CN107043719B - bacillus amyloliquefaciens HMB28353 and application thereof - Google Patents
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Abstract
The invention discloses a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain HMB28353 which is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 13209. The invention also discloses a microbial agent produced by using the strain. The bacillus amyloliquefaciens HMB28353 has high drug effect on cucumber target spot, and the average prevention effect is more than 90.0%; secondly, the HMB28353 is used for preventing and treating cucumber target spot diseases, so that the cucumber target spot diseases are not easy to generate drug resistance, and the drug effect durability is good; in addition, the invention has no problem of environmental pollution; the HMB28353 strain also has the characteristic of wide antibacterial spectrum, and has good inhibition effect on verticillium dahliae, fusarium oxysporum, verticillium solani or botrytis cinerea in addition to cucumber target spot pathogen.
Description
Technical Field
The invention belongs to the field of biological control, and particularly relates to bacillus amyloliquefaciens HMB28353, a microbial agent produced by using the bacillus amyloliquefaciens and application of the bacillus amyloliquefaciens to control of plant diseases such as cucumber target spot and the like.
Background
Target spot disease of cucumber (also known as Corynespora leaf spot disease, brown spot disease, "yellow spot disease", etc.) is caused by Corynespora spinosa (Berk & Curt) Wei), and is severe in protective areas such as overwintering greenhouses, winter-spring greenhouses, and the like. Symptoms of which are initially yellow water-like spots about 1 mm in diameter; the disease spots in the middle stage of disease attack are enlarged into round or irregular shapes, the holes are easy to perforate, the disease spots on the front surface of the leaf are rough and uneven, the whole disease spots are brown, and the center is grey white and semitransparent; the diameter of the later stage lesion spots can reach 10-15 mm, an obvious eye target center is arranged in the center of the lesion spots, and sparse gray black mildew-shaped substances can be generated on the lesion spots when the humidity is high and are in a ring shape. The cucumber target spot disease seriously affects the yield and quality of cucumbers, generally causes the yield reduction by 20 percent, and reaches 60 to 70 percent when the yield reduction is serious.
At present, the control method for the target spot disease of cucumber comprises the following steps: (1) and (3) cultivating disease-resistant varieties: the cultivation of disease-resistant varieties is the most economic and effective method for preventing and treating diseases, but the cultivation of disease-resistant varieties is difficult because the knowledge on resistant materials of cucumber target spot in domestic cucumber germplasm resources is less. (2) Agricultural cultivation measures are adopted for control: for example, the planting density is reduced, old plant leaves with diseases are cleaned in time, the humidity of a greenhouse is reduced by adopting drip irrigation under a film, and the like, but the prevention and treatment effect is poor. (3) Chemical control: the method is a main method for preventing and treating the target spot disease of the cucumber at present, and the target spot disease of the cucumber is mainly prevented and treated by spraying chemical bactericides such as chlorothalonil, hymexazol, thiram, prochloraz, mancozeb, tebuconazole, difenoconazole and the like. However, when a large amount of chemical pesticides are used for a long time, pathogenic bacteria easily generate drug resistance, so that the control effect is reduced; meanwhile, agricultural products and environment are easily polluted, and new diseases or secondary diseases are caused to occur greatly. (4) Biological control: biological control methods utilizing microorganisms and metabolites thereof are increasingly favored by people due to the advantages of strong specificity, difficult generation of drug resistance, no toxicity to people and livestock, environmental friendliness and the like.
At present, the microorganisms for preventing and treating cucumber target spot disease mainly comprise: bacillus simplex (CN105002121A), Bacillus subtilis (CN103131657A) and Bacillus amyloliquefaciens (CN 105950506A). Due to the diversity and co-evolutionary nature of organisms, different antagonistic microorganisms have different resistances against different pathogenic bacteria and different physiological races thereof, and therefore new microorganisms are continuously selected to cope with different pathogenic bacteria and different physiological races of cucumber target spot.
disclosure of Invention
The invention aims to provide a bacillus amyloliquefaciens strain which has the advantages of high efficiency, broad bactericidal spectrum and the like.
The invention also aims to provide a microbial agent produced by using the bacillus amyloliquefaciens.
The third object of the present invention is to provide a method for producing the microbial agent.
The fourth purpose of the invention is to provide the application of the bacillus amyloliquefaciens strain in preventing and treating plant diseases such as cucumber target spot and the like.
The fifth purpose of the invention is to provide the application of the microbial agent in preventing and treating plant diseases such as cucumber target spot.
The invention is realized by the following technical scheme:
A Bacillus amyloliquefaciens strain HMB28353 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 13209.
The active component of the microbial agent produced by the bacillus amyloliquefaciens HMB28353 is the bacillus amyloliquefaciens HMB28353 thallus; also included are extracellular metabolites thereof.
The microbial agent may be a liquid preparation.
The preparation method of the microbial agent comprises the following steps:
(1) activating strains: activating the HMB28353 strain stored at low temperature on an LB plate culture medium, selecting a single strain to be placed on an LB slant culture medium, and culturing for 10-16 hours at 25-35 ℃ 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 flour culture medium (pH value is 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 90% of the total quantity of the spores and the bacteria; the obtained liquid preparation is HMB 28353.
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 microbial agent has a viable count of HMB28353 higher than 18.0 × 108cfu/mL。
The bacillus amyloliquefaciens HMB28353 is applied to control of plant diseases such as cucumber target spot, cotton verticillium wilt, cotton fusarium wilt, cotton rhizoctonia solani or tomato gray mold.
The microbial agent is applied to preventing and treating plant diseases such as cucumber target spot, cotton verticillium wilt, cotton fusarium wilt, cotton rhizoctonia rot or tomato gray mold.
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 aim of preventing and treating the target spot disease of the cucumber can be achieved by spraying the leaves before the target spot disease of the cucumber occurs.
Separation and screening process of HMB28353 strain
HMB28353 is a strain obtained by collecting soil samples from five points beside a mountain lake of Basong measure lake of Gongjiang county, Basong measure lake of Linzhi city, autonomous region of Tibet, with an altitude of 3700 m at 2013, collected by plant protection institute of agricultural and forestry academy of Hebei province, 1 month of 2013, uniformly mixing, weighing 1g of the collected soil samples, placing the weighed soil samples into a 250mL sterile triangular flask, adding 100mL of sterile water, placing the sterile flask on a shaking bed, oscillating at 170r/min for 30min, standing for 2h, adding 10mL of supernatant into a 50mL sterile centrifuge tube, carrying out water bath at constant temperature of 80 ℃ for 30min, adding 9mL of sterile water into 1mL of the-3Multiplying the soil microorganism suspension, and then diluting the soil suspension to 10-4、10-5、10-6Taking 200 mu L of microorganism suspension with each concentration from the dilution liquid, coating the microorganism suspension on an LB culture medium plate, repeating each concentration for 3 times, culturing for 1d-3d at the constant temperature of 30 ℃, and separating and purifying bacteria. And screening the biocontrol bacteria by using the cucumber target spot as a target through a flat plate confronting method, a leaf disc floating method and a pot experiment method. And screening a strain with obvious control effect on the cucumber target spot disease, wherein the strain is named as HMB 28353.
Classification and identification of HMB28353 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 (edited by east elegans bead et al, science publishers, 2001), and it was preliminarily judged that the strain HMB28353 belongs to Bacillus (Bacillus).
(2) Identification and classification using 16S rDNA sequences
Performing PCR amplification by using genome DNA of HMB28353 as a template and F27 and R1492 as primers to obtain a PCR amplification product; the primer sequence is as follows:
F27：5’-AGAGTTTGATCATGGCTCAG-3’(SEQ ID No.1)，
R1492：5’-GGCTACCTTGTTACGACTT-3’(SEQ ID No.2)。
The PCR reaction system for 16S rDNA was 50. mu.L, 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 F27(10 μmol/L), and 1 μ L of R1492(10 μmol/L); 50ng of genomic DNA of HMB 28353; 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.3) of HMB 28353. Homology comparison is carried out on the obtained 16SrDNA sequence of the HMB28353 in Genbank, and as a result, the homology of the strain HMB28353 and the 16S rDNA of the bacillus reaches 99%; meanwhile, a phylogenetic tree (see fig. 1) is constructed by utilizing MEGA software (Molecular evolution Genetics Analysis), and HMB28353 is aggregated with Bacillus, so that the HMB28353 belongs to Bacillus.
(3) identification and classification using gyrB gene sequences
Performing PCR amplification by using HMB28353 genome DNA as a template and using Bacillus gyrB gene degenerate primers gyrB-F and gyrB-R 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’(SEQ ID No.4)，
gyrB-R：5’-TCCDCCSTCAGARTCWCCCTC-3’(SEQ ID No.5)。
the PCR amplification reaction system of gyrB is 50 μ L, 10 XPCR Buffer (Mg)2+)5 μ L, dNTP mix (2.5mM)5 μ L, Taq (5U/μ L)1 μ L, gyrB-F (10 μmol/L)1 μ L, gyrB-R (10 μmol/L)1 μ L, HMB28353 genomic DNA 50ng, 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.6) of the HMB28353 strain. The gyrB gene of the HMB28353 strain obtained is usedhomology comparison is carried out on the sequence in Genbank, and the result shows that the homology of HMB28353 and the gyrB gene sequence of the bacillus amyloliquefaciens is the highest and reaches 90.4 percent; meanwhile, a phylogenetic tree (see fig. 2) is constructed by utilizing MEGA software (Molecular evolution Genetics Analysis), and the HMB28353 strain and the Bacillus amyloliquefaciens are polymerized together, so that the HMB28353 is the Bacillus amyloliquefaciens and is a new strain.
By combining the morphological characteristics and the results of homology comparison analysis of 16S rDNA and gyrB gene sequences, HMB28353 is known to belong to Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), is different from the existing Bacillus amyloliquefaciens strain, and is a new Bacillus amyloliquefaciens strain.
The invention has the advantages and beneficial effects that: (1) the bacillus amyloliquefaciens HMB28353 has high drug effect on cucumber target spot, the average prevention effect is more than 90.0 percent, and the pertinence is strong; (2) the HMB28353 is used for preventing and treating cucumber target spot diseases, so that the cucumber target spot diseases are not easy to generate drug resistance, and the drug effect durability is good; (3) the HMB28353 strain has a wide antibacterial spectrum, and has a good inhibitory effect on verticillium dahliae, fusarium oxysporum, verticillium solani or botrytis cinerea and the like besides cucumber target spot germs; (4) the microbial agent is safe to people and livestock, and has no environmental pollution; (5) the preparation method is simple, low in cost and simple to use.
And (3) biological preservation: the Bacillus amyloliquefaciens strain HMB28353 disclosed by the invention is preserved in the China general microbiological culture Collection center in 2016, 10 and 28 days, 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. 13209.
drawings
FIG. 1 is a phylogenetic tree of the HMB28353 strain obtained from the 16S rDNA sequence.
FIG. 2 is a phylogenetic tree of HMB28353 strain obtained based on the gyrB gene sequence.
Detailed Description
the invention is further clearly 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 preparation of HMB28353 microbial Agents
the method comprises the following steps:
(1) Activating strains: activating (30 ℃) a bacillus amyloliquefaciens strain HMB28353 (the strain is stored in China general microbiological culture Collection center in 2016, 10 and 28 days, with the preservation number of CGMCC No.13209) stored at-80 ℃ on an LB plate culture medium (the components and the weight ratio of the components are tryptone 1g, yeast extract 0.5g, sodium chloride 1g, agar powder 15g and water 1000mL), and culturing a single strain on an LB inclined plane culture medium (the components and the weight ratio of the components are tryptone 1g, yeast extract 0.5g, sodium chloride 1g, agar powder 15g and water 1000mL) at 30 ℃ for 24-36 hours to obtain an activated strain.
(2) Preparing a seed solution: preparing LB liquid culture medium (the components and the weight ratio 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 the strain activated in the loop inoculation step (1) into each flask after the temperature is reduced to room temperature, and carrying out shaking culture for 24h under the conditions of 30 ℃ and the rotation speed of a shaking table of 190rpm to obtain 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 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%; co-fermenting and culturing for about 48h to obtain the liquid preparation of HMB 28353.
Example 2 antagonism of HMB28353 strain of the invention against cucumber Blastomyces cucumerinum
Sources of cucumber target spot germ strains CA-1 to be tested: the cucumber target spot pathogen CA-1 strain is collected from diseased leaves of cucumbers in the village of Susan county, full city, Baoding, Hebei province, and is separated and purified by a plant protection research institute of the academy of agriculture and forestry, Hebei university of agriculture, and the plant pathological system is identified as Corynespora polystachya (Corynespora cassicola), and the pathogenicity determination shows strong pathogenicity.
(II) test method:
Plate confrontation experiment: firstly, activating and culturing the cucumber target spot pathogen CA-1 strain on a PDA (personal digital Assistant) plate for 7 days, and then using a puncherPunching holes on the edge area of a colony to prepare a bacterial sheet, transferring the cucumber target spot pathogen bacterial sheet to the center of another PDA plate, then inoculating the activated bacillus amyloliquefaciens HMB28353 in the step (1) in the embodiment 1 at a position 2.0 cm away from the indication bacterial sheet, and setting a blank control (the growth condition of the cucumber target spot pathogen without inoculating the HMB28353 bacterial strain). Culturing at constant temperature of 25 deg.C, measuring control growth amount (colony radius) and treatment growth amount (growth inhibiting radius after inoculating HMB 28353) of cucumber leaf spot pathogen 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.
TABLE 1 results of experiments on the antagonistic effect of HMB28353 strain on cucumber leaf blight
The result (see table 1) shows that the inhibition rate of the bacillus amyloliquefaciens HMB28353 on the cucumber target spot germ reaches 73.68%; the transparent bacteriostatic bandwidth is 9.0 mm; the HMB28353 strain has obvious inhibition effect on cucumber target spot germs and biological control potential for preventing and treating cucumber target spot.
Example 3 comparative test of the prevention of cucumber target spot by the HMB28353 strain of the invention
Test materials (one):
(1) Cucumber varieties: jin you No. 1.
(2) Suspension of cucumber leaf blight bacteria CA-1 spores (the source of cucumber leaf blight bacteria CA-1 is the same as in example 2).
(II) test treatment:
(1) HMB28353 liquid formulation: a 100 fold aqueous dilution of the HMB28353 liquid formulation prepared in example 1.
(2) blank control: and (4) clear water.
(III) test method:
(1) Culturing cucumber plants: culturing cucumber seedlings of Jinyou No.1 in nutrition pots with the diameter of 6cm, wherein the seedling culture medium is vermiculite, two seeds are sown in each nutrition pot, and one strong seedling is left after seedling emergence. Transplanting the cucumber seedlings into a plastic flowerpot with the diameter of 25cm (a matrix prepared by vermiculite and garden soil in a ratio of 1:1 is filled in the flowerpot) when two true leaves grow out, and culturing at the constant temperature of 25 ℃ (lighting for 14 hours and darkness for 10 hours). Supporting and fixing cucumber seedlings by bamboo poles, continuously culturing, and removing cucumber buds to ensure the vegetative growth of plants.
(2) Preparation of cucumber target spot pathogen CA-1 spore suspension: activating and culturing cucumber target spot pathogen CA-1 strain on PDA plate for 7 days, and performing hole puncherpunching holes in the edge area of a bacterial colony to prepare a bacterial sheet, transferring the bacterial sheet of the cucumber target spot pathogen to the center of a PSA flat plate, culturing for 7-10 days at 25 ℃, flushing with sterile water after generating conidia to collect the conidia, and preparing the conidia with the concentration of 104Suspension per mL for use.
(3) The prevention and treatment effect is measured by a leaf disc floating method: taking cucumber leaves which are disease-free and have the same complete leaf positions in the step (1), treating the cucumber leaves, and spraying 100 times of water diluent of the HMB28353 liquid preparation prepared in the step (1) to the cucumber leaves in the step (1); treatment 2 was a blank control, sprayed with equal amount of clear water. Culturing in a 30 ℃ illumination incubator for 24 hours, cutting leaf discs with the diameter of 15mm by using a puncher, placing the leaf discs in culture dishes with sterile water, floating the leaf discs on the water surface by 10 leaf discs in each dish, and dripping 10ul of CA-1 spore suspension prepared in the step (2) into the middle of each leaf disc. Each treatment was repeated 4 times, each for 30 leaf disks. The culture was carried out in a 25 ℃ light incubator for 13 days, the disease grade was investigated and the control effect was calculated. Grading standard: grade 0, no scab; grade 1, a small amount of scabs and no conidia; grade 2, conidiophores are generated, and the lesion area is less than 50% of the leaf area; grade 3, conidiophores are generated, and the lesion area is more than 50 percent of the leaf area.
The result (see table 2) shows that the prevention effect of the HMB28353 liquid preparation on the cucumber target spot is 88.05%, which shows that the HMB28353 bacterial strain and the HMB28353 microbial agent thereof have the outstanding prevention and treatment effect on the cucumber target spot.
TABLE 2 comparative test results of the prevention of cucumber target spot disease by HMB28353 strain of the present invention
| treatment of | Index of disease condition | Control effect (%) |
| HMB28353 liquid formulation | 6.67b | 88.05 |
| Blank control | 55.83a | -- |
Example 4 comparative test of the prevention of cucumber target spot by the HMB28353 strain of the invention
(I) test treatment:
(1) HMB28353 microbial agent: a 100 fold aqueous dilution of the HMB28353 liquid formulation prepared in example 1.
(2) chemical agent control: 10% Difenoconazole Water dispersible granule (Shigao) (Suzhou crop protection Co., Ltd.) 1000 times diluted solution.
(3) Blank control: and (4) clear water.
(II) test method:
(1) Culturing cucumber plants: culturing cucumber seedlings of Jinyou No.1 in nutrition pots with the diameter of 6cm, wherein the seedling culture medium is vermiculite, two seeds are sown in each nutrition pot, and one strong seedling is left after seedling emergence. Transplanting the cucumber seedlings into a plastic flowerpot (a matrix prepared by vermiculite and garden soil according to the proportion of 1: 1) with the diameter of 15cm when the cucumber seedlings grow two true leaves, and culturing at the constant temperature of 25 ℃ (lighting for 14 hours and darkness for 10 hours). Supporting and fixing cucumber seedlings by bamboo poles, continuously culturing, and removing cucumber buds to ensure the vegetative growth of plants.
(2) A suspension of the spores of the pathogen cucumber target spot CA-1 was prepared (same as in example 3).
(3) Pot experiment determination of control effect: taking 4-6 pieces of true-leaf cucumber seedlings cultured in the step (1), treating the cucumber seedlings treated in the step (1), and spraying 100 times of water diluent of the HMB28353 liquid preparation prepared in the step (1); treating 2 with chemical agent, spraying 10% difenoconazole water dispersible granule (shigao) 1000 times water diluent; treatment 3 was a blank control, sprayed with equal amount of clear water. And (3) culturing for 24 hours in a constant-temperature culture room at the temperature of 30 ℃, and spraying the cucumber target spot pathogenic bacteria spore suspension prepared in the step (2). Each treatment was repeated 4 times, with 5 pots of cucumber seedlings each. Two batches of tests were performed. Culturing in 25 deg.C constant temperature incubator for 10-15 days (light 14 hr, dark 10 hr), examining disease grade after blank control is fully developed, and calculating control effect. The standard for grading the target spot of cucumber and the calculation method for preventing the target spot refer to GB/T17980.26-2000.
Results (see table 3) the disease index of HMB28353 liquid formulation treatment in the first trial was 5.92, which is significantly lower than the disease index 65.14 of the blank control, and the control effect of HMB28353 liquid formulation on cucumber target spot disease was 90.91%, which is equivalent to 88.36% of the control effect of chemical agents. In the second batch of test results, the index of illness of the HMB28353 liquid preparation is 3.72, which is obviously lower than the index of illness 52.63 of a blank control, and the control effect of the HMB28353 liquid preparation on cucumber target spot disease is 92.93 percent, which is equivalent to 97.64 percent of the control effect of a chemical agent. The above results show that the HMB28353 strain and the microbial agent thereof have outstanding control effects on cucumber target spot.
TABLE 3 comparative test results of the prevention of cucumber target spot disease by the strain HMB28353 of the present invention
Example 5 test of the inhibitory Effect of the HMB28353 Strain on various phytopathogenic fungi
Plant pathogenic fungi and sources thereof: verticillium dahliae (VD-1), Fusarium oxysporum (FOV-1), rhizoctonia solani (Rhizoctonia solani) RHs-15, and Botrytis cinerea (BC-32).
The 4 pathogenic fungi strains come from the plant disease biological control laboratory of the plant protection research institute of agriculture and forestry academy of sciences of Hebei province.
(II) test method:
the inhibition effect of the bacillus amyloliquefaciens HMB28353 strain on 4 plant pathogenic fungi is detected by adopting a confrontation culture method. The HMB28353 strain is activated for 24 hours at 30 ℃ on an LB slant culture medium to be detected. Activating 4 pathogenic fungi on a PDA culture medium for 5 days, punching a bacterial disc with consistent bacterial age at the edge of a bacterial colony by using a puncher with the diameter of 6mm, transferring hypha downwards to the center of another PDA flat plate, inoculating HMB28353 bacterial strain in a shape like a Chinese character 'pin' at a position 2cm away from the bacterial disc, repeatedly culturing for 5 days at a constant temperature of 25 ℃ after 4 times of treatment by taking non-inoculated bacteria as a control, measuring the radius of the treated bacterial colony and the distance (bacterial inhibition zone) between the inoculation point of the HMB28353 bacterial strain and the edge of the bacterial colony, determining the antibacterial degree, and calculating the antibacterial rate.
The bacteriostatic rate (%) (control growth amount-treated growth amount)/control growth amount × 100.
The results (see table 5) show that the inhibition rate of HMB28353 of the invention to 4 tested pathogenic fungi is 64.18% -82.23%, wherein the inhibition rate to cotton verticillium wilt is 82.23%, the inhibition rate to cotton verticillium wilt is 64.18%, and the results show that the Bacillus amyloliquefaciens strain HMB28353 of the invention has good control effects on cotton wilt, cotton verticillium wilt and tomato gray mold, and has wide inhibition spectrum.
TABLE 5 inhibition test results of HMB28353 Strain on plant pathogenic fungi
SEQUENCE LISTING
<110> institute of plant protection of academy of agriculture, forestry and science of Hebei province
<120> Bacillus amyloliquefaciens HMB28353 and application thereof
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<170> PatentIn version 3.5
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ccggggctaa taccggatgg ttgtctgaac cgcatggttc agacataaaa ggtggcttcg 180
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aggcgacgat gcgtagccga cctgagaggg tgatcggcca cactgggact gagacacggc 300
ccagactcct acgggaggca gcagtaggga atcttccgca atggacgaaa gtctgacgga 360
gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaag ctctgttgtt agggaagaac 420
aagtgccgtt caaatagggc ggcaccttga cggtacctaa ccagaaagcc acggctaact 480
acgtgccagc agccgcggta atacgtaggt ggcaagcgtt gtccggaatt attgggcgta 540
aagggctcgc aggcggtttc ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg 600
tcattggaaa ctggggaact tgagtgcaga agaggagagt ggaattccac gtgtagcggt 660
gaaatgcgta gagatgtgga ggaacaccag tggcgaaggc gactctctgg tctgtaactg 720
acgctgagga gcgaaagcgt ggggagcgaa caggattaga taccctggta gtccacgccg 780
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ttgaaaaagg tttaatggcc gcaagagcgc ggatggcggc gaaaaaagcc cgggaattga 840
cccggcgcaa aagtgcgctt gagatttcca atctgccggg caaactggcg gactgttctt 900
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aacca 965
Claims (8)
1. A Bacillus amyloliquefaciens strain HMB28353 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No. 13209.
2. The microbial agent produced by using the bacillus amyloliquefaciens HMB28353 according to claim 1, wherein the active ingredient is bacillus amyloliquefaciens HMB 28353.
3. The microbial inoculant according to claim 2, wherein said microbial inoculant is a liquid formulation; the preparation method of the microbial agent comprises the following steps:
(1) Activating the HMB28353 strain stored at low temperature on an LB plate culture medium, selecting a single strain to be placed on an LB slant culture medium, and culturing for 10-16 hours at 25-35 ℃ to obtain an activated strain;
(2) 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) Inoculating the seed solution obtained in the step (2) into a corn flour and soybean flour 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 90% of the total quantity of the spores and the bacteria; the obtained liquid preparation is HMB 28353.
4. The microbial inoculum according to claim 3, characterized in that the LB plate medium or LB slant medium in step (1) 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, 12-18 g of agar powder and 1000mL of water.
5. The microbial inoculant according to claim 3, wherein the LB liquid medium in step (2) 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.
6. The microbial inoculant according to claim 3, wherein the corn meal and soybean meal culture medium in step (3) 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.
7. The use of the bacillus amyloliquefaciens HMB28353 according to claim 1 for controlling cucumber target spot disease, cotton verticillium wilt, cotton fusarium wilt, cotton rhizoctonia rot and tomato gray mold.
8. the use of the microbial inoculant of claim 2 for controlling cucumber target spot, cotton verticillium wilt, cotton fusarium wilt, cotton rhizoctonia rot and tomato gray mold.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1946297A (en) * | 2004-02-27 | 2007-04-11 | 株式会社树 | Method of controlling plant disease damage by using bacillus and controlling agent |
| CN104560837A (en) * | 2015-02-02 | 2015-04-29 | 青岛蔚蓝生物集团有限公司 | Bacillus amyloliquefaciens and application thereof |
| CN105176893A (en) * | 2015-11-03 | 2015-12-23 | 保定微控生物科技有限公司 | Bacillus amyloliquefaciens for controlling seedling blight of crops and application thereof |
| CN105483054A (en) * | 2016-01-04 | 2016-04-13 | 河北省科学院生物研究所 | Bacillus amyloliquefaciens WS-8 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1946297A (en) * | 2004-02-27 | 2007-04-11 | 株式会社树 | Method of controlling plant disease damage by using bacillus and controlling agent |
| CN104560837A (en) * | 2015-02-02 | 2015-04-29 | 青岛蔚蓝生物集团有限公司 | Bacillus amyloliquefaciens and application thereof |
| CN105176893A (en) * | 2015-11-03 | 2015-12-23 | 保定微控生物科技有限公司 | Bacillus amyloliquefaciens for controlling seedling blight of crops and application thereof |
| CN105483054A (en) * | 2016-01-04 | 2016-04-13 | 河北省科学院生物研究所 | Bacillus amyloliquefaciens WS-8 and application thereof |
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