CN106591185B

CN106591185B - Bacillus amyloliquefaciens plant subspecies and preparation and application of microbial inoculum thereof

Info

Publication number: CN106591185B
Application number: CN201611145315.0A
Authority: CN
Inventors: 刘训理; 王晓辉; 晁强; 王爽; 安雪; 纪超; 隋君康; 洒荣波
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2016-12-13
Filing date: 2016-12-13
Publication date: 2020-09-29
Anticipated expiration: 2036-12-13
Also published as: CN106591185A

Abstract

The invention relates to a bacillus amyloliquefaciens plant subspecies and preparation and application of a microbial inoculum thereof; the XH-9 strain is identified as the Bacillus amyloliquefaciens plant subspecies through the combination of the thallus morphology, the colony characteristics, the physiological and biochemical indexes and 16S rDNA sequence analysis, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO. 13151; the strain has strong antagonistic action on pathogenic fungi such as fusarium oxysporum, bipolaris tritici, fusarium pseudograminearum, colletotrichum gloeosporum, poplar canker and alternaria; is capable of stimulating plant growth by producing indoleacetic acid and 1-aminocyclopropane-carboxylic Acid (ACC) deaminase; can degrade cellulose; can be stably colonized at the rhizosphere of crops such as wheat, corn, hot pepper and the like; the microbial inoculum prepared by the strain can be applied to the prevention and the treatment of root rot diseases or other soil-borne fungal diseases of crops such as wheat, corn, hot pepper and the like, and has the dual functions of preventing diseases and promoting growth; the preparation process of the microbial inoculum is simple, the fermentation period is short, the cost is low, and the industrial production and transportation are facilitated.

Description

Bacillus amyloliquefaciens plant subspecies and preparation and application of microbial inoculum thereof

Technical Field

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

Background

Root rot is a general term for rot diseases of plant roots and stem bases caused by fungi, bacteria and nematodes, and can damage hundreds of host plants such as grains, vegetables, cotton, grasses, oil crops and the like. In recent years, with continuous cropping of land, straw returning and large-area implementation of land no-tillage all the year round, the occurrence of root rot is increasingly serious.

Root rot belongs to soil-borne diseases, mainly harms seedlings, can also cause diseases in the adult plant stage, and is difficult to control. The reasons for this include: (1) the etiology is complex. China has wide territory, different crop types, planting seasons and planting systems, different pathogen types and infection laws and difficult control; (2) resistant varieties are lacking. Most of the crop varieties popularized in production are susceptible varieties, and resistant varieties are few and breeding is difficult; (3) the pathogen is difficult to eradicate. The pathogens can live through the winter in the soil or on the diseased residues of the crops, can survive for many years in the soil, has poor prevention and control effect in short-term crop rotation, and is difficult to implement in most areas of China in long-term crop rotation and fallow; (4) the difficulty of identification and diagnosis is high. The diseases mainly damage the roots and stem bases of plants, the symptoms are hidden, the early identification of the onset of the plants is difficult, and the effective prevention and control difficulty is high; (5) the research on biological control and ecological regulation mechanism is insufficient, and the research and development of related products are delayed; (6) the chemical bactericide has short control period, generally has better control effect in the early stage, and has poor control effect in the middle and later stages until the pesticide effect disappears.

At present, the prevention and control of root rot diseases mainly depend on chemical agents, and long-term use of the chemical agents causes increase of pathogen resistance, poor prevention and control effect and environmental pollution. The microbial fertilizer can increase soil nutrient elements, effectively improve the microbial community structure of the rhizosphere of crops, inhibit the quantity of pathogenic bacteria in soil or interfere the infection of the pathogenic bacteria to host plants, improve the ecological environment of the soil, has double effects of preventing diseases and promoting growth, and has important significance for effectively reducing the harm of root rot diseases of the crops and ensuring national food safety and effective supply of agricultural products. Meanwhile, the microbial fertilizer also has the advantages of low cost, high fertilizer efficiency, no pollution, energy conservation and the like, and is an effective substitute for chemical fertilizers and pesticides.

Disclosure of Invention

The invention aims to provide a bacillus amyloliquefaciens plant subspecies and preparation and application of a microbial inoculum thereof; can be used for preventing and treating root rot of crops such as wheat, hot pepper, corn and the like and promoting the growth of crops.

The inventor separates a broad-spectrum antagonistic bacterium from wheat rhizosphere soil of Korean village wheat field in villages and towns of Taian city in Shandong province; the strain is preserved in China general microbiological culture Collection center (CGMCC) at 2016, 10 months and 26 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 NO. 13151. And (3) classification and naming: bacillus amyloliquefaciens subsp. plantarum; the 16S rDNA sequence is shown in SEQ. NO. 1.

The strain has a wider antagonistic spectrum, and has stronger inhibiting effects on fusarium, bipolaris tritici, colletotrichum gloeosporioides, alternaria alternata and the like which cause root rot diseases of crops such as wheat, corn, pepper and the like; capable of producing Indole Acetic Acid (IAA) and 1-aminocyclopropane-carboxylic Acid (ACC) deaminase; can degrade cellulose; can be stably colonized at the rhizosphere of crops such as wheat, corn, pepper and the like.

The active component of the microbial agent produced by using the bacillus amyloliquefaciens subspecies XH-9 is bacillus amyloliquefaciens subspecies XH-9 spores, and the microbial agent is a liquid microbial agent or a solid microbial agent.

A preparation method of a bacillus amyloliquefaciens subspecies XH-9 microbial inoculum comprises the following steps:

(1) activating strains: streaking or coating the bacillus amyloliquefaciens subspecies XH-9 preserved at low temperature on an LB solid culture medium plate, and culturing for 24-36h at 30-32 ℃; and selecting a single colony, streaking again, transferring to an LB solid culture medium plate, and culturing at 30-32 ℃ for 16-24h for later use. The LB solid medium comprises the following components: 10g of peptone, 5g of yeast powder, 10g of NaCl10g, 10g of agar and 1L of deionized water.

(2) Preparing a first-level seed solution: scraping the activated XH-9 strain lawn in the two-ring step 1) by using an inoculating ring, inoculating the bacterial lawn in an LB liquid culture medium, culturing at the temperature of 30-32 ℃ and 200-220r/m for 16h by shaking to obtain a first-level seed solution. The LB liquid culture medium comprises the following components: 10g of peptone, 5g of yeast powder, 10g of NaCl and 1L of deionized water.

(3) Preparing a secondary seed liquid: inoculating the primary seed liquid prepared in the step 2) and a secondary seed culture medium into a fermentation tank filled with the secondary seed culture medium according to the proportion of 1-2% (volume percentage) for fermentation culture; the rotation speed is 120r/m, the temperature is 30-32 ℃, and the ventilation volume is 6m³H, filling pressure of 0.05-0.06MPa, and culturing for 12-16h to obtain secondary seed liquid.

The secondary seed culture medium comprises the following components: 30g of bran, 10g of peptone, (NH)₄)₂SO₄1.5g，KH₂PO₄0.75g， CaCO₃6.0g，MgSO₄1.5g，NaCl 2.0g，MnSO₄0.25g, 1L of water; and (3) sterilization conditions: 121 ℃, 30min and 0.2MPa pressure.

(4) Fermentation culture: inoculating the secondary seed liquid obtained in the step 3) and a fermentation culture medium into a fermentation tank filled with the fermentation culture medium according to the proportion of 10 percent (volume percentage) for fermentation culture; the rotation speed is 120r/m, the temperature is 30-32 ℃, and the ventilation volume is 60m³And h, filling pressure is 0.05-0.06MPa, and the culture time is 24-28h, so that the XH-9 strain liquid microbial inoculum is obtained.

The fermentation medium comprises the following components: 30g of bran, 10g of peptone, (NH)₄)₂SO₄1.5g，KH₂PO₄0.75g，CaCO₃6.0g，MgSO₄1.5g，NaCl 2.0g，MnSO₄0.25g, 1L of water; and (3) sterilization conditions: 121 ℃, 30min and 0.2MPa pressure.

(5) Preparing a solid microbial inoculum: centrifuging the XH-9 strain liquid microbial inoculum obtained in the step 4) by a disc centrifuge 6000-containing 8000r/min, collecting precipitates, pumping the precipitates into a stirrer, slowly adding corn flour serving as a carrier matrix into the stirrer, wherein the amount of the added corn flour is 6 percent of the weight ratio of the XH-9 strain liquid microbial inoculum, uniformly stirring, conveying to an LPG80L centrifugal spray dryer, dispersing by an atomizer (about 60 ℃) and quickly evaporating to dryness by high-temperature air (181 ℃), and collecting solid matters in feed liquid by a cyclone separator to obtain the XH-9 strain solid microbial inoculum, wherein the number of living bacteria can reach 1800 hundred million/g.

Uniformly mixing the XH-9 strain liquid bacterial agent prepared in the step 4) with water according to the proportion of 1:100 (volume percentage), and irrigating roots; irrigating roots of the XH-9 strain solid microbial inoculum prepared in the step 5) according to the using amount of 4-6 kg/mu; the prepared XH-9 strain liquid microbial inoculum and the prepared XH-9 strain solid microbial inoculum can be used as biological organic fertilizer and compound microbial fertilizer strains.

The invention has the following advantages:

1. the XH-9 strain has wide antagonistic spectrum and can be stably colonized at the rhizosphere of crops such as wheat, corn, pepper and the like.

2. The microbial inoculum produced by the XH-9 strain can effectively prevent and treat root rot diseases or other soil-borne fungal diseases of various crops such as wheat, corn, hot pepper and the like caused by fusarium, wheat root rot hyphomycete, anthrax, alternaria and the like.

3. The XH-9 strain can produce growth promoting matters such as siderophin, indoleacetic acid (IAA), ACC deaminase and the like, and has the function of promoting the growth of crops while preventing and treating diseases.

4. The XH-9 strain has the capacity of degrading cellulose, is applied to returning wheat and corn straws to the field directly, and is favorable for the quick decomposition of the straws.

5. The XH-9 strain has good fermentation character, simple microbial inoculum production process, short period and low cost, and is beneficial to industrial production and transportation.

Drawings

FIG. 1 is a phylogenetic tree of XH-9 strain constructed using Mega4.0 analysis based on the 16SrDNA sequence. As can be seen from FIG. 1, 16SrDNA homology of the XH-9 strain and a known strain Bacillus amyloliquefaciens subsp.plantarum (CP000560) reaches 100 percent, and the strain is identified as a Bacillus amyloliquefaciens subsp.plantarum by combining the measurement results of thallus morphology, colony characteristics and physiological and biochemical indexes of the XH-9 strain.

FIG. 2 is an antibacterial spectrum of the XH-9 strain, which shows that the XH-9 strain can effectively inhibit plant pathogenic fungi such as fusarium oxysporum, colletotrichum gloeosporioides, alternaria alternata, helminthosporium cerealis, fusarium pseudograminearum, and populus tremula.

In fig. 2: a is the antagonism of XH-9 strains against Fusarium oxysporum; b is the antagonism of XH-9 strain to colletotrichum gloeosporioides; c shows the antagonistic effect of XH-9 strain on Poplar canker; d is the antagonism of XH-9 strain against Fusarium pseudograminearum; e picture shows antagonism of XH-9 strain to Helminthosporium tritici; f is antagonistic action of XH-9 strain on Alternaria alternata;

FIG. 3a is a proteolytic clearing circle of the XH-9 strain, indicating that the XH-9 strain is capable of producing proteinoid enzymes.

FIG. 3b is a cellulose Congo Red hydrolysis clearing circle of the XH-9 strain, indicating that the XH-9 strain is capable of producing cellulose-based hydrolase.

FIG. 3c is a clear band of β -glucan hydrolysis by the XH-9 strain, indicating that the XH-9 strain is able to efficiently hydrolyze β -glucan, one of the major components of the fungal cell wall.

FIG. 3d shows the hydrolysis test results of filter paper strips of XH-9 strain, which indicates that XH-9 strain can synthesize and secrete cellulose hydrolase and effectively hydrolyze cellulose substances during liquid fermentation.

FIG. 4a is a standard curve for measuring protease activity of XH-9 strain; determining the coefficient R²0.9923, meeting the measurement requirement; p < 0.0001, indicating that the model is extremely significant; the obtained linear regression equation is that y is 0.0492x +0.0038, which shows that the tyrosine concentration and the absorbance are in a linear relation, and the linear regression equation can be used for measuring the protease activity of the XH-9 strain.

FIG. 4b is a standard curve of enzyme activity measurement of XH-9 strain cellulase, determining coefficient R²0.9961, meets the measurement requirement; p < 0.0001, indicating that the model is extremely significant; the obtained linear regression equation is that y is 0.0014x-0.3858, which shows that the glucose concentration and the absorbance are in a linear relation, and the linear regression equation can be used for measuring the enzyme activity of the XH-9 strain cellulase.

FIG. 5 shows the results of experiments on the secretion of indoleacetic acid by XH-9 strain, which indicates that XH-9 strain can synthesize and secrete indoleacetic acid to directly promote plant growth.

FIG. 6 shows the test results of the XH-9 strain synthesizing and secreting ACC deaminase, which indicates that the XH-9 strain can synthesize and secrete ACC deaminase, hydrolyze a large amount of ethylene precursor substances accumulated by plants under adversity stress, and indirectly promote the growth of crops.

FIG. 7 shows the results of experiments on the production of siderophins by XH-9 strains, which indicates that XH-9 strains are capable of producing siderophins and promoting the absorption of trace iron ions in soil by crops, thereby indirectly promoting the growth of crops.

FIG. 8 is a quantitative PCR standard curve for dynamic monitoring of Fusarium oxysporum by real-time fluorescent quantitative PCR technique. Determining the coefficient R²0.9965, meets the measurement requirement; p < 0.0001, indicating that the model is extremely significant; the obtained linear regression equation is y-2.293 x +32.886, which shows that the concentration of the constructed recombinant plasmid of the fusarium oxysporum and the qRT-PCR cycle number are in a linear relationship, and the method can be used for monitoring the quantity of the fusarium oxysporum in soil in real time.

The specific implementation mode is as follows:

example one

1. Isolation of XH-9 Strain

Taking rhizosphere soil sample of wheat of Korean village and village and village of Taian city, Shandong province, taking 10g of soil sample under aseptic condition, dissolving in 90mL of sterile distilled water, performing shaking culture for 30min, sucking 1mL of sterile water and mixing with 9mL of sterile water to obtain 10^-11mL of the diluted solution of (1) to 10^-1The diluted solution is mixed with 9mL of sterile water to obtain 10^-2The dilution of (1) is prepared by analogy to dilution, and 10^-3， 10^-4，1^0-5，10^-6，10^-7And a series of dilutions. The selected dilution is 10^-5、10^-6、10^-7The soil diluent is respectively absorbed by 100uL of a liquid transfer machine in the center of a PDA culture medium flat plate, the diluent is evenly coated by a coater, and the soil diluent is cultured for 1 to 3 days at the constant temperature of 30 to 32 ℃. Picking single colony, streaking to obtain pure culture, and storing. The PDA culture medium comprises the following components: 200g of potato, 5.0g of beef extract, 20g of glucose, 15g of agar, (NH4)₂SO₄1.0g，MgSO₄1.0g，KH₂PO₄0.6g，CaCO₃3.0g, pH 6.8-7.2, 1L deionized water.

And (3) carrying out a flat plate confrontation test on the obtained pure cultures, observing the bacteriostasis condition, measuring the width of the bacteriostasis band, and screening to obtain the antagonistic bacterium XH-9 strain with wide antibacterial spectrum and strong antibacterial effect.

2. Identification of XH-9 strains

(1) Morphology and colony characteristics of thallus

Bacterial colonies of the XH-9 bacterial strain on the PDA plate are yellow, the surface is dry, the edge is irregular, tiny wrinkles are formed on the surfaces of the bacterial colonies along with the prolonging of the culture time, the middle of the bacterial colonies is slightly raised, and the shape of the bacterial body observed under a microscope is rod-shaped.

(2) Physiological and biochemical characteristics

Physiological and biochemical characteristics of the XH-9 strain are shown in Table 1. The strain has positive oxygen demand in reactions of facultative anaerobism, catalase, nitrate reduction, gelatin liquefaction, starch hydrolysis, citrate utilization and the like; V-P determination, methyl Red test, H₂The S test, the indole test, the glucose gas production test and the MR test are negative; can grow in liquid LB liquid culture medium with NaCl concentration of 2% and 5%.

TABLE 1 physiological and biochemical characteristics of the XH-9 Strain

(3) Sequence analysis of 16S rDNA of strain

The 16S rDNA sequence of the XH-9 strain is as follows:

CGACTTCGGG TGTTACAAAC TCTCGTGGTG TGACGGGCGG TGTGTACAAG GCCCGGGAAC 60

GTATTCACCG CGGCATGCTG ATCCGCGATT ACTAGCGATT CCAGCTTCAC GCAGTCGAGT 120

TGCAGACTGC GATCCGAACT GAGAACAGAT TTGTGGGATT GGCTTAACCT CGCGGTTTCG 180

CTGCCCTTTG TTCTGTCCAT TGTAGCACGT GTGTAGCCCA GGTCATAAGG GGCATGATGA 240

TTTGACGTCA TCCCCACCTT CCTCCGGTTT GTCACCGGCA GTCACCTTAG AGTGCCCAAC 300

TGAATGCTGG CAACTAAGAT CAAGGGTTGC GCTCGTTGCG GGACTTAACC CAACATCTCA 360

CGACACGAGC TGACGACAAC CATGCACCAC CTGTCACTCT GCCCCCGAAG GGGACGTCCT 420

ATCTCTAGGA TTGTCAGAGG ATGTCAAGAC CTGGTAAGGT TCTTCGCGTT GCTTCGAATT 480

AAACCACATG CTCCACCGCT TGTGCGGGCC CCCGTCAATT CCTTTGAGTT TCAGTCTTGC 540

GACCGTACTC CCCAGGCGGA GTGCTTAATG CGTTAGCTGC AGCACTAAGG GGCGGAAACC 600

CCCTAACACT TAGCACTCAT CGTTTACGGC GTGGACTACC AGGGTATCTA ATCCTGTTCG 660

CTCCCCACGC TTTCGCTCCT CAGCGTCAGT TACAGACCAG AGAGTCGCCT TCGCCACTGG 720

TGTTCCTCCA CATCTCTACG CATTTCACCG CTACACGTGG AATTCCACTC TCCTCTTCTG 780

CACTCAAGTT CCCCAGTTTC CAATGACCCT CCCCGGTTGA GCCGGGGGCT TTCACATCAG 840

ACTTAAGAAA CCGCCTGCGA GCCCTTTACG CCCAATAATT CCGGACAACG CTTGCCACCT 900

ACGTATTACC GCGGCTGCTG GCACGTAGTT AGCCGTGGCT TTCTGGTTAG GTACCGTCAA 960

GGTGCCGCCC TATTTGAACG GCACTTGTTC TTCCCTAACA ACAGAGCTTT ACGATCCGAA1020

AACCTTCATC ACTCACGCGG CGTTGCTCCG TCAGACTTTC GTCCATTGCG GAAGATTCCC1080

TACTGCTGCC TCCCGTAGGA GTCTGGGCCG TGTCTCAGTC CCAGTGTGGC CGATCACCCT1140

CTCAGGTCGG CTACGCATCG TCGCCTTGGT GAGCCGTTAC CTCACCAACT AGCTAATGCG1200

CCGCGGGTCC ATCTGTAAGT GGTAGCCGAA GCCACCTTTT ATGTCTGAAC CATGCGGTTC1260

AGACAACCAT CCGGTATTAG CCCCGGTTTC CCGGAGTTAT CCCAGTCTTA CAGGCAGGTT1320

ACCCACGTGT TACTCACCCG TCCGCCGCTA ACATCAGGGA GCAA 1364

performing Blast analysis comparison on the sequence and a sequence in a Genbank database, finding that strains with higher homology belong to the genus Bacillus, selecting 7 strains with higher sequence similarity with the XH-9 strain to perform phylogenetic analysis, and constructing a phylogenetic tree (shown in figure 1) based on a 16SrDNA complete sequence by using Mega4.0 software and adopting a Neighbor-Joining method. The 16SrDNA sequence of the XH-9 strain has 100 percent of homology with the publicly published 16SrDNA of Bacillus amyloliquefaciens subsp.plantarum (CP000560), and is identified as a Bacillus amyloliquefaciens subsp.plant (Bacillus amyloliquefaciens subsp.plantarum) XH-9 by combining the thallus morphology, the colony characteristics and the physiological and biochemical characteristics.

Example two

Determination of the bacteriostatic Profile of the XH-9 Strain

A loop of purified thallus is picked by using inoculating loops, the thallus is spotted at the positions 3cm away from the two ends of the center of a PDA flat plate, 8 pathogenic fungi including fusarium oxysporum, colletotrichum gloeosporum, alternaria alternata, bipolaris tritici, fusarium graminearum, fusarium pseudograminearum, and staphylococcus are taken as targets, a bacterial disc with the diameter of 5mm is cut by a puncher and inoculated to the center of the PDA flat plate, and the bacterial disc is cultured for 2-3d at 28 ℃, so that the test result shows that the XH-9 bacterial strain has better antagonistic effect on 6 pathogenic fungi including fusarium oxysporum, colletotrichum gloeosporum, fusarium pseudograminearum, alternaria alternata and the like, the test result is shown in a table 2, and the antagonistic spectrum of the XH-9 bacterial strain is shown in an attached figure 2.

TABLE 2 relative inhibition rates of XH-9 strains on various pathogenic fungi

EXAMPLE III

1. Determination of colonization ability of XH-9 strain in rhizosphere of wheat, pepper and corn

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

Scraping the XH-9 strain lawn cultured by the 2-ring plate, inoculating the lawn into LB liquid culture medium without rifampicin, culturing at 30-32 ℃ for 24h at 200-220r/m, and taking the lawn as seed liquid. Inoculating the seed liquid into LB liquid culture medium containing 5 mug/mL rifampicin (Rif) with the inoculation amount of 2% (volume ratio), and performing shake flask culture 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; by analogy, the rifampicin concentration was gradually increased from 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL, 80. mu.g/mL, 150. mu.g/mL to 300. mu.g/mL, and the strains resistant to rifampicin were cultured. Inoculating the obtained rifampicin resistant strain to LB liquid culture medium containing 300 mug/mL rifampicin and 5 mug/mL spectinomycin (Spe), screening the dual-resistant strain simultaneously resisting rifampicin and spectinomycin, wherein the concentration of rifampicin in LB liquid culture medium is 300 mug/mL, the concentration of spectinomycin is increased and the screening method is the same as the screening method of rifampicin resistant strain, and finally obtaining the initial dual-resistant labeled strain simultaneously resisting rifampicin and spectinomycin.

The screened double-antibody marker strain is alternately cultured in LB solid culture medium and LB liquid culture medium without rifampicin and spectinomycin for 3-5 generations, and then respectively returned to LB liquid culture medium with rifampicin 300 mug/mL and spectinomycin 300 mug/mL for culture, so as to verify the drug resistance stability of XH-9 strain, and finally obtain the double-antibody marker strain which can stably inherit and simultaneously resist rifampicin and spectinomycin.

(2) Colonization test

Scraping the XH-9 dual-resistant marker strain lawn cultured by the 2-ring plate, inoculating the lawn to an LB liquid culture medium, and performing shaking culture at the temperature of 30-32 ℃ and the pressure of 200-. Inoculating the seed liquid into LB liquid culture medium containing 300 mug/mL rifampicin and 300 mug/mL spectinomycin at the same time in an inoculation amount of 2% (volume ratio), and performing shaking culture at 30-32 ℃ and 200-220r/m for 18-20 h; then inoculating the culture solution into LB liquid culture medium with the inoculation amount of 2 percent (volume ratio), and carrying out shaking culture at the temperature of 30-32 ℃ and the temperature of 200-.

According to the invention, the colonization ability of the XH-9 strain on the rhizosphere of wheat, pepper and corn is researched through a pot experiment. The inner diameter of the flowerpot is 22cm, 5 wheat plants are planted in each pot, 3 pepper plants are planted in each pot, 1 corn plant is planted in each pot, and each crop is repeated for 3 times. And (3) removing surface soil of the roots of the seedlings when the plant height of the seedlings is about 10cm, respectively inoculating cultured XH-9 double-antibiotic marked strain bacterial solutions, inoculating 20mL bacterial solution in each pot, irrigating roots, and covering the surface soil. And respectively taking rhizosphere soil samples of crops such as wheat and the like at 10 th, 20 th, 30 th, 40 th and 50 th days after the inoculation of the bacterial liquid, and detecting the colonization condition of the double antibiotic marked XH-9 bacterial strain.

Weighing 10g of rhizosphere soil, adding the rhizosphere soil into a triangular flask filled with 90mL of sterile water, and oscillating for 30min at normal temperature to fully disperse soil particles and microorganisms. Sucking 1mL of soil suspension by a pipettor, adding into 9mL of sterile water, shaking and mixing uniformly to obtain 10^-2Soil dilution from 10^-2Absorbing 1mL of the soil diluent, adding the soil diluent into 9mL of sterile water, and fully and uniformly mixing to obtain 10^-3The soil dilution is analogized by analogy to prepare 10^-2、10^-3、10^-4Serial gradient soil dilution liquid is sucked by pipette 10^-4100uL of dilution, coating on PDA plate culture medium containing both spectinomycin 300 mug/mL and rifampicin 300 mug/mL, placing in 30 deg.C constant temperature incubator for 28-36h, counting bacterial colony, converting into bacterial number of dry soil per gram, the test result is shown in Table 3, the bacterial number in the inoculated 10 th day, wheat, corn and pepper rhizosphere soil is 5.70 × 10 respectively⁵cfu/g、3.22×10⁵cfu/g、4.63×10⁵cfu/g, 20d, the number of rhizobacteria of wheat is 4.08 × 10⁵cfu/g, the number of bacteria is slightly reduced, and the number of rhizosphere bacteria of the corn is 3.53 × 10⁵cfu/g, the number of bacteria is basically kept unchanged, and the number of rhizosphere soil-like bacteria of the pepper is reduced to 2.88 × 10⁴cfu/g, 30d, the number of rhizobacteria of wheat is reduced to 6.10 × 10⁴cfu/g, the number of corn rhizobacteria is 2.89 × 10⁵cfu/g, the number of bacteria is slightly reduced, and the number of bacteria at the rhizosphere of the pepper is 3.00 × 10⁴cfu/g, basically maintained, 40d, the number of rhizosphere of wheat is 4.35 × 10⁴cfu/g, basically maintained, and the number of rhizosphere bacteria of the corn is reduced to 8.02 × 10⁴cfu/g, the number of bacteria in the rhizosphere of pepper was 2.65 × 10⁴cfu/g, re-sampling at 50d, the number of rhizosphere of wheat is 6.26 × 10⁴cfu/g, the number of corn rhizobacteria is reduced to 1.02 × 10⁵cfu/g, the number of bacteria in the rhizosphere of Capsicum was 3.19 × 10⁴cfu/g. Tracking the amount of XH-9 colonization of the rhizosphere of wheat, corn and pepper by the XH-9 strain over multiple days indicates that the XH-9 strain is capable of stable colonization of the rhizosphere of wheat, corn and pepper.

TABLE 3 colonization results (cfu/g dry soil) of the XH-9 strain on the rhizosphere of wheat, maize and capsicum

Example four

Disease prevention and growth promotion mechanism of XH-9 strain

Antagonism is obtained. Antagonism is an important mechanism for antagonistic bacteria to prevent and treat diseases. Antagonistic bacteria inhibit or kill pathogenic microorganisms by synthesizing and secreting antibacterial substances to act on cell walls, cell membranes, protein synthesis systems, energy metabolism systems and the like of pathogenic fungi. Antibacterial substances mainly include two main classes: antibiotics (peptides, bacteriocins, etc.) and hydrolases (chitinase, protease, cellulolytic enzymes, dextranase, lysozyme, etc.).

The XH-9 strain provided by the invention can synthesize and secrete cellulose hydrolase, protease and β -glucan hydrolase, the XH-9 strain is respectively spotted and inoculated to a functional detection flat plate for culturing β -glucan, congo red cellulose and skim milk, and the like, the inverted culture is carried out for 2-3d at 30-32 ℃, the observation result shows that the XH-9 strain can generate obvious hydrolysis transparent rings on a cellulose congo red culture medium plate, a skim milk culture medium plate and a β -glucan culture medium plate, the test result shows that the XH-9 strain can generate cellulose hydrolase, protease and KH 36-glucan hydrolase, the test result shows that the XH-9 strain is inoculated to a Hocken culture medium added with XH-9 strain bars, the test result shows that the XH-9 strain can generate KH hydrolase, the strain is inoculated to a filter paper, the test result shows that the XH-9 strain does not inoculate cellulase, the strain can generate KH-200 fester strain per min, the strain is inoculated to 24h, 36h, the strain is inoculated to a WK-48 h, the strain is further inoculated to a filter paper, the enzyme activity test result shows that the XH-9 strain can be further tested by a test paper strain fermentation test method for testing cellulose hydrolase, the cellulose hydrolase degradation test method for further testing the cellulose hydrolase of a cellulose fermentation test method for testing the strain of a cellulose fermentation test paper, the test paper strain of 0.5-9 strain, the test paper strain of the₂PO₄0.1g，MgSO₄0.02g，(NH₄)₂SO₄0.3g, 1L deionized water; the liquid fermentation medium for protease activity determination comprises: glucose 5g, Na₂HPO₄0.1g, peptone 5g, Tween-801 mL, CaCl₂0.1g, deionized water 1L.

Beta-glucan medium: peptone 1.0g, beef extract 0.5g, NaCl0.5g, beta-glucan 0.005g, Congo red 0.01g, deionized water 1L and agar 25 g.

Heckson medium: KH (Perkin Elmer)₂PO₄1.0g，CaCl₂0.1g，MgSO₄·7H₂O 0.3g，NaCl 0.1g，FeCl₃0.01g， NaNO₃2.5g, pH 7.2-7.3, deionized water 1L, 121 deg.C, 20min sterilizing.

Promoting growth. Substances for promoting plant growth generally include indoleacetic acid (IAA), Gibberellin (GA), Cytokinin (CTK), cytokinin (BA), etc., and the XH-9 strain provided by the present invention is capable of producing indoleacetic acid (IAA). The specific implementation steps are as follows: the activated XH-9 strain was inoculated into TSB medium containing 5mmol/L tryptophan, cultured with shaking at 30 ℃ and 170rpm for 12 hours and 10000rpm at 4 ℃ for 5 minutes, 0.5mL of the sterile supernatant was taken, 1mL of Salkawski's color developer was added, and the mixture was allowed to stand at room temperature for 30 minutes to develop color, with the medium as a blank, and 3 replicates were performed. If IAA is produced, the color changes from yellowish to pink. The supernatant obtained by centrifuging the XH-9 strain fermentation broth, after adding the color-developing agent, changed the solution from yellow to pink, while the control group remained yellow, as shown in FIG. 5, it is shown that the XH-9 strain is capable of producing indoleacetic acid.

The TSB medium comprises the following components: 2.5g of glucose, 17g of tryptone, 3g of soybean peptone, 5g of NaCl and K₂HPO₄2.5g, 1L deionized water.

The Salkowski's color developing solution is as follows: 1mL of 0.5mol/LFeCl₃Adding to 50mL of 35% HCIO₄In solution.

ACC deaminase is produced. 1-aminocyclopropane-carboxylic Acid (ACC) deaminase, ACC deaminase for short, is one of indexes of plant growth promoting ability of plant growth promoting bacteria. Ethylene is an important plant hormone that regulates or stimulates different biological processes and plays a role as a signaling molecule in biological systems. The ethylene increase can be caused by environmental stresses such as high salinity, drought, waterlogging, heavy pollution, pathogen invasion and the like, but the excessive production of the ethylene can cause the growth and development of plants to be blocked or the plants to be dead, especially the plant seedlings. If rhizosphere functional bacteria can synthesize and secrete ACC deaminase, the direct precursor ACC of ethylene can be decomposed, so that the ethylene concentration is reduced, adverse reactions of plants to environmental stress are relieved, the growth and development of the plants are promoted, and the stress resistance of the plants is improved.

Experiments prove that the XH-9 strain provided by the invention can synthesize and secrete ACC deaminase. The specific implementation steps are as follows: after sterilization, 3mmol/L ACC (1-aminocyclopropane-carboxylic acid) was added to DF mineral salt medium without ammonium sulfate, fresh XH-9 single colonies were inoculated to this medium for 3 replicates, and a blank medium inoculated with an equal amount of sterile water was used as a control, and shaking cultured at 30 ℃ and 220r/min for 48h, and the results were observed. The results showed that the culture broth of XH-9 strain was significantly turbid compared to the control, see FIG. 6, i.e.it was shown that the XH-9 strain was able to produce ACC deaminase. The DF inorganic salt culture medium comprises: (NH)₄)₂SO₄2.0g，K₂HPO₄4.0g，Na₂HPO₄6.0g，MgSO₄·7H₂O 0.2g，FeSO₄·7H₂O 1.0mg，H₃BO₃10ug，MnSO₄·H₂O10ug，ZnSO₄70ug，CuSO₄·7H₂O 50ug，MoO₃·H₂O10 ug, 1L deionized water.

Competition effect. The competitive action comprises ecological niche competition and nutrient element competition. For example, biocontrol bacteria produce siderophore, also called siderophore, which can combine with the rare trivalent iron ion (Fe) in soil³⁺) So that the pathogenic bacteria lack iron nutrition and can not grow and reproduce normally, and the growth of the pathogenic bacteria is inhibited indirectly. The XH-9 strain provided by the invention can generate siderophin, thereby indirectly inhibiting the growth of pathogenic fungi. The XH-9 strain is spotted and inoculated to a CAS detection plate, the uninoculated CAS plate is used as a control, the culture is inverted at the temperature of 30 ℃ for 2-3d, and yellow halo appears if siderophin is produced. Spotting XH-9 strainsWhen cultured on CAS plates for 48h, yellow halos appear, as shown in FIG. 7, indicating that the XH-9 strain is capable of producing siderophiles and thereby indirectly inhibiting the growth and reproduction of pathogenic fungi. Preparation of CAS detection plate: 1mL of 20% sucrose, 3mL of 10% hydrolyzed casein, and 1mmol/L CaCl₂100uL，1mmol/L MgSO₄·7H₂02 mL, agar 1.8g, deionized water 100mL, autoclaving at 121 ℃ for 20min, cooling the culture medium to 60 ℃, slowly adding 0.1mol/L phosphate buffer solution with pH6.8 and 5mL of CAS detection solution respectively, mixing uniformly, and pouring into a flat plate. When the plate is solidified, point sample inoculation is carried out on the XH-9 strain.

Preparation of CAS detection solution:

solution A: 0.07g CAS is dissolved in 50mL deionized water, 10mL of 1mmol/L FeCl is added₃(containing 10mmol/L hydrochloric acid solution);

solution B: 0.06g of cetyltrimethylammonium bromide was dissolved in 40mL of deionized water;

solution C: and slowly adding the solution A into the solution B along the wall of the beaker, and fully and uniformly mixing the solution A and the solution B to obtain the CAS blue detection solution.

0.1mol/L phosphate buffer (pH 6.8): weighing Na₂HPO₄·12H₂O 2.427g，NaH₂PO₄·2H₂O0.5905g， KH₂PO₄0.075g，NH₄0.250g of Cl, 0.125g of NaCl and 100mL of deionized water, and the solution is diluted by 10 times when used.

EXAMPLE five

Optimization of bacterial fermentation medium of XH-9 strain

(1) Selection of basal Medium

The basic formula of the XH-9 strain is selected based on the principles of simple composition of the culture medium, convenient source, low cost and the like, and the basic culture medium formula obtained by screening the basic culture medium is as follows: 30g of bran, 15g of peptone, 1g of calcium chloride, 5g of sodium chloride and 1L of deionized water.

(2) Screening of organic Nitrogen sources

On the basis of the basic culture medium formula, peptone, yeast powder, beef extract, soybean cake powder, soybean meal and the like with the mass concentrations of 15g/L, 30g/L and 4g/L are selected,The results show that when peptone is used as the organic nitrogen source, the number of spores in the fermentation liquid is the highest and reaches 41.0 × 10⁸cfu/mL。

(3) Screening of inorganic Nitrogen sources

On the basis of a basic culture medium formula, NH with the mass concentration of 0.6g/L, 1.0g/L, 1.25g/L and 0.8g/L is selected₄NO₃、(NH₄)₂SO₄、NaNO₃、NH₄Cl is an inorganic nitrogen source, a fermentation medium without the inorganic nitrogen source is used as a reference, and the influence of the addition of different inorganic nitrogen sources in the medium on the yield of spores is compared. The results show that: (NH)₄)₂SO₄When the fermentation liquor is used as an inorganic nitrogen source, the number of spores in the fermentation liquor is the highest and reaches 50.0 × 10⁸cfu/mL。

(4) Screening of carbon sources

Respectively selecting lactose, maltose, glucose and sucrose with the addition concentrations of 5g/L, 10g/L, 15g/L and 5g/L (mass ratio) as quick-acting carbon sources, selecting bran, starch and corn flour with the concentrations of 20g/L, 30g/L and 40g/L to replace slow-acting carbon sources of a primary culture medium, taking a fermentation culture medium without a carbon source as a control, and comparing the influence of the addition of different carbon sources in the culture medium on the yield of spores⁸cfu/mL。

(5) Influence of different carbon and nitrogen source concentrations on spore number

The selected bran, peptone and ammonium sulfate are taken as three factors, each factor is set to three levels, as shown in table 4, and the concentrations of carbon and nitrogen sources in the fermentation medium are further optimized, as shown in table 5. Data were analyzed using SAS 9.0. The analysis result shows that the influence of the bran on the spore number is at an extremely significant level, (NH)₄)₂SO₄The effect on spore number was significant, while peptone affected spore number nearly significantly, see table 6.

TABLE 4 test factors and level design

TABLE 5 orthogonal experimental design and results of the influence of carbon and nitrogen source concentrations on the spore production number of XH-9 strains

Note: the data in the table are mean ± sd of 3 replicates. The data suffixes in the same row differ alphabetically to indicate significant differences (p <0.05) between different treatment groups, and the letters are the same to indicate insignificant differences between different treatment groups.

TABLE 6 SAS analysis table of test results of the influence of carbon and nitrogen source concentrations on the spore production number of XH-9 strain

(6) Effect of different inorganic salts on spore number

Selecting KH on the basis of the screened optimal nitrogen source and optimal carbon source₂PO₄、CaCO₃、MgSO₄、NaCl、MnSO₄The five inorganic salts were designed orthogonally, the experimental factors and the horizontal design are shown in Table 7, and the results of the optimization of the inorganic salts in the XH-9 strain fermentation medium are shown in Table 8. The influence of different inorganic salts on the spore production quantity of the fermentation medium is shown by the SAS analysis of the orthogonal test design result, and the influence degree of each factor on the spore production quantity of the fermentation medium is as follows: MgSO (MgSO)₄＞CaCO₃＞MnSO₄＞ KH₂PO₄Greater than NaCl. Wherein, MgSO4, CaCO₃、MnSO₄The influence on the spore production quantity of the culture medium at the level of 0.01 reaches an extremely remarkable level, KH₂PO₄NaCl was produced at 0.05 level on the mediumThe number of spores reached a significant level. The optimum inorganic salt combination is treated by the treatment 8, and the optimum spore-forming fermentation medium composition of the XH-9 strain is finally obtained: 30g of bran, 10g of peptone, (NH)₄)₂SO₄1.5g，KH₂PO₄0.75g，CaCO₃6.00g，MgSO₄1.5g，NaCl 2g，MnSO₄0.25g of deionized water and 1L of deionized water, the germination rate is more than 90 percent after fermentation culture for 36h, and the final counting result is 58.0 × 10⁸cfu/mL, SAS analysis results are shown in Table 9.

TABLE 7 test factors and level settings

TABLE 9 SAS analysis of test results of the effect of different inorganic salts on the spore production number of XH-9 strains

TABLE 8 orthogonal experimental design and results for the effect of different inorganic salts on the sporulation number of XH-9 strains

EXAMPLE six

Preparation of Bacillus amyloliquefaciens plant subspecies XH-9 microbial inoculum

(1) Activating strains: streaking or coating a low-temperature-stored bacillus amyloliquefaciens sub XH-9 plant on an LB solid culture medium flat plate, and culturing for 24-36h at the temperature of 30-32 ℃; and selecting a single colony, streaking again, transferring to an LB solid culture medium plate, and culturing at 30-32 ℃ for 16-24h for later use.

(2) Preparing a first-level seed solution: scraping two rings of activated bacterial lawn of the XH-9 bacterial strain obtained in the step 1) by using an inoculating ring, inoculating the bacterial lawn in an LB liquid culture medium, culturing at the temperature of 30-32 ℃ and 200-220r/m for 16h by shaking to obtain a first-level seed solution.

(3) Preparing a secondary seed liquid: mixing the first-stage seed liquid prepared in the step 2) and the second-stage seed culture medium according to the proportion of 1-2 percent (body)Volume percent) is inoculated into a fermentation tank filled with a secondary seed culture medium for fermentation culture; the rotation speed is 120r/m, the temperature is 30-32 ℃, and the ventilation volume is 6m³H, filling pressure of 0.05-0.06MPa, and culturing for 12-16h to obtain secondary seed liquid.

The formula of the secondary seed culture medium is consistent with that of the fermentation culture medium in the step 4).

(4) Fermentation culture: inoculating the secondary seed liquid obtained in the step 3) and a fermentation culture medium into a fermentation tank filled with the fermentation culture medium according to the proportion of 10 percent (volume percentage) for fermentation culture; the rotation speed is 120r/m, the temperature is 30-32 ℃, and the aeration amount is 60m³And h, filling pressure is 0.05-0.06MPa, and the culture time is 24-28h, so that the XH-9 strain liquid microbial inoculum is obtained.

The fermentation medium comprises the following formula components: 30g of bran, 10g of peptone, (NH)₄)₂SO₄1.5g，KH₂PO₄0.75g，CaCO₃6.0g，MgSO₄1.5g，NaCl 2.0g，MnSO₄0.25g, 1L of water; and (3) sterilization conditions: 121 ℃, 30min and 0.2MPa pressure.

Uniformly mixing the XH-9 strain liquid bacterial agent prepared in the step 4) with water according to the proportion of 1:100 (volume percentage), and irrigating roots; irrigating roots of the XH-9 strain solid microbial inoculum prepared in the step 5) according to the using amount of 4-6 kg/mu; both the two microbial agents can be used as biological organic fertilizer and compound microbial fertilizer strains.

EXAMPLE seven

Application of XH-9 microbial inoculum

(1) Pot test 1: antagonistic effect of XH-9 microbial inoculum on wheat rhizosphere fusarium oxysporum

Washing semen Tritici Aestivi with clear water, soaking in 75% ethanol for 10min, soaking in 30% sodium hypochlorite for 30-60s, washing with sterile water for 5-6 times, and air drying. Selecting uniform wheat seeds, sowing the wheat seeds in a plastic basin with the inner diameter of 22cm, repeating the steps for 3 times per basin, watering thoroughly, and cultivating conventionally. When the wheat seedlings grow to about 10cm, selecting the wheat seedlings with consistent growth vigor, applying a microbial inoculum for treatment, and setting a blank control without adding the microbial inoculum.

The preparation method of the fusarium oxysporum spore suspension comprises beating fusarium oxysporum cultured on a PDA plate into uniform bacterial cakes with a hole puncher with a diameter of 7cm, inoculating three bacterial cakes in each bottle into a Chaudou culture medium, performing constant-temperature shaking culture at 30-32 ℃ for about one week at 200-220r/m, filtering, and diluting with sterile water to a concentration of 1.2 × 10⁶cfu/mL。

A total of 3 treatments were designed for this trial. Treatment group: adding 20mL of each of fusarium oxysporum spore suspension and XH-9 liquid microbial inoculum; control CK 1: adding fusarium oxysporum spore suspension and 20mL of blank fermentation medium which is not inoculated with the XH-9 strain respectively; control CK 2: adding 20mL of fusarium oxysporum spore suspension and inactivated XH-9 liquid microbial inoculum respectively. After adding the fusarium in the soil, taking a wheat rhizosphere soil sample every 10 days, extracting total DNA of the soil, monitoring the quantity of the fusarium oxysporum in the soil by a real-time fluorescent quantitative PCR technology, wherein the test result is shown in a table 10, and a qRT-PCR standard curve for detecting the quantity of the fusarium oxysporum is shown in a table 8.

TABLE 10 real-time fluorescent quantitation PCR-Fusarium oxysporum number monitoring results

From the test results, the numbers of the control groups CK1 and CK2 of wheat rhizosphere fusarium oxysporum show the situation that the number of the wheat rhizosphere fusarium oxysporum firstly decreases and then increases at 10d, 20d, 30d, 40d and 50d, because the number of the fusarium oxysporum in the wheat rhizosphere soil is maintained at a higher concentration in a short time after a high-concentration fusarium oxysporum spore suspension is added into the wheat rhizosphere soil; with the time being prolonged, the pathogenic bacteria can be partially killed in the colonization process, and the number of the pathogenic bacteria is sharply reduced; when the colonization reaches the stationary phase, the surviving fusarium oxysporum starts to breed at the rhizosphere of the wheat, the quantity of the fusarium oxysporum is increased again, and the fusarium oxysporum is maintained at a relatively stable level. The SAS analysis of the test data shows that the number of fusarium in wheat seedlings at 10d, 20d and 30d in the control group CK1 and CK2 after the bacteria adding treatment is remarkably different, but the number of fusarium in wheat seedlings at 40d and 50d after the bacteria applying treatment is not remarkably different, probably because some secondary metabolites generated by the XH-9 strain in the fermentation process in the control group CK2 treatment still have high activity after the strain is inactivated, the fusarium oxysporum is inhibited after being applied to soil, and as the test time is prolonged, antagonistic substances are inactivated or consumed, the number of fusarium oxysporum begins to increase, and finally the number of fusarium oxysporum is consistent with that of the blank control CK 1. The wheat rhizosphere soil obtained by the treatment group is sampled at 5 different time points, and the monitored quantity of fusarium is obviously lower than that of CK1 and CK2 of the control group, so that the colonization of the XH-9 strain in the wheat rhizosphere can obviously inhibit the quantity of fusarium oxysporum in the wheat rhizosphere soil.

(2) Pot experiment 2: growth promotion test of XH-9 microbial inoculum

The treatment and sowing of wheat seeds were the same as in experiment 1. Let 2 groups of treatments, treatment group: inoculating XH-9 liquid microbial inoculum for sowing wheat in 20 mL/pot for 6 times; control CK: and (3) inoculating an inactivated XH-9 liquid microbial inoculum in 20 mL/pot for 6 times during wheat sowing. And when the wheat seedlings grow to 30 days, taking out the whole pots of the wheat seedlings of the treatment group and the control group, respectively and randomly selecting 20 wheat seedlings, carefully shaking off soil samples attached to the wheat rhizosphere, collecting the soil samples, washing the wheat rhizosphere with tap water, airing, and measuring the dry, fresh and heavy growth indexes of the wheat, wherein the test results are shown in a table 11. The results show that compared with the control group, the wheat in the treatment group has long root system and many branches, the average length of the root system reaches 13.0cm, the overground part is stronger than that of the blank control group, the leaf surface is wide and long, the color is dark green, the average height of the plant is 18.5cm, and the total fresh weight of 20 seedlings is 3.50 g; the average root length of wheat in the blank control group is 9.83cm, the average plant height is 16.20cm, and the total fresh weight of 20 seedlings is 3.01 g. Separating the overground part and the underground part of each group of randomly selected wheat seedlings, putting the wheat seedlings into a drying oven, drying the wheat seedlings to constant weight at the temperature of 80 ℃, and respectively measuring the dry weight and the root cap ratio of the wheat seedlings of the treatment group to the control group. The results show that the total dry weight of wheat seedlings of the treated group and the control group is 0.563g and 0.503g respectively, and the root-cap ratio of the wheat seedlings is 0.392 and 0.352 respectively. SAS analysis is carried out on all the data, and data analysis results show that the plant height, root length, fresh weight, dry weight and root cap of the wheat seedlings of the treatment group and the control group are obviously different from 5 growth amount indexes.

TABLE 11 measurement results of wheat seedlings growing 30d in treatment group and blank control group

The result of the experiment pot experiment 1 shows that the XH-9 microbial inoculum has effective disease prevention effect, and the result of the experiment pot experiment 2 shows that the XH-9 microbial inoculum has growth promotion effect, namely the XH-9 microbial inoculum provided by the invention has double effects of disease prevention and growth promotion.

(3) Plot experiment in the field

Pathogenic bacteria to be tested: helminthosporium tritici and Fusarium pseudograminearum are provided by Henan university of agriculture, and Fusarium oxysporum is stored in the laboratory. The solid culture medium of the above 3 pathogenic fungi comprises the following components: 65% of bran, 10% of soybean meal, 18% of rice hull, 5% of corn flour and CaCl₂0.2%, material: stirring with water at ratio of 10:8, sterilizing at 121 deg.C for 30min, and sterilizing intermittently for 3 times. Cooling, inoculating pathogenic fungi at a volume ratio of 10%Sub-suspensions (0.2-0.6 × 10)⁸Hundred million/mL) are stirred evenly in a sterile operation, the mixture is placed in a constant-temperature incubator to be cultured in a dark place, the samples are turned over and supplemented with proper moisture during the culture, hypha grows on the surface of the samples, the mixture is stirred and evenly mixed again, and the mixture is naturally air-dried or dried at 60 ℃.

The test was carried out at the experimental station of Shandong university of agriculture. Set 4 treatments in total, treatment 1: inoculating wheat root rot helminthosporium umbilicifolium and an XH-9 microbial inoculum; and (3) treatment 2: inoculating fusarium pseudograminearum, fusarium oxysporum and an XH-9 microbial inoculum; CK 1: inoculating wheat root rot helminthosporium umbilicifolium and an inactivated XH-9 microbial inoculum; CK 2: inoculating fusarium pseudograminearum, fusarium oxysporum and an inactivated XH-9 microbial inoculum. Each process is repeated 3 times, namely 12 cells are divided, and the area of each cell is set to be 9.0m²。

Application amount of pathogenic bacteria: 3.0kg of wheat root rot helminthosporium umbiliciformis solid culture per cell; fusarium oxysporum and Fusarium pseudograminearum solid cultures 2.0 kg/cell. Application amount of XH-9 liquid microbial inoculum: according to 80 mL/cell.

10 months in 2015, harvesting the corn, uniformly mixing the pathogenic bacteria solid culture into soil along with the whole land before wheat sowing, diluting XH-9 liquid microbial inoculum or inactivated microbial inoculum according to the volume ratio of 1:100 after wheat sowing for about 1 week, and irrigating roots. During the growth and development period (seedling emergence, green turning, jointing and maturation period) of wheat, the disease condition of wheat was investigated by chessboard sampling.

TABLE 12 field control test results for XH-9 inoculants

As can be seen from Table 12, the XH-9 fungicide has better control effect on both wheat root rot and stem base rot. The control effect on the wheat root rot is the highest at the seedling stage and reaches 84.20 percent, and then at the green turning stage, the jointing stage and the mature stage, the control effects are respectively 79.67 percent, 72.46 percent and 70.23 percent; the control effect on the stem base rot is that the control effects in the seedling emergence stage, the green turning stage, the jointing stage and the mature stage are respectively as follows: 78.49%, 67.53%, 63.00% and 64.56%.

From the field control effect test of wheat, the XH-9 microbial inoculum prepared by the Bacillus amyloliquefaciens subspecies XH-9 bacterial strain provided by the invention has the field control effect on the root rot and stem root rot of wheat in each growth period of the wheat kept above 63%. The XH-9 strain and the bacterial agent thereof have better inhibiting effect on pathogenic fungi such as fusarium oxysporum, bipolaris tritici, fusarium pseudograminearum, anthrax, poplar canker and the like, and have good market application prospect for effectively preventing and treating fungal diseases of crops.

<110> Shandong university of agriculture

<120> bacillus amyloliquefaciens plant subspecies and preparation and application of microbial inoculum thereof

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Claims

1. A bacillus amyloliquefaciens subsp. plantarum plant with the preservation number of CGMCC NO.13151 is XH-9, is preserved in the China general microbiological culture Collection center in 2016, 10 and 26 days, and has a 16S rDNA sequence shown in SEQ ID NO. 1;

bacterial colonies of Bacillus amyloliquefaciens subsp. plantarum) XH-9 on a PDA (personal digital assistant) plate are yellow, the surfaces of the bacterial colonies are dry, the edges of the bacterial colonies are irregular, tiny wrinkles are formed on the surfaces of the bacterial colonies along with the prolonging of culture time, the middles of the bacterial colonies are slightly raised, and the shapes of the bacteria observed under a microscope are rod-shaped;

the aerobic nature of the Bacillus amyloliquefaciens subsp. plantarum) XH-9 is facultative anaerobic; has salt tolerance and can grow in liquid LB culture medium with NaCl concentration of 2% and 5%.

2. A microbial agent comprising Bacillus amyloliquefaciens subsp.

3. The microbial agent of claim 2, for use in the control of wheat or corn root rot diseases.

4. The use of the microbial inoculant of claim 2 for the production of indoleacetic acid and 1-aminocyclopropane-carboxylic Acid (ACC) deaminase to stimulate plant growth and to control diseases caused by fusarium oxysporum, fusarium pseudograminearum, helminthosporium tritici, alternaria alternata, colletotrichum gloeosporioides, populus tremula.

5. The method for preparing a microbial agent according to claim 2, comprising the steps of:

1) activating strains: inoculating XH-9 of Bacillus amyloliquefaciens subsp (plant) with preservation number of CGMCC NO.13151 preserved at low temperature on an LB solid culture medium plate in a streaking way or a coating way, and culturing for 24-36h at 30-32 ℃; selecting single colony, streaking again, transferring to LB solid culture medium plate, and culturing at 30-32 deg.C for 16-24 hr;

2) preparing a first-level seed solution: scraping two rings of the activated bacterial lawn of the XH-9 strain in the step 1) by using an inoculating ring, inoculating the bacterial lawn in an LB liquid culture medium, culturing at the temperature of 30-32 ℃ and 200-220r/m for 16h by shaking to obtain a first-level seed solution;

3) preparing a secondary seed liquid: inoculating the primary seed liquid prepared in the step 2) and a secondary seed culture medium into a fermentation tank filled with the secondary seed culture medium according to the volume percentage of 1-2% for fermentation culture; the rotation speed is 120r/m, the temperature is 30-32 ℃, the ventilation volume is 6m3/h, the irrigation pressure is 0.05-0.06MPa, and the culture is carried out for 12-16h to be used as secondary seed liquid;

4) fermentation culture: will step withInoculating the secondary seed liquid obtained in the step 3) and a fermentation culture medium into a fermentation tank filled with the fermentation culture medium according to the volume percentage of 10% for fermentation culture; the rotation speed is 120r/m, the temperature is 30-32 ℃, and the ventilation volume is 60m³H, filling pressure is 0.05-0.06MPa, and culture time is 24-28h, so as to obtain an XH-9 strain liquid microbial inoculum;

5) preparing a solid microbial inoculum: centrifuging the XH-9 strain liquid bacterial agent obtained in the step 4) by a disc centrifuge 6000-containing 8000r/min, collecting precipitates, pumping the precipitates into a stirrer, slowly adding corn flour serving as a carrier matrix into the stirrer, wherein the amount of the added corn flour is 6% of the weight ratio of the XH-9 strain liquid bacterial agent, uniformly stirring, conveying to an LPG80L centrifugal spray dryer, dispersing by an atomizer at 60 ℃ and quickly evaporating to dryness by high-temperature air at 181 ℃, and collecting solid matters in feed liquid by a cyclone separator to obtain the XH-9 strain solid bacterial agent;

the LB solid medium in the step 1) comprises the following components: 10g of peptone, 5g of yeast powder, 10g of NaCl, 10g of agar and 1L of deionized water;

the LB liquid culture medium in the step 2) comprises the following components: 10g of peptone, 5g of yeast powder, 10g of NaCl and 1L of deionized water;

the components of the secondary seed culture medium in the step 3) are as follows: 30g of bran, 10g of peptone, (NH)₄)₂SO₄1.5g，KH₂PO₄0.75g，CaCO₃6.0g，MgSO₄1.5g，NaCl 2.0g，MnSO₄0.25g, 1L of water; and (3) sterilization conditions: at 121 deg.C for 30min under 0.2 MPa;

the fermentation medium in the step 4) comprises the following components: 30g of bran, 10g of peptone, (NH)₄)₂SO₄1.5g，KH₂PO₄0.75g，CaCO₃6.0g，MgSO₄1.5g，NaCl 2.0g，MnSO₄0.25g, 1L of water; and (3) sterilization conditions: 121 ℃, 30min and 0.2MPa pressure.