim 3, wherein the microbial agent is a liquid formulation.
5. The use of the microbial inoculant of claim 3 or 4 for the control of Botrytis cinerea, Blastomyces tabaci, Phyllostachys Mali, Populus tremula, Sclerotinia Juglandis, and Phyllostachys Juglandis.
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| CN111748498A (en) * | 2020-07-08 | 2020-10-09 | 云南省烟草公司曲靖市公司 | Bacillus compound microbial inoculum with functions of preventing and treating powdery mildew and brown spot as well as preparation method and application thereof |
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| CN113025529B (en) * | 2021-03-29 | 2022-08-09 | 江南大学 | Method for producing cercospora bacteriocin by co-culture fermentation |
| CN113846039B (en) * | 2021-11-20 | 2023-10-03 | 江苏科技大学 | Bacillus bailii and application thereof |
| CN114480220B (en) * | 2022-03-15 | 2023-02-14 | 浙江大学 | Bacillus belgii, method for inhibiting botrytis cinerea by using same and application of bacillus belgii |
| CN116121123B (en) * | 2022-12-09 | 2024-04-09 | 青岛农业大学 | Bacillus bailii and application thereof in grape gray mold and grape fresh-keeping |
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| CN110283742A (en) * | 2019-06-17 | 2019-09-27 | 北京农业生物技术研究中心 | The Bei Laisi bacillus of one plant of broad-spectrum disease resistance and its application |
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| CN110283742A (en) * | 2019-06-17 | 2019-09-27 | 北京农业生物技术研究中心 | The Bei Laisi bacillus of one plant of broad-spectrum disease resistance and its application |
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