CN107099475B

CN107099475B - Bacillus methylotrophicus and preparation and application of microbial inoculum thereof

Info

Publication number: CN107099475B
Application number: CN201710395966.3A
Authority: CN
Inventors: 刘训理; 刘朝阳; 杨涛; 王晓辉; 纪超; 安雪; 王爽; 隋君康; 王昌乾; 李倩
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2017-05-31
Filing date: 2017-05-31
Publication date: 2021-03-30
Anticipated expiration: 2037-05-31
Also published as: CN107099475A

Abstract

The invention provides a methylotrophic bacillus and preparation and application of a microbial inoculum thereof, belonging to the technical field of agricultural microorganisms. The bacillus methylotrophicus HZ-9 is preserved in China general microbiological culture Collection center in 2017, 4 and 13 months, and the preservation number is CGMCC No. 14022. The bacterial strain has a wide antibacterial spectrum, has a strong inhibition effect on pathogenic bacteria causing anthurium bacterial leaf spot disease and pathogenic bacteria such as grape anthracnose pathogen, fusarium oxysporum, poplar canker pathogen, rhizoctonia solani, botrytis cinerea and the like, has a particularly remarkable inhibition effect on pathogenic bacteria causing anthurium bacterial leaf spot disease, and can stably colonize on the surfaces of anthurium rhizosphere, leaves and petioles; the microbial inoculum prepared by the strain can be applied to the prevention and control of diseases such as anthurium bacterial leaf spot and the like.

Description

Bacillus methylotrophicus and preparation and application of microbial inoculum thereof

Technical Field

The invention provides a methylotrophic bacillus and preparation and application of a microbial inoculum thereof, belonging to the technical field of agricultural microorganisms.

Background

Anthurium also called as anthurium, lucky flower, anthurium and the like is a plant of the genus anthurium in the family of the Araceae, has the origin in tropical rainforest areas in south America, and is widely cultivated in Asia, Europe, Africa and the like. The crop belongs to perennial evergreen herbaceous flowers, has peculiar flower type, bright and rich color and wide application range, is one of the famous and precious flowers which are very popular and popular in the world, has very high economic value, and is second to orchid in the global tropical flower trade.

In recent years, anthurium bacterial diseases show a remarkable increasing trend in the cultivation process of anthurium, and mainly comprise bacterial leaf spot, bacterial leaf blight and bacterial wilt. Especially, the growth trend of bacterial leaf spot is most obvious, the diffusion speed is high, the harm is large, and the great harm is caused to the anthurium industry. Anthurium bacterial leaf spot is a diseased heterogeneous species caused by the mosaic of the xanthomonas carpiXanthomonas axonopodis pv.dieffenbachiae) Causing a class of bacterial diseases. The disease has the main symptoms that water stain-shaped pit spots are distributed on the anthurium leaves at the beginning of infection, the disease spots are semitransparent at the initial stage of infection, wherein the disease spots of new leaves appear between veins, the spots are brown in color and are accompanied with clear edges, and the disease spots of old leaves usually appear at the edges of leaf parts and are arranged in small dots. When the air humidity is higher, the disease spots are rapidly increased, spots filled with brown pyogenic bacteria can be seen on the leaf back of the new leaf, when the air humidity is reduced, the pyogenic bacteria on the leaf back of the new leaf disappear, and the disease spots are changed into brown or black from brown and are dried up. Pathogenic bacteria infection can be spread to vascular bundles at the stem base from vascular bundles of the petioles, the vascular bundles are brown after infection, pus appears in the petioles, and the petioles are cut off, so that the pus can overflow from the vascular bundles. At this time, the severely affected anthurium leaves will be wilted and withered. If the pathogen infestation is transmitted to the base of the stem, it is likely that the entire plant will die. Bacterial leaf spot of anthuriumThe rapid onset and the high mortality rate, the morbidity rate of the anthurium bacterial leaf spot in the serious area reaches 100 percent, and the mortality rate also reaches more than 80 percent. The prevalence of the disease seriously affects the development of anthurium industry and restricts the production of anthurium.

At present, the main measures for preventing and treating the anthurium bacterial diseases are still chemical pesticide prevention and treatment. Although chemical control has the advantages of wide antimicrobial spectrum, low cost, convenient use, high efficiency, stability and the like, the problems of soil and air pollution, ecological balance damage, food safety threat, enhancement of resistance of pathogenic bacteria to pesticides and the like caused by the large-scale use of chemical bactericides are increasingly serious, so that the biological control method which is efficient, non-toxic, free of environment pollution and difficult to generate drug resistance is increasingly emphasized by people for controlling plant diseases. But biological control preparations and methods aiming at anthurium bacterial diseases are quite lacking.

Disclosure of Invention

The invention aims to provide a methylotrophic bacillus capable of preventing and controlling anthurium bacterial leaf spot and preparation and application of a microbial inoculum thereof.

The invention separates and obtains a strain of bacteria from anthurium rhizosphere soil, and the strain is identified as bacillus methylotrophicus (Bacillus methylotrophicus)Bacillus methylotrophicus) (ii) a The invention is named as bacillus methylotrophicus HZ-9. The strain is preserved in China general microbiological culture Collection center in 2017, 4 months and 13 days, and the preservation address is as follows: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences. The preservation number is CGMCC number 14022.

The bacillus methylotrophicus HZ-9 disclosed by the invention has a rod-shaped thallus, is of a facultative anaerobic fermentation type, is positive in reactions such as catalase, nitrate reduction, starch hydrolysis, V-P determination, gelatin liquefaction, citrate utilization, nitrogen source utilization and the like, is negative in methyl red test and phenylalanine deaminase test, can grow on LB solid culture media with NaCl mass concentration of 2% and 5%, and has a raised colony after growing for 12 hours, a dry surface and a white and opaque round shape.

The length of the 16SrDNA sequence of the bacillus methylotrophicus HZ-9 is 1454bp, and the specific sequence is shown as SEQ ID NO. 1.

The methylotrophic bacillus HZ-9 has a wide antibacterial spectrum, and has strong inhibition effects on pathogenic bacteria causing anthurium bacterial leaf spot disease and pathogenic bacteria such as grape anthracnose pathogen, fusarium oxysporum, poplar canker, rhizoctonia solani, botrytis cinerea and the like; the inhibitor has a remarkable effect on inhibiting pathogenic bacteria causing anthurium bacterial leaf spot, and can stably colonize the surfaces of anthurium roots, leaves and petioles; therefore, can be used for preventing and controlling anthurium bacterial leaf spot.

The bacillus methylotrophicus HZ-9 can be prepared into a liquid microbial inoculum or a solid microbial inoculum for application. Therefore, the invention also provides a method for producing a microbial agent by utilizing the bacillus methylotrophicus HZ-9, wherein the secondary seed solution of the bacillus methylotrophicus HZ-9 is inoculated to a fermentation culture medium for fermentation culture to obtain a liquid microbial agent, and the fermentation conditions are as follows: the rotating speed is 100-: 0.5-1.6, 0.05-0.06MPa pressure and 24-28h culture time.

The method comprises the steps of centrifuging the obtained liquid microbial inoculum at 6000-.

In the above method, the secondary seed solution of Bacillus methylotrophicus HZ-9 can be obtained by conventional method, or by the following steps:

(1) activating strains: inoculating bacillus methylotrophicus HZ-9 to an LB solid culture medium plate, and culturing at 30 ℃ for 20-24 h; selecting single colony, streaking again, transferring to LB solid culture medium plate, and culturing at 30 deg.C for 16-20 hr;

(2) preparing a first-level seed solution: inoculating the activated HZ-9 strain into an LB liquid culture medium, and performing shake culture at 30 ℃ for 16h at 200r/min for later use;

(3) preparing a secondary seed liquid: inoculating the first-stage seed solution into a fermentation culture medium at a rotation speed of 200-.

In the above-mentioned method, the first step is,

the ventilation amount is the ratio of the volume of air introduced per minute to the volume of fermentation liquor;

LB solid medium: 10g of peptone, 10g of sodium chloride, 5g of yeast powder, 15g of agar and 1L of distilled water, and sterilizing at 121 ℃ for 30 min;

LB liquid medium: 10g of peptone, 10g of sodium chloride, 5g of yeast powder and 1L of distilled water, and sterilizing at 121 ℃ for 30 min;

fermentation medium: 10g of glucose, 10g of starch, 5g of soluble corn steep liquor, 1.5g of ammonium sulfate, 0.5g of monopotassium phosphate, 1g of calcium carbonate and 1L of distilled water, and sterilizing at 121 ℃ for 30 min.

The microbial agent prepared by the invention is a liquid microbial agent or solid powder, and the active ingredients of the microbial agent are spores of bacillus methylotrophicus HZ-9 and extracellular metabolites thereof.

The invention also provides a specific use method of the microbial agent, which comprises the following steps: uniformly mixing the liquid microbial inoculum and water according to the volume ratio of 1:100, and irrigating roots; the solid microbial inoculum is applied by irrigating roots according to the dosage of 4-6 kg/mu; both the two microbial agents can be used as biological organic fertilizer and compound microbial fertilizer strains.

The invention has the following advantages:

1. the bacillus methylotrophicus HZ-9 strain has a wider antibacterial spectrum and can stably colonize in anthurium rhizosphere.

2. The microbial inoculum produced by the methylotrophic bacillus HZ-9 can effectively prevent and treat anthurium bacterial leaf spot, grape anthracnose, poplar rot, cucumber fusarium wilt and the like.

3. The bacillus methylotrophicus HZ-9 has no pollution, no ecological toxicity, good safety and stable passage antibacterial property.

4. The bacillus methylotrophicus HZ-9 has good fermentation character, the microbial inoculum production process is simple, the period is short, the cost is low, and the method is beneficial to industrial production and transportation.

Preservation information:

the preservation unit: china general microbiological culture Collection center;

address: the institute of microbiology, national academy of sciences, west road No.1, north Chen, Chaozhou, Chaoyang;

the preservation date is as follows: 13/4/2017;

the preservation number is: CGMCC No. 14022;

and (3) classification and naming: bacillus methylotrophicus (A), (B) and (C)Bacillus methylotrophicus)。

Drawings

FIG. 1 is a phylogenetic tree of the HZ-9 strain constructed using Mega5.05 analysis based on the 16SrDNA sequence; FIG. 1 shows that the HZ-9 strain and the known strainsBacillus methylotrophicus(JF899288) has homology of 16SrDNA up to 99%, and is identified as Bacillus methylotrophicus by combining the measurement results of the cell morphology, the colony characteristics and the physiological and biochemical indexes of the HZ-9 strain ((Bacillus methylotrophicus) ((JF 899288)Bacillus methylotrophicus)。

FIG. 2 shows the bacterial inhibition spectrum of Bacillus methylotrophicus HZ-9.

Detailed Description

In the following examples:

PDA solid medium: 200g of potato, 5g of beef extract, 20g of glucose, 15g of agar, (NH4)₂SO₄1g、MgSO₄1g、KH₂PO₄0.6g、CaCO₃3g of agar powder, 15g of agar powder and 1L of distilled water, wherein the pH value is 6.8-7.2, and the mixture is sterilized for 30min at 121 ℃;

LB solid medium: 10g of peptone, 10g of sodium chloride, 5g of yeast powder, 15g of agar powder and 1L of distilled water, and sterilizing at 121 ℃ for 30 min;

LB liquid medium: 10g of peptone, 10g of sodium chloride, 5g of yeast powder and 1L of distilled water, and sterilizing at 121 ℃ for 30 min.

Example 1

1. Isolation of HZ-9 Strain

Collecting 5g of Anthurium rhizosphere soil sample under sterile condition, dissolving in 45mL of sterile water, oscillating at 28 deg.C and 220r/min for 1h, sucking 0.5mL and 4.5mLMixing with water to obtain 10^-10.5mL of 10^-1The diluted solution is mixed with 4.5mL of sterile water to obtain 10^-2The dilution of (1) is prepared by analogy to dilution, and 10^-3、10^-4、10^-5、10^-6And a series of dilutions. Will 10^-4、10^-5、10^-6The dilution liquid is respectively sucked by a pipette to be 100 mu L and added into the center of a PDA solid culture medium plate, the dilution liquid is evenly smeared on each PDA solid culture medium plate by a spreader, and the PDA solid culture medium plate is cultured for 1 to 3 days at the constant temperature of a biochemical incubator at 37 ℃. Single colonies of each strain were picked with an inoculating loop in a plate, streaked on an LB solid medium plate to form a pure culture, and stored.

Carrying out a plate confrontation test on a plurality of pure cultures, observing the bacteriostasis condition by taking anthurium bacterial leaf spot germs as indicator bacteria, measuring the width of the bacteriostasis band, screening to obtain a strain with strong bacteriostasis effect, namely an HZ-9 strain, and sending the strain to the common microorganism center of China Committee for culture Collection of microorganisms and strains for preservation in 2017, 4 and 13 days, wherein the preservation number is CGMCC No. 14022.

2. Identification of HZ-9 Strain

(1) Morphology and colony characteristics of thallus

After the HZ-9 strain grows for 12h on LB solid medium: the colony is raised, and the surface is dry and is white and non-transparent round; the thallus is rod-shaped and has no flagellum.

(2) Physiological and biochemical characteristics

The physiological and biochemical characteristics of the HZ-9 strain are shown in Table 1, the strain is gram-positive bacteria, is of a facultative anaerobic fermentation type, and is positive in reactions such as catalase, nitrate reduction, starch hydrolysis, V-P determination, gelatin liquefaction, citrate utilization, nitrogen source utilization and the like; methyl red test and phenylalanine deaminase test are negative; can grow in LB liquid culture medium with NaCl concentration of 2% and 5%.

TABLE 1 physiological and biochemical characteristics of HZ-9 Strain

Note: "+" indicates positive, and "-" indicates negative.

(3) Sequence analysis of 16S rDNA of strain

The length of the 16S rDNA sequence of the HZ-9 strain is 1454bp, as shown in SEQ ID NO.1, the sequence is compared with the sequence in a GenBank database by Blast analysis, and the strains with higher homology with the sequence are all found to belong to the bacillus methylotrophicus. 10 strains with higher sequence similarity with the HZ-9 strain are selected for phylogenetic analysis, and a Neighbor-Joining method is adopted to construct a phylogenetic tree (shown in figure 1) based on a 16S rDNA sequence by utilizing Mega5.05 software. 16S rDNA sequence of HZ-9 strain is publishedBacillus methylotrophicus(JF899288) has homology of 16S rDNA up to 99%, and is identified as bacillus methylotrophicus by combining the bacterial morphology, the bacterial colony characteristics and the physiological and biochemical characteristicsBacillus methylotrophicus）。

Example 2

Determination of the bacteriostatic Profile of the HZ-9 Strain

A loop of purified thallus is picked by inoculating loops, the thallus is spotted at the position 3cm away from the two ends of the center of a PDA flat plate, 5 pathogenic fungi of grape anthracnose pathogen, fusarium oxysporum, poplar rot pathogen, rhizoctonia solani and botrytis cinerea stored in a laboratory are taken as targets, a bacterial disc with the diameter of 5mm is cut by a puncher and inoculated in the center of the PDA flat plate, and the PDA flat plate is cultured for 3-5 days at 28 ℃, and the test result shows that the HZ-9 strain has good antagonistic effect on the grape anthracnose pathogen, fusarium oxysporum, poplar rot pathogen, rhizoctonia solani and botrytis cinerea, and the test result is shown in a table 2 and an attached figure 2.

TABLE 2 antagonistic Effect of HZ-9 strains on various plant pathogenic fungi

Note: "+" indicates the width of the bacteriostatic band is less than 2mm, "+" indicates the width of the bacteriostatic band is 2-5mm,

"+ + + +" indicates a width of the zone of inhibition >5 mm.

Example 3

1. Determination of colonization ability of HZ-9 strain in anthurium rhizosphere and surfaces of leaves and petioles

(1) Double antibiotic screening and stability detection of HZ-9 strain

The HZ-9 strain is inoculated into an LB liquid culture medium and is subjected to shaking culture at 30 ℃ and 200r/min for 24h to serve as a seed solution. Inoculating the seed liquid into LB liquid culture medium containing 5 mug/mL rifampicin with the inoculation amount of 2% (volume percentage), and shaking-culturing at 30 ℃ and 200r/min for 24 h; inoculating the culture solution into LB liquid culture medium containing 10 ug/mL rifampicin with an inoculum size of 2% (volume percentage), and shake culturing for 24 h; and by analogy, gradually increasing the concentration of rifampicin from 5 [ mu ] g/mL, 10 [ mu ] g/mL, 20 [ mu ] g/mL, 40 [ mu ] g/mL, 80 [ mu ] g/mL and 150 [ mu ] g/mL to 300 [ mu ] g/mL, and culturing to obtain the rifampicin-resistant strain.

Inoculating the obtained rifampicin resistant strain to an LB liquid culture medium containing 300 mug/mL rifampicin and 5 mug/mL spectinomycin, and screening the rifampicin and spectinomycin resistant double-resistant strain, wherein the concentration of rifampicin in the LB liquid culture medium is 300 mug/mL all the time in the screening process, and the concentration of spectinomycin is gradually increased to 300 mug/mL from 5 mug/mL, 10 mug/mL, 20 mug/mL, 40 mug/mL, 80 mug/mL and 150 mug/m, so that an initial double-resistant marker strain simultaneously resistant to rifampicin and spectinomycin is finally obtained.

And alternately culturing the screened double-antibody marker mutant strain in an LB solid culture medium and an LB liquid culture medium which do not contain rifampicin and spectinomycin for 3-5 generations, and then returning the strain to the LB liquid culture medium containing rifampicin 300 mug/mL and spectinomycin 300 mug/mL for detection to verify the stability of drug resistance of the strain, thereby finally obtaining the double-antibody marker strain which can resist rifampicin and spectinomycin and can be stably inherited.

(2) Colonization test

Inoculating the HZ-9 strain marked by double antibodies into an LB liquid culture medium, and culturing at 30 ℃ and 200r/min for 18-20h to obtain a seed solution. Inoculating the seed solution into LB liquid culture medium containing 300 mug/mL of rifampicin and 300 mug/mL of spectinomycin at the same time in an inoculation amount of 2% (volume percentage), and performing shaking culture at 30 ℃ and 200r/min for 18-20 h; then inoculating the culture solution into LB liquid culture medium with the inoculation amount of 2% (volume percentage), and carrying out shaking culture at 30 ℃ for 18-20h at 200r/mim to obtain the HZ-9 strain liquid marked by double antibodies.

According to the invention, the colonization ability of the HZ-9 strain on the anthurium rhizosphere, the surfaces of the leaves and the petioles is researched through a pot experiment.

Colonization test of HZ-9 strain in anthurium rhizosphere

The inner diameter of the flowerpot is 22cm, 1 anthurium is planted in each pot, and the planting is repeated for 3 pots. Removing surface soil at the root of anthurium andraeanum, inoculating 5mL of cultured double-antibody-labeled HZ-9 strain bacterial liquid into each pot, and then covering the surface soil well again. Collecting the anthurium rhizosphere soil samples respectively on 15 days, 30 days, 45 days and 60 days after the inoculation of the bacterial liquid. Weighing 5g of anthurium rhizosphere soil in an aseptic environment, adding the 5g of anthurium rhizosphere soil into a triangular flask filled with 45mL of sterile water, uniformly mixing, oscillating at a constant temperature of 28 ℃ for 1h to fully disperse bacterial cells, sucking 0.5mL of sterile water and uniformly mixing with 4.5mL of sterile water to obtain 10^-1Diluting the solution; to a small tube containing 4.5mL of sterile water was added 0.5mL10^-1Diluting the solution, and mixing to obtain 10^-2Diluting the solution; and so on to obtain 10^-3、10^-4、10^-5、10^-6、10^-7、10^-8And a series of dilutions. And (3) respectively adding 100 mu L of diluent with different concentrations into the center of a double-antibody flat plate, uniformly coating the double-antibody flat plate by using an applicator, and counting colonies after culturing for 2d in an incubator at 30 ℃, wherein the test results are shown in Table 3. As is clear from Table 3, the number of HZ-9 colonies in the rhizosphere soil of anthurium was 3.2X 10 at day 15 after inoculation of the HZ-9 strain⁸cfu/g; on day 30, the number of HZ-9 colonies in the anthurium rhizosphere soil was 5.2X 10⁷cfu/g; on the 45 th day, the number of HZ-9 colonies in the anthurium rhizosphere soil is slightly reduced; at day 60, the number of HZ-9 colonies in the anthurium rhizosphere soil was 2.0X 10⁷cfu/g, basically maintains stable. According to the tracking of the number of colonies colonized by the HZ-9 strain in the rhizosphere of anthurium for 60 days, the HZ-9 strain is shown to be capable of stably colonizing the rhizosphere of anthurium.

TABLE 3 colonization results of the HZ-9 strain at the anthurium rhizosphere

Colonization test of HZ-9 strain on surfaces of anthurium leaf and petiole

The inner diameter of the flowerpot is 22cm, 1 anthurium is planted in each pot, and the planting is repeated for 3 pots. And uniformly spraying the cultured double-antibody marked HZ-9 strain liquid on the surfaces of the leaves and the petioles by using a small sprayer, and spraying 5mL of the double-antibody marked HZ-9 strain liquid on each plant. And respectively collecting leaf blade and petiole samples on 7 days, 14 days, 21 days and 28 days after the bacterial liquid is sprayed. Weighing 5g and 1g of leaves and petioles respectively in a sterile environment, adding the leaves and the petioles respectively into triangular flasks filled with 45g and 9g of sterile water, oscillating the mixture at a constant temperature of 28 ℃ for 30min, diluting and coating the liquid in a gradient manner on a double-antibody flat plate (the method is the same as a rhizosphere colonization test), and counting colonies after culturing the liquid in an incubator at 30 ℃ for 2 days, wherein the test results are shown in Table 4. As is clear from Table 4, the number of colonies colonized on the leaf was 2.2X 10 on day 7 after inoculation with the HZ-9 strain⁵cfu/g, petiole colonizing colony number 5.0X 10⁴cfu/g; on day 14, the number of colonized colonies on the leaf was 9.6X 10⁴cfu/g, the number of petiole colonizing colonies decreased to 4.2X 10²cfu/g; on day 21, the number of colonized colonies on the leaves decreased to 5.0X 10³cfu/g, the number of petiole colonizing colonies decreased to 3.1X 10²cfu/g; on day 28, the number of colonized colonies on leaves was 1.5X 10³cfu/g, petiole colonizing colony number 2.9X 10²cfu/g, basically maintains stable. According to the tracking of the cloning quantity of the HZ-9 strain on 28 days of anthurium leaves and petioles, the HZ-9 strain is shown to be capable of stably cloning on the surfaces of the anthurium leaves and the petioles.

TABLE 4 colonization results of anthurium leaf and petiole by HZ-9 strain

Time (sky)	Colony number of colonized leaf (cfu/g)	Colony count for petiole colonization (cfu/g)
			7	2.2×10⁵	5.0×10⁴
14	9.6×10⁴	4.2×10²
			21	5.0×10³	3.1×10²
28	1.5×10³	2.9×10²

Example 4

Optimization of HZ-9 strain optimal spore-producing fermentation medium formula

The basic formula of the HZ-9 strain is selected based on the principles of convenient culture medium material source, low cost, simple components and the like, and is obtained by screening from the laboratory empirical fermentation formula. Glucose is selected as a quick-acting carbon source, starch and corn steep liquor are selected as a slow-acting carbon nitrogen source, and three inorganic salts, namely calcium carbonate, ammonium sulfate and potassium dihydrogen phosphate, are respectively selected to carry out 6-factor 3-level orthogonal test design, wherein the test factors and the level setting are shown in table 5.

Inoculating HZ-9 strain into a triangular flask containing 50mL LB liquid medium, culturing at 30 deg.C and 200r/min to logarithmic scaleAnd then inoculating the bacillus subtilis into a fermentation medium (the formula is shown in table 5), wherein the inoculation amount is 2 percent (volume percentage) of the fermentation medium, culturing at 30 ℃ at 200r/min until the spore conversion rate is observed to be more than 95 percent under a microscope, coating a flat plate, counting, and calculating the number of spores in the fermentation liquid. The analysis of the spore-forming fermentation results of the HZ-9 strain is shown in Table 6, and it can be seen from Table 6 that the number of spores in fermentation medium No. 17 reached 4.0X 10⁹cfu/mL, there was a significant difference in the number of colonies from other fermentation media. The optimal spore-producing fermentation medium formula obtained finally is as follows: 10g of glucose, 10g of starch, 5g of corn steep liquor, 1.5g of ammonium sulfate, 0.5g of monopotassium phosphate, 1g of calcium carbonate and 1L of water.

TABLE 5 setting of factors and levels

TABLE 6 analysis of optimized data for spore-forming fermentation medium of HZ-9 strain

Note: the capital letters after the data are the multiple comparisons between treatments, α = 0.01.

Example 5

Preparation of bacillus methylotrophicus HZ-9 microbial inoculum

(1) Activating strains: streaking or coating the HZ-9 strain stored at low temperature on an LB solid culture medium plate, and culturing at 30 ℃ for 24-36 h; and selecting a single colony, streaking again, transferring to an upper plate of an LB solid culture medium, and culturing at 30 ℃ for 16-24h for later use.

(2) Preparing a first-level seed solution: scraping two rings of activated HZ-9 strain lawn by using an inoculating ring, inoculating the lawn into 50mL LB liquid culture medium, and shaking-culturing at 30 ℃ and 200r/min for 16h for later use.

(3) Preparing a secondary seed liquid: inoculating the first-stage seed liquid obtained in the step (2) into a 2-ton fermentation tank according to the inoculation amount of 1-2% (volume percentage)The second-stage seed liquid prepared by large-scale culture has rotation speed of 120r/min, temperature of 30-32 deg.C, and ventilation capacity of 6m³Culturing for 12-16h under 0.05-0.06MPa to obtain secondary seed solution; the culture medium is fermentation medium.

(4) Fermentation culture: inoculating the secondary seed liquid obtained in the step (3) into a 20-ton fermentation tank according to the inoculation amount of 10% (volume percentage) for fermentation, wherein the rotation speed is 120r/min, the temperature is 30-32 ℃, and the aeration amount is 60m³H, the tank pressure is 0.05-0.06MPa, the culture time is 24-28h, the spore yield is more than 90%, and the liquid microbial inoculum of the HZ-9 strain is obtained; the culture medium is fermentation medium.

(5) Preparing a solid microbial inoculum: centrifuging fermented HZ-9 strain fermentation liquor by a disc centrifuge 6700r/min, collecting all precipitates, pumping into a stirrer, slowly adding corn flour serving as a carrier matrix, adding 6-10% (w/w) of the corn flour to the precipitates, uniformly stirring, conveying to a centrifugal spray dryer, dispersing by an atomizer, quickly evaporating to dryness by high-temperature air to form a powdery product from solid matters in feed liquid, and finally collecting by a cyclone separator to obtain a solid microbial inoculum of the HZ-9 strain; the number of living bacteria in the obtained solid microbial inoculum can reach 1200 hundred million cfu/g.

Example 6

Prevention and control effect of bacillus methylotrophicus HZ-9 on anthurium leaf spot

Preparing a bacterial solution of pathogenic bacteria of the anthurium leaf spot: scraping off Physalospora leaf spot with inoculating ring, adding into sterile water, and mixing to reach concentration of 1 × 10⁶cfu/mL. Selecting healthy anthurium seedlings with consistent growth and development, and dividing the healthy anthurium seedlings into three test treatments: (1) CK blank: no pathogenic bacteria and microbial inoculum are applied; (2) pathogen control: applying pathogenic bacteria without applying microbial inoculum; (3) and (3) treating a microbial inoculum: applying pathogenic bacteria and microbial inoculum. Each treated 20 seedlings of anthurium. Wherein, the pathogenic bacteria control group and the microbial inoculum treatment group are prepared by soaking the wounded root of the anthurium andraeanum seedling in a solution with the concentration of 1 × 10⁶cfu/mL of pathogenic bacteria liquid for 30 min; CK blank group, the damaged root of the anthurium andraeanum seedling is soaked in the same amount of clear water. Transplanting the anthurium seedlings into a plastic pot with the diameter of 22cm after soaking; microbial inoculum treatment group applied microbial inoculum (prepared in example 6)The liquid microbial inoculum is diluted by 50 times), each pot is 100mL, and the same amount of clear water is applied to a CK blank group and a pathogen control group; repeatedly applying the same amount of microbial inoculum and clear water every 7 days; during the test period, the management measures of each treated potted plant are consistent, the morbidity of each treated potted plant is observed every day, and when the morbidity of the pathogenic bacteria control group reaches more than 80%, the disease index of each treated potted plant is investigated, the plant height is measured, and the relative prevention effect is calculated. By tracking observation, when the anthurium andraeanum seedlings are transplanted for 20 days, the CK blank group has no disease, the pathogenic bacteria control group and the microbial inoculum treatment group start to have the disease, and the incidence of the anthurium andraeanum seedlings in the pathogenic bacteria control group reaches over 80 percent after the anthurium andraeanum seedlings are transplanted for 35 days. Investigating the disease index of each treatment, wherein the disease index of a pathogenic bacteria control group is 25.32, the relative prevention effect is 0, and the disease index of a microbial inoculum treatment group is 12.64, and the relative prevention effect is 50.1%; the results are shown in Table 7. The test result shows that the HZ-9 strain microbial inoculum has better prevention and treatment effect on anthurium bacterial leaf spot.

TABLE 7 prevention and control Effect of Bacillus methylotrophicus HZ-9 Strain on Anthurium leaf Spot

<110> Shandong university of agriculture

<120> bacillus methylotrophicus and preparation and application of microbial inoculum thereof

<160>1

<210>1

<211>1454

<212>DNA

<213> Artificial Synthesis

<400>1

AGGGCGGGCG TGCTAATACA TGCAAGTCGA GCGGACAGAT GGGAGCTTGC TCCCTGATGT 60

TAGCGGCGGA CGGGTGAGTA ACACGTGGGT AACCTGCCTG TAAGACTGGG ATAACTCCGG 120

GAAACCGGGG CTAATACCGG ATGGTTGTCT GAACCGCATG GTTCAGACAT AAAAGGTGGC 180

TTCGGCTACC ACTTACAGAT GGACCCGCGG CGCATTAGCT AGTTGGTGAG GTAACGGCTC 240

ACCAAGGCGA CGATGCGTAG CCGACCTGAG AGGGTGATCG GCCACACTGG GACTGAGACA 300

CGGCCCAGAC TCCTACGGGA GGCAGCAGTA GGGAATCTTC CGCAATGGAC GAAAGTCTGA 360

CGGAGCAACG CCGCGTGAGT GATGAAGGTT TTCGGATCGT AAAGCTCTGT TGTTAGGGAA 420

GAACAAGTGC CGTTCAAATA GGGCGGCACC TTGACGGTAC CTAACCAGAA AGCCACGGCT 480

AACTACGTGC CAGCAGCCGC GGTAATACGT AGGTGGCAAG CGTTGTCCGG AATTATTGGG 540

CGTAAAGGGC TCGCAGGCGG TTTCTTAAGT CTGATGTGAA AGCCCCCGGC TCAACCGGGG 600

AGGGTCATTG GAAACTGGGG AACTTGAGTG CAGAAGAGGA GAGTGGAATT CCACGTGTAG 660

CGGTGAAATG CGTAGAGATG TGGAGGAACA CCAGTGGCGA AGGCGACTCT CTGGTCTGTA 720

ACTGACGCTG AGGAGCGAAA GCGTGGGGAG CGAACAGGAT TAGATACCCT GGTAGTCCAC 780

GCCGTAAACG ATGAGTGCTA AGTGTTAGGG GGTTTCCGCC CCTTAGTGCT GCAGCTAACG 840

CATTAAGCAC TCCGCCTGGG GAGTACGGTC GCAAGACTGA AACTCAAAGG AATTGACGGG 900

GGCCCGCACA AGCGGTGGAG CATGTGGTTT AATTCGAAGC AACGCGAAGA ACCTTACCAG 960

GTCTTGACAT CCTCTGACAA TCCTAGAGAT AGGACGTCCC CTTCGGGGGC AGAGTGACAG 1020

GTGGTGCATG GTTGTCGTCA GCTCGTGTCG TGAGATGTTG GGTTAAGTCC CGCAACGAGC 1080

GCAACCCTTG ATCTTAGTTG CCAGCATTCA GTTGGGCACT CTAAGGTGAC TGCCGGTGAC 1140

AAACCGGAGG AAGGTGGGGA TGACGTCAAA TCATCATGCC CCTTATGACC TGGGCTACAC 1200

ACGTGCTACA ATGGACAGAA CAAAGGGCAG CGAAACCGCG AGGTTAAGCC AATCCCACAA 1260

ATCTGTTCTC AGTTCGGATC GCAGTCTGCA ACTCGACTGC GTGAAGCTGG AATCGCTAGT 1320

AATCGCGGAT CAGCATGCCG CGGTGAATAC GTTCCCGGGC CTTGTACACA CCGCCCGTCA 1380

CACCAGAGAG TTTGTAACAC CCGAAGTCGG TGAGGTAACC TTTATGGAGC CAGCCGCCGA 1440

AAGGTGACCA AGGT 1454

Claims

1. A methylotrophic Bacillus HZ-9 capable of preventing and controlling anthurium bacterial leaf spot is classified and named as Bacillus methylotrophicus with the preservation number of CGMCC No. 14022.

2. The Bacillus methylotrophicus HZ-9 according to claim 1, wherein the bacterial cells are rod-shaped, nonflagellated, facultative anaerobic fermentation type, catalase, nitrate reduction, starch hydrolysis, V-P assay, gelatin liquefaction, citrate utilization and nitrogen source utilization reaction are positive, methylred test and phenylalanine deaminase test are negative, and the bacterial colonies grow on LB solid medium with NaCl concentration of 2% and 5% by mass, and after 12 hours of growth, the colonies swell, and are dry on the surface, and are white, opaque and round.

3. The bacillus methylotrophicus HZ-9 according to claim 1 or 2, wherein the 16SrDNA sequence is as shown in SEQ ID No. 1.

4. Use of bacillus methylotrophicus HZ-9 according to any one of claims 1 to 3 for the prevention and control of bacterial leaf spot of anthurium.

5. A method for producing a microbial agent by using the Bacillus methylotrophicus HZ-9 of claim 1, 2 or 3, wherein a secondary seed solution of the Bacillus methylotrophicus HZ-9 is inoculated into a fermentation medium for fermentation culture to obtain a liquid microbial agent, and the fermentation medium comprises the following specific components: 10g of glucose, 10g of starch, 5g of soluble corn steep liquor, 1.5g of ammonium sulfate, 0.5g of monopotassium phosphate, 1g of calcium carbonate and 1L of distilled water; the fermentation conditions are as follows: the rotating speed is 100-: 0.5-1.6, 0.05-0.06MPa pressure and 24-28h culture time.

6. A microbial inoculant produced by the method of claim 5.

7. The microbial agent according to claim 6, wherein the active ingredient is a spore of Bacillus methylotrophicus HZ-9 and an extracellularly metabolite thereof.