CN108085284B - Bacillus methylotrophicus, and preparation method and application of microbial inoculum containing same - Google Patents
Bacillus methylotrophicus, and preparation method and application of microbial inoculum containing same Download PDFInfo
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Abstract
The invention discloses Bacillus methylotrophicus Z-1(Bacillus methylotrophicus Z-1) with the preservation number of CGMCC NO.15004, a microbial agent containing the Bacillus methylotrophicus Z-1, and application of the Bacillus methylotrophicus or the microbial agent in preventing and treating strawberry blight. The bacillus methylotrophicus and the microbial agent thereof provided by the invention have a remarkable antagonistic effect on strawberry fusarium wilt, can prevent and treat the strawberry fusarium wilt, and have the prevention and treatment efficiency of over 80%. The strain and the use of the microbial agent thereof can reduce the application of chemical fertilizer and pesticide in the production process of strawberries, avoid the problems of soil hardening, organic matter reduction, environmental pollution, ecological imbalance, drug residue, food safety and the like caused by excessive use of the chemical fertilizer and pesticide, and have good ecological and social benefits.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a novel bacillus methylotrophicus Z-1 strain with antagonistic action on pathogenic bacteria of strawberry blight, a preparation method and application of a microbial agent containing the strain.
Background
Bacillus methylotrophicus Z-1(Bacillus methylotrophicus Z-1) belongs to cell organisms (Cellular organisms), bacterial domains (bacillia), Firmicutes (Firmicutes), Bacilli (Bacillus), Bacillales (Bacillales), Bacillaceae (Bacillaceae) and Bacilli (Bacillus) in taxonomy, is widely distributed in soil and other natural environments, and is one of soil and plant microecological dominant populations. In recent years, with the intensive research on bacillus methylotrophicus, it has been found that bacillus methylotrophicus has various biological activities such as insecticidal activity, antibacterial activity, biodegradation activity and the like, and is considered to be one of the most important species in bacillus having biocontrol application value.
Strawberry (Fragaria ananassa Duch) belongs to perennial herbaceous plants in the genus of strawberry of the family Rosaceae, is one of important berries with high economic value, is known to be more than 50 in the world, integrates the values of delicious food, health care and medicine, is rich in vitamin C, tetrahydrofolic acid, phenols and other bioactive substances, can effectively prevent cardiovascular and cerebrovascular diseases, and has multiple effects of oxidation resistance, tumor resistance, beauty treatment and the like. In recent years, with the continuous expansion of the cultivation area of strawberry facilities, strawberry blight commonly occurs in some main planting areas, and the damage degree is increased year by year. When the strawberries are planted by continuous cropping, the incidence of disease of the plants can reach 89.2 percent. Once the strawberry blight occurs, the results of reduction of fruit setting rate, abnormal fruit enlargement, quality reduction, great reduction of yield and the like can be caused, and the economic benefit and sustainable development of the strawberry industry are seriously influenced. In the past, chemical bactericides are used for controlling strawberry blight, but the chemical bactericides are used in a large area and easily cause the problems of environmental pollution, ecological imbalance, drug residues, food safety and the like, so the biological method which is low in cost, high in efficiency, environment-friendly and free of drug residues is concerned. The bacillus methylotrophicus is a bacterium with broad-spectrum bacteriostatic activity, has strong secondary metabolite generation capacity, can generate multiple bacteriostatic substances, is used for preparing a microbial agent, and is a technical means with the most application prospect in future biological control of strawberry blight.
Disclosure of Invention
The invention aims to provide a bacillus methylotrophicus and a microbial inoculum capable of efficiently antagonizing strawberry fusarium wilt, and the microbial inoculum is applied to the rhizosphere of strawberries and can effectively prevent and treat strawberry fusarium wilt, so that the use of chemical pesticides in strawberry production is reduced, the problems of environment and food safety brought by the chemical pesticides are reduced, and the yield and the quality of strawberries are improved.
In order to achieve the aim, the invention provides the bacillus methylotrophicus Z-1, wherein the bacillus methylotrophicus Z-1 is preserved by China general microbiological culture Collection center (CGMCC for short) in 2017, 12 and 4 months, and the preservation number is CGMCC NO. 15004. CGMCC No. 3 of No.1 Xilu of Beijing, Chaoyang.
The bacillus methylotrophicus Z-1 is used for preventing and treating antagonistic strawberry fusarium wilt.
The bacillus methylotrophicus Z-1 disclosed by the invention has an antagonistic effect on sphacelotheca cucurbitacearum, Chinese cabbage anthracnose pathogen, tomato leaf mold pathogen, watermelon fusarium wilt pathogen and phytophthora capsici.
The invention also provides a preparation method of the microbial agent containing the bacillus methylotrophicus, which comprises the following specific steps:
s1, inoculating Bacillus methylotrophicus Z-1 into a seed liquid culture medium, culturing for 24h at 30 ℃ to obtain a Bacillus methylotrophicus Z-1 seed liquid, wherein the concentration of the Bacillus methylotrophicus Z-1 in the seed liquid is 1.0-1.5 multiplied by 107～ 8CFU/mL; inoculating the seed solution into a fermentation culture medium according to the volume ratio of 2-5.0%, and culturing at 30 ℃ for 48h to obtain a bacillus methylotrophicus Z-1 fermentation broth;
s2, uniformly mixing the fermentation liquor prepared in the step S1 and the plant carrier according to the volume-to-mass ratio of 1:2 (mL/g); and (5) airing until the water content is less than or equal to 25 percent to obtain the microbial agent containing the bacillus methylotrophicus.
The mixture contains fermentation liquid 0.5mL/g and plant carrier about 10 bacteria/g8One per gram.
Preferably, the seed liquid culture medium of step S1 comprises: peptone 1.6% (m/V), yeast powder 1.0% (m/V), sodium chloride 0.5% (m/V), pH 7.0-7.2;
the composition of the large amount of fermentation medium is as follows: glucose 2.8% (m/V), peptone 1.0% (m/V), yeast extract 0.5% (m/V), potassium dihydrogen phosphate 0.35% (m/V) and calcium carbonate 0.25% (m/V), pH 7.2.
The "m/V" of the present invention is "g/ml".
Preferably, the vegetal carrier in step S2 is 60-mesh corn cob meal or rice husk meal.
The invention also provides application of the microbial agent containing the bacillus methylotrophicus for preventing and treating strawberry blight.
In conclusion, compared with the existing products, the invention has the advantages that:
1. the microbial inoculum can effectively prevent and treat strawberry blight: the microbial agent contains bacillus methylotrophicus Z-1 capable of efficiently antagonizing strawberry fusarium wilt, and the bacillus methylotrophicus can secrete a plurality of secondary metabolites such as surfactin, iturin and camelina and the like, and the substances are strong fungal inhibitors, so that the bacillus methylotrophicus Z-1 can effectively perform biological control on the strawberry fusarium wilt.
2. The microbial inoculum can improve the yield of strawberries, improve the quality of strawberries: the bacillus methylotrophicus Z-1 is used as an important plant rhizosphere growth promoting bacterium, can generate plant hormones such as auxin, cytokinin, gibberellin and the like, induces plants to generate systemic resistance, and accelerates the synthesis and transformation of nutrients required by the plants, so that the growth and development of strawberries are promoted, and the quality of the strawberries is improved.
3. The invention is safe without drug residue: the microbial agent disclosed by the invention is composed of bacillus methylotrophicus Z-1 and a carrier material (selected from 60-mesh corncob powder or rice hull powder), has no toxic or harmful components, avoids the problems of pesticide residue, food safety and the like caused by pesticide use in strawberry production, and meets the requirement of green sustainable development of strawberry planting industry.
Deposit description
The preservation information of the biological material sample related to the present invention: the related microorganism strain is Z-1, is classified and named as Bacillus methylotrophicus (Bacillus methylotrophicus), and is preserved by China general microbiological culture Collection center (CGMCC for short) in 2017, 12 and 4 months with the preservation number of CGMCC NO. 15004. CGMCC No. 3 of No.1 Xilu of Beijing, Chaoyang.
Drawings
The invention will be further described with reference to the following examples with reference to the accompanying drawings.
FIG. 1 is a colony morphology of Bacillus methylotrophicus Z-1 according to the present invention.
FIG. 2 is a colony morphology chart of strawberry blight pathogenic bacteria.
FIG. 3 is a diagram showing the growth of a plate of pathogenic bacteria of Bacillus methylotrophicus Z-1 and strawberry wilt.
FIG. 4 is a gel electrophoresis diagram of a PCR amplified fragment of Bacillus methylotrophicus Z-1 according to the present invention.
FIG. 5 is a schematic diagram of a phylogenetic tree of Bacillus methylotrophicus Z-1 based on the 16S rRNA gene sequence according to the present invention.
Detailed Description
The technical solution of the present invention is explained in detail below with reference to preferred embodiments. The following examples are only for illustrating and explaining the present invention and do not constitute a limitation to the technical solution of the present invention.
Example 1: separation, screening and identification of bacillus methylotrophicus Z-1
The strain is separated from the soil of strawberry growing area in national agriculture science and technology park of Dalian Jinzhou.
1. Isolation and screening of Bacillus methylotrophicus Z-1
(1) Weighing 10g of the collected soil sample, adding into a triangular flask containing 90mL of PBS buffer solution, adding glass beads (which help the sample to be uniformly dispersed in the PBS buffer solution), shaking on a shaker at 30 deg.C and 160rpm for 30min, standing for 10min, and preparing soil bacterial suspension (i.e. 10 min)-1Soil dilution); then 10-fold gradient dilution was performed with PBS buffer to obtain 10-2、10-3、10-4、10-5、10-6、10-7And (3) diluting the suspension, respectively taking 100 mu L of soil bacterium suspensions with different concentration gradients, uniformly coating the soil bacterium suspensions on a broth agar plate (NA), and culturing for 48h in a constant-temperature incubator at 30 ℃. Individual colonies with significantly different culture characteristics were picked and streaked onto NA plates for purification (FIG. 1). The purified strains were transferred to NA slants and stored at 4 ℃.
The NA flat plate solid medium comprises the following components in percentage by weight: 10g of peptone, 3g of beef extract powder, 5g of sodium chloride, 15g of agar and 1000mL of deionized water, wherein the pH value is 7.4, and the peptone is used after being sterilized at 121 ℃ for 20 min.
(2) The pathogenic bacteria of strawberry blight (figure 2) are used as indicator bacteria, and the improved opposing culture method is adopted to screen out the bacterial strains which have the inhibiting effect on the pathogenic bacteria of strawberry blight. The method comprises the steps of culturing strawberry blight pathogenic bacteria on a PDA (personal digital assistant) flat plate at 28 ℃ for 5-7 days for activation, punching a newly activated strawberry blight pathogenic bacteria circular bacterial cake by using a sterilization puncher (the diameter is 5mm), inoculating the circular bacterial cake to the center of the PDA flat plate, culturing in a constant-temperature incubator at 28 ℃ for 1 day, then, inoculating antagonistic bacteria to be detected at positions 20mm away from the pathogenic bacteria bacterial cake respectively at four points with sterilization toothpicks at equal intervals, repeating the opposite experiments of each group for 3 times, continuously performing constant-temperature inverted culture at 28 ℃ for 3-5 days, measuring and recording the diameter of an antibacterial ring, and calculating the average inhibition rate.
The PDA flat solid culture medium comprises the following components in percentage by weight: 200g of potato, 20g of glucose, 15-20 g of agar, 1000mL of deionized water, pH7.2, and sterilizing at 121 ℃ for 20 min.
The result shows that the strain with the number of the bacillus methylotrophicus Z-1 has the strongest antagonistic action on pathogenic bacteria of strawberry blight (figure 3), the diameter of a bacteriostasis ring of the strain is 28.5mm, the average inhibition rate is 75.3 percent, and the strain is preserved with the preservation number of CGMCC NO. 15004.
2. Molecular biological identification of Bacillus methylotrophicus Z-1
The molecular biological identification of the bacillus methylotrophicus Z-1 adopts a method for directly amplifying 16S rRNA by using thalli. The method comprises the following specific steps:
(1) extraction of strain genome DNA: extracting the genome DNA of the strain by adopting a genome DNA extraction kit of Tiangen Biochemical technology (Beijing) Co.
(2) PCR amplification of 16S rRNA sequences: the 16S r RNA gene of Bacillus methylotrophicus was PCR-amplified using the extracted total DNA as a template and the universal primers 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-TACCTTGTTACGACTT-3') for the bacterial 16S r RNA gene.
And (3) PCR reaction system: 2 Taq Master Mix 12.5. mu.L, DNA template and upstream and downstream primers 1. mu.L each, ddH2O9.5. mu.L, 25. mu.L for PCR reaction system.
And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 deg.C for 30s, annealing at 50 deg.C for 1min, extension at 72 deg.C for 2min, and circulation for 30 times; 10min at 72 ℃.
(3) Detection and sequencing of PCR amplification: taking the PCR amplification product in the step (2) to carry out electrophoresis detection by using 1% agarose gel, sending the PCR product with the PCR band size of about 1500bp to Shanghai Biotechnology Limited company for sequencing as shown in figure 4, carrying out Blast comparison on the obtained DNA sequence on NCBI, selecting the sequence with higher homology and constructing a phylogenetic tree by using a Neighbor-Joining method as shown in figure 5.
The results show that the strain is determined to be Bacillus methylotrophicus by combining morphological observation of the strain and homology analysis in a phylogenetic tree.
Example 2: preparation of microbial agent
(1) Preparing a seed solution: streaking and transferring the bacillus methylotrophicus Z-1 strain stored on the NA inclined plane into an NA flat plate, placing the NA flat plate into a constant-temperature incubator at 30 ℃ for culturing for 24h, picking out a single colony by using a sterilized inoculating ring to inoculate the single colony into a seed liquid culture medium, and performing shaking culture on a shaking table at 30 ℃ for 14h to obtain a seed liquid. The formula of the seed liquid culture medium is as follows: peptone 1.6% (m/V), yeast powder 1.0% (m/V), sodium chloride 0.5% (m/V), pH 7.0-7.2.
(2) Preparing fermentation liquor: inoculating the cultured seed liquid into a large amount of fermentation medium at a volume ratio of 5.0%, culturing at 30 deg.C for 48 hr to obtain fermentation liquid, counting the number of viable bacteria by plate bacteria to 1.0 × 109CFU/mL or above for use. The formula of the large-scale fermentation medium is as follows: glucose 2.8% (m/V), peptone 1.0% (m/V), yeast extract 0.5% (m/V), potassium dihydrogen phosphate 0.35% (m/V) and calcium carbonate 0.25% (m/V), pH 7.2.
(3) Preparation of the microbial inoculum: taking rice hull powder sieved by a 60-mesh sieve as a carrier, and fully and uniformly mixing the fermentation liquor prepared in the step (2) and the carrier according to the mass-to-volume ratio of 1:2 to ensure that the volume ratio of the fermentation liquor to the carrier reaches 0.5mL/g (about 10)8One/gram), then airing indoors, preparing the microbial inoculum into the microbial inoculum with the water content of less than 25%, and storing the microbial inoculum in a cool and dry place for later use.
Example 3: microbial agent for preventing and treating strawberry blight
Selecting a Fengxiangyi strawberry variety as an experimental object, sowing the strawberry seeds into a seedling tray after the strawberry seeds begin to accelerate germination, dipping the strawberry seedlings to 1.0 x 10 after cotyledons of the strawberries are unfolded7Each spore/mL of strawberry blight germ spore liquid is cultured for 20min, and then the strawberry blight germ spore liquid is transplanted into a plastic pot filled with strawberry planting soil. Diluting the microbial agent with irrigation water to viable count of 1 x 106CFU/mL～1ⅹ107CFU/mL, each at 1m3Mu, 3m3Mu and 5m3The amount per mu is applied to the soil near the roots of the strawberry plants, and the application of clear water is used as a blank control, and each treatment is repeated for 3 times. The interval irrigation time is 15d, the period of the biocontrol experiment is 2 months, and the morbidity and the biocontrol effect are observed.
The morbidity and control efficacy are shown in the following table:
control effect of different treatments on strawberry blight
From the above results, it can be seen that the screen of the present inventionThe selected Bacillus methylotrophicus Z-1 strain has good antagonistic effect on strawberry blight, and the microbial agent can effectively prevent and treat strawberry blight according to the length of 3m3The microbial agent has the best prevention and treatment effect per mu.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Sequence listing
<110> university of Large Community
<120> Bacillus methylotrophicus, preparation method and application of microbial inoculum containing same
<130> Biotechnology
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1441
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<213> Bacillus methylotrophicus
<400> 1
cgggcgtgct atacatgcag tcgagcggac agatgggagc ttgctccctg atgttagcgg 60
cggacgggtg agtaacacgt gggtaacctg cctgtaagac tgggataact ccgggaaacc 120
ggggctaata ccggatggtt gtttgaaccg catggttcag acataaaagg tggcttcggc 180
taccacttac agatggaccc gcggcgcatt agctagttgg tgaggtaacg gctcaccaag 240
gcaacgatgc gtagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc 300
agactcctac gggaggcagc agtagggaat cttccgcaat ggacgaaagt ctgacggagc 360
aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa 420
gtgccgttca aatagggcgg caccttgacg gtacctaacc agaaagccac ggctaactac 480
gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa 540
gggctcgcag gcggtttctt aagtctgatg tgaaagcccc cggctcaacc ggggagggtc 600
attggaaact ggggaacttg agtgcagaag aggagagtgg aattccacgt gtagcggtga 660
aatgcgtaga gatgtggagg aacaccagtg gcgaaggcga ctctctggtc tgtaactgac 720
gctgaggagc gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta 780
aacgatgagt gctaagtgtt agggggtttc cgccccttag tgctgcagct aacgcattaa 840
gcactccgcc tggggagtac ggtcgcaaga ctgaaactca aaggaattga cgggggcccg 900
cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg 960
acatcctctg acaatcctag agataggacg tccccttcgg gggcagagtg acaggtggtg 1020
catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1080
cttgatctta gttgccagca ttcagttggg cactctaagg tgactgccgg tgacaaaccg 1140
gaggaaggtg gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc 1200
tacaatggac agaacaaagg gcagcgaaac cgcgaggtta agccaatccc acaaatctgt 1260
tctcagttcg gatcgcagtc tgcaactcga ctgcgtgaag ctggaatcgc tagtaatcgc 1320
ggatcagcat gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac 1380
gagagtttgt aacacccgaa gtcggtgagg taacctttag gagccagccg ccgaaggttg 1440
a 1441
Claims (4)
1. The bacillus methylotrophicus Z-1 is characterized in that the bacillus methylotrophicus is used for preventing and treating strawberry fusarium wilt, the strain name is Z-1, and the bacillus methylotrophicus is classified and named as bacillus methylotrophicus (Bacillus methylotrophicus Z-1)Bacillus methylotrophicus) In 2017, 12 and 4 months, China general microbiological culture Collection centerPreservation, CGMCC for short, preservation number CGMCC number 15004.
2. A method for preparing a microbial agent comprising Bacillus methylotrophicus Z-1 of claim 1, which comprises the following steps:
s1, inoculating Bacillus methylotrophicus Z-1 into a seed liquid culture medium, culturing for 24h at 30 ℃ to obtain a Bacillus methylotrophicus Z-1 seed liquid, wherein the concentration of the Bacillus methylotrophicus Z-1 in the seed liquid is 1.0-1.5 multiplied by 107~8CFU/mL; inoculating the seed solution into a fermentation culture medium according to the volume ratio of 2-5.0%, and culturing at 30 ℃ for 48h to obtain a bacillus methylotrophicus Z-1 fermentation broth;
s2, uniformly mixing the fermentation liquor prepared in the step S1 and the plant carrier according to the volume mass ratio of 1ml to 2 g; and (5) airing until the water content is less than or equal to 25 percent to obtain the microbial agent containing the bacillus methylotrophicus.
3. The method according to claim 2, wherein the seed culture medium of step S1 comprises the following components in percentage by mass: 1.6% of peptone, 1.0% of yeast powder and 0.5% of sodium chloride, and the pH value is 7.0-7.2;
the mass fermentation culture medium comprises the following components in percentage by mass and volume: 2.8 percent of glucose, 1.0 percent of peptone, 0.5 percent of yeast extract, 0.35 percent of monopotassium phosphate and 0.25 percent of calcium carbonate, and the pH value is 7.2.
4. The method of claim 2, wherein the plant-derived carrier used in step S2 is 60-mesh corn cob meal or rice husk meal.
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