CN112266881B

CN112266881B - Bacillus amyloliquefaciens and application thereof in preventing and treating apple continuous cropping obstacle

Info

Publication number: CN112266881B
Application number: CN202011124878.8A
Authority: CN
Inventors: 尹承苗; 段亚楠; 毛志泉; 刘鑫; 陈学森; 沈向
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2020-10-20
Filing date: 2020-10-20
Publication date: 2021-11-19
Anticipated expiration: 2040-10-20
Also published as: CN112266881A

Abstract

The invention discloses a bacillus amyloliquefaciens and application thereof in preventing and treating apple continuous cropping obstacles, wherein the bacillus amyloliquefaciens is preserved in the China general microbiological culture Collection center in 2018, 1 month and 25 days, and the biological preservation number is as follows: CGMCC NO. 15307. The strain provided by the invention has an inhibiting effect on various pathogenic bacteria, and particularly has a very strong antagonistic effect on fusarium oxysporum, fusarium verticillium, fusarium exserotina and fusarium solani causing apple continuous cropping obstacle. The strain can also promote the growth of the overground part and the underground part of the continuous cropping apple tree seedlings; the content of bacteria in the soil is improved, and the ratio of bacteria to fungi is improved; improving the activity of soil urease, phosphatase, sucrase and catalase in the apple continuous cropping soil; has excellent effect on relieving apple continuous cropping obstacles, and can be used for preventing and treating apple continuous cropping obstacles.

Description

Bacillus amyloliquefaciens and application thereof in preventing and treating apple continuous cropping obstacle

Technical Field

The invention relates to the technical field of agricultural microorganisms, in particular to a bacillus amyloliquefaciens and application thereof in preventing and treating apple continuous cropping obstacles.

Background

The apple cultivation area, the total output, the per-capita occupation and the export of the apples are all the first in the world in China, and become the biggest apple production and consumption countries in the world. At present, the apple cultivation area of China is 3700 ten thousand mu, the apple yield is 4380 ten thousand tons, but apple orchards in traditional dominant cultivation areas are mainly established in the 80 th and 90 th of the last century, most of the apple orchards enter the aging period, and only Shandong province has nearly 6.7-10 ten thousand hm in 5 years in the future²Apple orchards are subject to renewal. Due to limited land resources, continuous cropping cultivation of apple trees cannot be avoided when old orchards are updated, and continuous cropping obstacles are caused to have universality. Compared with a normal orchard, continuous cropping obstacles can reduce the acre yield of apples by 20-50%, bring huge loss to orchard operators, and seriously restrict the sustainable development of fruit tree industry. How to effectively relieve or overcome the continuous cropping obstacle becomes an important work to be urgently solved for the sustainable development of the apple industry in China.

The pathogenic factors of apple continuous cropping obstacle are very complex, including two main categories of biological factors and non-biological factors, and biological factors mainly comprising harmful fungi dominate. The harmful fungi in the continuous cropping soil in different areas and different orchards are different. Major harmful fungi related to apple continuous cropping obstacle have been reported to be of the genera Stylosporium, Fusarium, Rhizoctonia, Phytophthora, Pythium, and the like. Franke-Whittle et al have considered that the genus Podospora and Fusarium are the major harmful bacteria causing apple continuous cropping obstacles. Tewoldemedhin et al found that Pythium species fungi are the major harmful fungi causing apple replant disorder. When Van Schoor et al investigated the continuous cropping of apple orchard in south Africa, Fusarium, Cylindrocarpon and Pythium were found to be the main causes of continuous cropping disorders. Kelderer et al have shown that Fusarium solani, Fusarium oxysporum, Cylindrocarpon and Rhizoctonia dinucleae are the major pathogens responsible for apple stubble disorders in Italy. The abiotic factors comprise soil phenolic acid substance accumulation, soil physicochemical property deterioration, soil nutrient imbalance and the like. Previous researches believe that phenolic acids play an important role in apple continuous cropping obstacles, and in a continuous cropping soil environment, the apple continuous cropping obstacles are aggravated by the combined action of phlorizin and fusarium moniliforme, and further researches show that the phlorizin can promote the rapid increase of the number of the fusarium moniliforme and accelerate the hypha division speed of the fusarium moniliforme. At present, the control of apple continuous cropping diseases mainly depends on soil fumigation, such as soil disinfection of methyl bromide, chloropicrin, dazomet, metam and formalin. While chemical disinfection is effective in controlling soil-borne diseases, there are several disadvantages to using these chemical disinfection methods, including difficulty of application, high cost, and the environmental pollution and health hazards to some chemical disinfectants. The biological control has been widely applied to various agriculture and forestry because of its characteristics of green, environmental protection, low cost, sustainable development and the like. Therefore, it is very important to select biological alternative chemical disinfection to control the continuous cropping diseases of apples so as to achieve the effects of controlling the occurrence of diseases and chemical disinfection.

Bacillus is used as a new biological control agent, and one of the functions of the Bacillus is to control various diseases of plants. Bacteria belonging to the genus bacillus are considered safe microorganisms and have the remarkable ability to meet the agricultural and industrial demands for the synthesis of large amounts of beneficial substances. Bacillus amyloliquefaciens is a nonpathogenic bacterium, can synthesize various metabolites with activity against plant pathogens, and has been found by a great deal of research to induce systemic immunity and generate resistant endospores, thereby having wide application in practical production. However, few reports of Bacillus amyloliquefaciens for preventing and treating apple continuous cropping obstacles exist at present.

Disclosure of Invention

Aiming at the prior art, the bacillus amyloliquefaciens QSB-6 is obtained by separating from healthy fruit tree rhizosphere soil of an old apple orchard, and has obvious antagonistic action on fusarium oxysporum, fusarium verticillium, fusarium sporogenes and fusarium solani; has degradation effect on phenolic acid substances causing apple continuous cropping obstacle; can also promote the growth of continuous cropping apple trees; can effectively prevent, control and relieve the continuous cropping problem of the apples.

Specifically, the invention relates to the following technical scheme:

the invention provides a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6 which is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 of Xilu-1. of the sunward area of Beijing) in 1 month and 25 days in 2018, and the biological preservation number is as follows: CGMCC NO. 15307.

The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6 is separated from rhizosphere soil of healthy fruit trees in old apple orchards and has the following characteristics:

the colonies are cultured on an LB plate culture medium at 36 ℃ for 48 hours, and the surfaces of the colonies are opaque and yellowish, rough in surface and irregular in edge. Wrinkles and aerobic bacteria are often formed when grown in liquid media. The gram-stained bacteria show bluish purple color, and are gram-positive bacteria, under the scanning electron microscope, the QSB-6 thalli are rod-shaped, have two blunt ends, have no flagella, and have the size of (0.6-0.8) Mumx (1.5-2.8) Mum. The cells were in the shape of short rods when observed under a fluorescence microscope at 100X/1.30 oil microscope.

In a second aspect of the invention, there is provided a fermentation broth, bacterial suspension and/or supernatant of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6.

The fermentation liquor can be prepared by the following method: inoculating Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6 into a fermentation culture medium, and inoculating at 30 ℃ for 200 r-min^-1Performing shake fermentation culture for 24 h;

the fermentation medium comprises the following components: 20.0g of cane sugar, 15.0g of yeast extract,MnSO₄ 1.0g，NaH₂PO₄·2H₂O 2.0g，Na₂HPO₄·2H₂o4.0 g, and 1000mL of distilled water.

The bacterial suspension can be prepared by the following method: and (4) centrifuging the fermentation liquor, and then resuspending the thallus precipitate with sterile water to obtain a bacterial suspension.

The supernatant can be specifically prepared by the following method: centrifuging the fermentation liquor, taking the supernatant, and filtering to obtain the supernatant.

In a third aspect of the present invention, there is provided a use of a fermentation broth, a bacterial suspension and/or a supernatant of the above-mentioned Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6 or Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6 in at least one of the following (1) to (6):

(1) inhibiting phytopathogens;

(2) preparing a product for inhibiting phytopathogens;

(3) preventing and controlling diseases caused by plant pathogenic bacteria;

(4) preparing a product for preventing and treating diseases caused by phytopathogens;

(5) degrading phenolic acid substances;

(6) preparing the product for degrading phenolic acid substances.

Preferably, the plant pathogenic bacteria are one or more of fusarium oxysporum, fusarium verticillium, fusarium exserotinae, fusarium solani, penicillium citrinum, colletotrichum, alternaria zeae and apple tree rot.

Preferably, the phenolic acid substance is one or more of phlorizin, benzoic acid, ferulic acid, vanillic acid, gallic acid and phloretin.

In a fourth aspect of the invention, the application of fermentation liquor, bacterial suspension and/or supernatant of the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6 or the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6 in promoting the growth of continuous cropping soil fruit trees is provided.

In a fifth aspect of the present invention, there is provided a use of a fermentation broth, a bacterial suspension and/or a supernatant of the above Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6 or Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6 in any one of the following (1) or (2):

(1) reducing continuous cropping obstacles of apple trees;

(2) preparing a biocontrol agent or bacterial manure for relieving continuous cropping obstacles of apple trees.

In a sixth aspect of the invention, a biocontrol agent for alleviating continuous cropping obstacles of apple trees is provided, and the biocontrol agent takes fermentation liquor, bacterial suspension and/or supernatant of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6 or Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6 as an active ingredient.

Preferably, the biocontrol agent is in the form of wettable powder, water dispersant, water suspension or dispersible oil suspension.

Preferably, the biocontrol agent also comprises an agriculturally and pharmaceutically acceptable auxiliary material, and the agriculturally and pharmaceutically acceptable auxiliary material is selected from one or more of a dispersing agent, a wetting agent, a disintegrating agent, a binder and an antifoaming agent. The sources of the agriculturally and pharmaceutically acceptable auxiliary materials are not particularly limited, and the agriculturally and pharmaceutically acceptable auxiliary materials are generally commercially available products.

The seventh aspect of the invention provides a bacterial fertilizer containing Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6, which is prepared by the following method:

inoculating Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6 into a fermentation medium, wherein the fermentation medium comprises the following components: 20.0g of cane sugar, 15.0g of yeast extract and MnSO₄ 1.0g，NaH₂PO₄·2H₂O 2.0g，Na₂HPO₄·2H₂O4.0 g and 1000mL of distilled water; 30 ℃ and 200r min^-1Performing shake fermentation culture for 24h to obtain a liquid microbial inoculum;

mixing the liquid microbial inoculum and the sterilized carrier according to the proportion of 1 ml: (8-12) g, uniformly mixing, and standing for 12-24h at 35-38 ℃; then putting into a closed container, and fermenting for 14-16 days.

Preferably, the carrier is formed by mixing cow dung and crop straws according to the weight ratio of 3: 1.

In an eighth aspect of the present invention, there is provided a use of the biocontrol agent or bacterial manure as described above in at least one of the following (1) to (4):

(1) promoting the growth of overground parts and underground parts of the continuous cropping apple seedlings;

(2) inhibiting the growth of pathogenic bacteria causing apple continuous cropping obstacles in soil;

(3) the content of bacteria in the soil is improved, and the ratio of bacteria to fungi is improved;

(4) improving the activity of soil urease, phosphatase, sucrase and catalase in the apple continuous cropping soil.

In a ninth aspect of the present invention, there is provided a method for alleviating continuous cropping obstacles of apple trees, comprising the steps of:

applying fermentation liquor, bacterial suspension and/or supernatant of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6 or Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6 to rhizosphere soil of apple tree plants.

The invention has the beneficial effects that:

according to the invention, a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) QSB-6 is separated from rhizosphere soil of healthy fruit trees in an old apple orchard for the first time, and the strain has an inhibiting effect on various pathogenic bacteria, and particularly has a strong antagonistic effect on Fusarium oxysporum, Fusarium verticillatum, Fusarium sporogenes and Fusarium solani which cause apple continuous cropping obstacles; the strain has a degradation effect on phenolic acid substances causing apple continuous cropping obstacles. The strain can also promote the growth of the overground part and the underground part of the continuous cropping apple tree seedlings; the content of bacteria in the soil is improved, and the ratio of bacteria to fungi is improved; improving the activity of soil urease, phosphatase, sucrase and catalase in the apple continuous cropping soil; has excellent effect on relieving apple continuous cropping obstacles, and can be used for preventing and treating apple continuous cropping obstacles.

Drawings

FIG. 1: the morphology of the strain QSB-6; wherein, A: the colony morphology is obtained after culturing for 48 hours at 36 ℃ on an LB plate culture medium; b: scanning an electron microscope image; c: morphology under fluorescence microscope after gram staining.

FIG. 2: test of the confrontation of the Strain QSB-6 with eight pathogenic bacteria (PDA Medium).

FIG. 3: phylogenetic tree of the 16S rDNA sequence of strain QSB-6.

FIG. 4: phylogenetic tree of the gyrA sequence of strain QSB-6.

FIG. 5: phylogenetic tree of the gyrB sequence of strain QSB-6.

FIG. 6: phylogenetic tree of rpoB sequence of strain QSB-6.

FIG. 7: biolog physiological and biochemical identification results of QSB-6 strains.

FIG. 8: and (3) measuring the degradation capability of the strain QSB-6 on phenolic acid substances (liquid chromatography).

FIG. 9: influence of QSB-6 strains on soil microorganisms.

FIG. 10: effect of different treatments on copy number of 4 fusarium genes in soil.

FIG. 11: influence of different treatments on the respiration rate of the root system of Malus hupehensis Rehd.

FIG. 12: influence of different treatments on the activity of protective enzymes of the root system of Malus hupehensis Rehd.

FIG. 13: effect of different treatments on the activity of soil protective enzymes in the field.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As described in the background section, due to limited land resources, continuous cropping of apple trees is inevitable when old orchards are updated, and continuous cropping obstacles are common. Compared with a normal orchard, continuous cropping obstacles can reduce the acre yield of apples by 20-50%, bring huge loss to orchard operators, and seriously restrict the sustainable development of fruit tree industry.

The inventor makes clear that the main harmful fungus causing apple continuous cropping obstacle in China is Fusarium in the early period by constructing and analyzing a clone library of continuous cropping apple orchard soil fungi in two large production areas of Bohai Bay and northwest loess plateau and adopting a method combining correlation analysis and inoculation verification, and the pathogenicity test result shows that the conidia, the beads, the rotten skin and the layered Fusarium are main pathogenic strains.

Aiming at the problem of apple continuous cropping in China, the invention uses a dilution plate method to separate bacteria QSB-6 from the rhizosphere soil of healthy fruit trees in a 15-year-old apple orchard. The bacteria are identified by a Biolog microorganism identification system in combination with 16S rDNA and gyrA gene sequence analysis, biochemical physiological characteristics and morphological characteristics, and a evolutionary tree of QSB-6 strains is constructed by MEGA 7.0.26 software, so that the strains are identified as bacillus amyloliquefaciens. On a potato culture medium, the confronting experiments are respectively carried out on Bacillus amyloliquefaciens QSB-6 and Fusarium oxysporum, Fusarium roseum, Fusarium verticillarum, Fusarium solani, Penicillium citrinum, colletotrichum botrytis, alternaria zeae and apple tree canker, and the results show that the Bacillus amyloliquefaciens QSB-6 has a good inhibiting effect on apple continuous cropping Fusarium and has a broad-spectrum antibacterial characteristic. The pot experiment result shows that compared with continuous cropping soil control, the QSB-6 bacterial manure obviously promotes the plant height and the dry weight of the Malus hupehensis seedling, the plant height and the dry weight are respectively increased by 1.74 and 5.93 times, the number of soil bacteria is increased after the QSB-6 fertilizer is treated, the propagation of harmful fungi in the soil is inhibited, the copy number of four Fusarium genes in the soil is detected by using a real-time fluorescent quantitative PCR technology, the gene copy number of Fusarium grown in the soil treated by the QSB-6 bacterial manure is obviously reduced, and the growth of the Bacillus amyloliquefaciens QSB-6 can be obviously inhibited. The soil phenolic acid data show that compared with a blank carrier T1, the content of phlorizin, phloretin, cinnamic acid, p-hydroxybenzoic acid and benzoic acid in the soil treated by QSB-6 bacterial manure in 9 months is reduced most obviously and is respectively reduced by 49.17%, 41.26%, 25.34%, 65.89% and 49.80%, and the degradation rate of the strain QSB-6 in 15 days is determined to be 90.20% by high performance liquid chromatography. The field test data show that the strain QSB-6 can obviously improve the biomass and the soil enzyme activity of Fuji/M9T 337. In conclusion, the QSB-6 strain can reduce the apple continuous cropping diseases to a certain extent and has good field development potential.

In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments. If the experimental conditions not specified in the examples are specified, the conditions are generally conventional or recommended by the reagent company; reagents, consumables, and the like used in the following examples are commercially available unless otherwise specified. Wherein:

yeast extract peptone agar medium LB: tryptone 10.0g, yeast extract 5.0g, NaCl 10.0g, agar 15.0g, pH 7.

Fermentation medium: 20.0g of cane sugar, 15.0g of yeast extract and MnSO₄ 1.0g，NaH₂PO₄·2H₂O 2.0g，Na₂HPO₄·2H₂O4.0 g, and 1000mL of distilled water.

Improving a PDA culture medium: 200.0g of potato, 5.0g of beef extract, 20.0g of glucose, 20.0g of agar and 1000mL of distilled water.

An oxygen demand determination culture medium, a glucose oxidation fermentation culture medium, a starch hydrolysis culture medium, a nitrate reduction culture medium, a V-P culture medium and an M.R. culture medium are prepared according to a method of a common bacteria system identification manual (Dongxu pearl, Chuia Miaoying editions).

Inorganic salt culture medium: NH (NH)₄Cl 0.5g/L，NaCl 1.0g/L，K₂HPO₄·3H₂O 1.3g/L，MgSO₄·7H₂O 0.4g/L。

Example 1: isolation and characterization of strains

1. Separating and purifying the strains:

rhizosphere bacteria were isolated using a modified dilution-spread plate method. And (4) passing the fresh soil sample through a sieve with the diameter of 3-4 mm, and removing impurities in the soil. Weighing 5g rhizosphere soil, adding into a triangular flask (with glass beads) containing 45mL sterile water, oscillating at 180rpm for 30min, standing for 5min, and diluting with sterile water to 1 × 10^-4100. mu.L of the supernatant from the gradient dilution was applied to an LB plate, repeated three times, and cultured in an inverted state in a constant temperature incubator at 28 ℃. After the single colony grows out in the plate, the single colony with different forms is picked and streaked and purified on a new LB plate. Meanwhile, the purified strain was inoculated in liquid LB medium, constantly shaken at 180rpm at 28 ℃ for 12h, and at 4 ℃ for 100Centrifuging at 00r for 10min, pouring out supernatant, adding 15% glycerol, and storing at-80 deg.C.

2. Screening and bacteriostasis of the strains:

the antagonism of the separated strain to four kinds of Fusarium (Fusarium oxysporum, Fusarium verticillum, Fusarium sporotrichioides and Fusarium solani) is determined by plate opposition method, i.e. firstly, the bacteria is streaked and inoculated on LB plate, a single colony is selected and inoculated in a fermentation culture medium (100 mL in a 250mL conical flask), the constant oscillation culture is carried out for 48h at 180rpm under 28 ℃, then, the bacteria are obtained by centrifugation for 20min at 10000r, the bacteria are re-suspended in PBS buffer solution (pH 7.0, 1/25 volume, 10 mmol. L^-1) In (1X 10), the concentration is adjusted to⁹CFU·mL^-1. A5 mm diameter Fusarium cake was inoculated into the center of the PDA plate, 4 sterile filter paper discs (6 mm diameter) were placed equidistantly around the cake, 10. mu.L of bacterial suspension was dropped onto each filter paper disc, and the experiment was repeated three times with Sterile Distilled Water (SDW) as a control. All plates were incubated at 28 ℃ for 7 days, the width of the zone of inhibition was measured and the rate of inhibition was calculated. The calculation formula is as follows: inhibition rate ═ control colony radius-treated colony radius)/control colony radius × 100%.

133 strains of bacteria are detected from rhizosphere soil of 3 old apple orchards of Shahezhen town (H), Jintown (D) and Su Jia town (Q) of Su Jia town of Suxia city of Taizhou city by a dilution coating plate method, a plate confronting experiment is carried out after separation and purification, wherein 46 strains of bacteria have an inhibiting effect on Fusarium, an inhibition zone is formed around the Fusarium, and QSB-6 of the bacteria separated from the rhizosphere soil of the Su Jia town of Suxia city is finally determined as a trial antagonistic strain by comparing the size of the inhibition zone.

3. And (3) carrying out classification identification on the antagonistic strain QSB-6:

3.1 morphological and physiological biochemical identification:

culturing the separated bacteria on an LB culture medium at 36 ℃ for 48h, and observing the morphological characteristics of colonies when single colonies appear. Gram staining is firstly carried out by ammonium oxalate crystal violet staining, then Lugol iodine solution is used as mordant to be treated, and then 95% ethanol is used as decolorant to be treated, so that gram-positive bacteria are blue-violet, gram-negative bacteria are colorless. The shape and size of the bacteria are observed by using a Nikon fluorescence microscope BX-51 and a scanning electron microscope SU-8010, and the preparation process of the scanning electron microscope sample refers to the methods of Linying and the like. Analysis of physiological and biochemical characteristics was performed according to methods of Bergey's Manual of identification of bacteria (second edition) and Manual of identification of commonly used bacteria systems, each index was examined 3 times, and the experiment was repeated 2 times.

As shown in FIG. 1, when QSB-6 strain was cultured on LB plate medium at 36 ℃ for 48h, the colony surface was opaque and yellowish, the surface was rough, and the edge was irregular (FIG. 1A). Wrinkles and aerobic bacteria are often formed when grown in liquid media. The gram-stained bacteria showed bluish purple color, and were gram-positive, and the cells were in the shape of short rods when observed under a fluorescence microscope 100X/1.30 oil microscope (FIG. 1C). Under scanning electron microscope, QSB-6 thallus is rod-shaped, with round ends and no flagella, and has size of (0.6-0.8) μm x (1.5-2.8) μm (FIG. 1B).

14 physiological and biochemical indexes of the QSB-6 strain are detected, and the results are shown in Table 1. The results show that: the method comprises the following steps of (1) catalase reaction, hydrogen peroxide reaction, hydrogen sulfide reaction, starch hydrolysis reaction, arginine double hydrolysis reaction, methyl red reaction, citrate utilization, V-P reaction and nitrate reduction to be positive; indole test, urease reaction and gelatin liquefaction test are negative; glucose and sucrose may be utilized. According to the physiological and biochemical results of the QSB-6 strain, the QSB-6 strain is preliminarily presumed to belong to the genus Bacillus (Bacillus spp.) by referring to Bergey's Manual of bacteria identification and general Manual of bacteria System identification.

Table 1: physiological and biochemical characteristics of QSB-6 strains

Note: "+" indicates a positive reaction or availability; "-" indicates a negative reaction.

3.216 amplification, sequence analysis and phylogenetic analysis of S rDNA and gyrA fragments:

QSB-6 strain is inoculated in liquid LB culture medium, and is cultured for 12h under the condition of 30 ℃ and the speed of 200r/min in a shaking way, and the whole genome DNA of the strain is extracted by adopting a bacterial genome extraction kit. Then, the total DNA of the bacteria QSB-6 is taken as a template, 16S rDNA gene and gyrA gene are adopted for PCR amplification, 1 percent agarose gel electrophoresis detection is carried out, and the DNA is sent to Shanghai biological engineering technology company Limited for sequencing.

The 16S rDNA gene amplification selects a universal primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3' are provided. PCR reaction (50. mu.L): 3 μ L of total DNA template, 1 μ L of each primer, 1 μ L of dNTP, 5 μ L of 10 XPCR buffer, 0.6 μ L of Taq DNA polymerase, ddH₂O38.4. mu.L (Weisburg et al, 1991). PCR amplification reaction procedure: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 deg.C for 1.5 min; annealing at 55 deg.C for 1 min; extending for 1.5min at 72 ℃; the total elongation is 72 ℃, 10min and the temperature is kept at 4 ℃ for 30 cycles.

Primers 42f: 5'-CAGTCAGGAAATGCGTACGTCCTT-3' and 1066r: 5'-CAAGGTAATGCTCCAGGCATTGCT-3' were selected for amplification of the gyrA gene. PCR reaction (30. mu.L): taq PCR Master Mix15 μ L, ddH2O 12 μ L, 1 μ L each of primers, 1 μ L of DNA template. And (3) PCR reaction system: pre-denaturation at 95 ℃ for 5 min; denaturation at 94 deg.C for 1 min; annealing at 62 deg.C for 1 min; extending for 2min at 72 ℃; the total elongation is 72 ℃, 10min and the temperature is kept at 4 ℃ for 30 cycles.

Primers up1f: GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA and up2r: AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT are selected for amplification of gyrB gene. PCR reaction (30. mu.L): taq PCR Master Mix 15. mu.L, ddH₂O12. mu.L, primers 1. mu.L each, and DNA template 1. mu.L. PCR amplification reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s; annealing at 60 ℃ for 30 s; extending for 1min at 72 ℃; the total elongation is 72 ℃, 10min and the temperature is kept at 4 ℃ for 30 cycles.

Primers 2292F: AGGTCAACTAGTTCAGTATGGAC and 3354R: AAGAACCGTAACCGGCAACTT were used for rpoB gene amplification. PCR reaction (50. mu.L): 5 × PrimeSTAR Buffer (Mg)²⁺Plus)10μL，dNTP Mixture(2.5mM each)4μL，PrimeSTAR HS DNA Polymerase(2.5U/ul)0.5μL，ddH₂O31.5. mu.L, primers 1. mu.L each, and DNA template 2. mu.L. PCR amplification reaction procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 15 s; annealing at 56 ℃ for 30 s; extension at 72 ℃ for 30 s; total 30 cycles, total extension 72 ℃, 10min, 4 DEG CAnd (5) preserving the heat.

The sequencing results were submitted to GenBank of National Center for Biotechnology Information (NCBI) of the United states, subjected to homology alignment by the BLAST program, and used to construct phylogenetic trees using MEGA 7.0.26 software, and subjected to 1000 replicate tests for bootstrap value.

The length of the 16S rDNA gene sequence of the QSB-6 strain is 1452bp, and the 16S rDNA sequence is shown in SEQ ID NO. 1. BLAST alignment of this sequence in the NBCI GenBank database revealed 99% similarity of the 16S rDNA gene sequence of QSB-6 strain to Bacillus amyloliquefaciens B.amyloliquefaciens (KY 685066.1). A phylogenetic tree (figure 3, figure 4, figure 5 and figure 6) is constructed by utilizing 16S rDNA, gyrA, gyrB and rpoB gene sequences in combination with MEGA 7.0.26, and the result shows that the 16S rDNA gene sequence of the strain QSB-6 and the 16S rDNA gene sequence of Bacillus amyloliquefaciens (MH188056.1) belong to the same branch in the phylogenetic tree and have the highest homology (100%); the gyrA gene sequence of the strain QSB-6 and the gyrA gene sequence of Bacillus amyloliquefaciens (KF803276.1) belong to the same branch in the evolutionary tree, and have the highest homology (100%); the gyrB gene sequence of the strain QSB-6 and the gyrB gene sequence of Bacillus amyloliquefaciens (AB972386.1) belong to the same branch in an evolutionary tree, and have the highest homology (100%); the rpoB gene sequence of the strain QSB-6 and the rpoB gene sequence of the bacillus amyloliquefaciens (KF803276.1) belong to the same branch in a evolutionary tree, and have the most homology (100%). The combination of the above analyses indicates that the QSB-6 strain is Bacillus amyloliquefaciens B.

3.3Biolog automatic analysis system for microorganisms:

the Biolog GEN III system is used for detecting absorbance change caused by the combination of a redox product generated by aerobic metabolism activity of bacteria by utilizing 95 carbon sources on a micropore plate and a chromogenic substance and turbidity difference caused by the growth of microorganisms, generating a characteristic fingerprint, and comparing the characteristic fingerprint with a standard strain spectrum database to identify strains. When the bacteria are cultured for 16-24 h, the SIM value is more than or equal to 0.50, the system automatically gives an identification result, and the closer the SIM value is to 1.00, the more accurate the identification result is.

Biolog microbial identification systems perform strain identification by utilization of different carbon sources by bacteria. The Biolog microorganism identification system method based on the carbon utilization analysis strain has the PROB value of 0.522, the SIM value of 0.522 and the DIST value of 7.148, and shows that the identification result is a relatively reliable matching result. FIG. 7 identifies that the strain QSB-6 is Bacillus amyloliquefaciens.

In conclusion, the isolated strain QSB-6 was identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). And is preserved in the China general microbiological culture Collection center in 2018, 1 month and 25 days, with the preservation number: CGMCC No. 15307.

Example 2: broad-spectrum bacteriostatic effect of strain QSB-6

In order to further verify whether the QSB-6 strain has broad-spectrum bacteriostatic action on the plant-derived diseases, 4 other common pathogenic fungi are selected for further antibacterial test. The results are shown in Table 2.

The results of the confrontation test show that the QSB-6 strain has strong inhibition effect on the growth of 4 pathogenic fusarium hyphae. Has strong inhibiting effect on penicillium citrinum, colletotrichum gloeosporioides, alternaria zeae and apple tree canker. Therefore, the strain QSB-6 has broad-spectrum bacteriostatic action on plant pathogenic bacteria under the in-vitro culture dish test condition.

Table 2: inhibitory Effect of Strain QSB-6 on 8 pathogenic bacteria

Note: -, no zone of inhibition; +, the inhibition zone is less than 5mm, the inhibition effect is weak, and hyphae stop growing at the edge of bacteria; a medium inhibition zone with a bacteriostasis zone of 5-10 mm; + + + +, strong inhibition, bacteriostatic zone >10 mm. Different lower case letters indicate significant differences at the 0.05 level (Duncan's test).

Example 3: optimization of fermentation conditions for Strain QSB-6

1. The test method comprises the following steps:

transferring the preserved strain to LB solid culture medium, and culturing at 37 deg.C for 24 h. Picking activated single colony for inoculationInto a triangular flask containing 50ml of LB liquid medium, 160 r.min^-1Shaking and culturing at 37 deg.C until OD600 is about 1. Inoculating the seed liquid into a triangular flask containing a culture medium according to the inoculation amount of 2 percent; at 200 r.min^-1And cultured in a shaker at 37 ℃ for 24 hours.

Fermentation culture studies were performed in a one-factor experiment.

1.1 carbon source: respectively using 20 g.L^-1The carbon source of the basic culture solution is equivalently replaced by the sucrose, maltose, glucose, lactose and soluble starch. After screening different concentrations of 0.1%, 0.5%, 1%, 1.5% and 2%, the optimum carbon source was determined by measuring the viable count of the fermentation broth after 24h using shake flask fermentation culture.

1.2. Nitrogen source: sequentially using 20 g.L^-1The yeast extract, peptone, ammonium sulfate, beef extract, ammonium nitrate, urea and ammonium chloride replace the nitrogen source of the basic fermentation liquid. After screening different concentrations of 0.1%, 0.5%, 1%, 1.5% and 2%, the optimum nitrogen source was determined by measuring the viable count of the fermentation broth after 24h using shake flask fermentation culture.

1.3 inorganic salts: sequentially using 0.5 g.L^-1NaCl, CaCl of₂·2H₂O、MgSO₄·7H₂And the equal amount of O, manganese sulfate and KCl replaces the inorganic salt of the basic fermentation liquor. After screening different concentrations of 0.01%, 0.03%, 0.05%, 0.07% and 0.09%, the optimal inorganic salt species is determined by measuring the viable count of the fermentation liquor after 24h by adopting shake flask fermentation culture. Each treatment was repeated 3 times.

1.4 culture condition optimization design:

and (3) fixing other fermentation conditions of shake flask culture by using the screened optimal culture medium formula, and optimizing the fermentation conditions such as initial pH, liquid loading amount, temperature, inoculation amount, fermentation time and the like. The initial pH value is adjusted to 5.5, 6.0, 6.5, 7.0, 7.5, 8.5 and 9.5 in sequence; the inoculation amount is respectively adjusted to be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10% of the liquid loading amount; 25mL, 50mL, 75mL, 100mL, 125mL and 150mL of fermentation medium were respectively filled in a 250m L triangular flask; setting the fermentation time to 12h, 24h, 48h and 60h respectively; the temperatures were 25 deg.C, 30 deg.C, 35 deg.C, 37 deg.C, 40 deg.C and 50 deg.C, respectively(ii) a The rotation speeds are respectively set to 100 r.min^-1、150r·min^-1、200r·min^-1、220r·min^-1And 250 r.min^-1. Each treatment was repeated 3 times.

1.5 determination method: and (3) taking a blank culture medium as a reference, and measuring the thallus fermentation liquor by adopting a turbidimetric method, namely centrifuging 10-15 mL of fermentation liquor for 2min at 2000 r.min < -1 >, measuring the OD600 value (the light absorption value of the solution at the wavelength of 600 nm) of the bacterial liquid, and measuring the pH value of the bacterial liquid by using a pH meter.

2. The experimental results are as follows:

screening different conditions respectively, and fermenting with the same other components. As shown by experiments, the best carbon source for the growth of the strain QSB-6 is sucrose (concentration 20 g.L)^-1) The best nitrogen source is yeast extract (concentration 15 g.L)^-1) The most preferred inorganic salt is manganese sulfate (1 g. L concentration)^-1) pH 7.5, optimum inoculum size 5%, liquid loading 100mL, fermentation time 24h, temperature 30 deg.C, rotation speed 200 r.min^-1。

Example 4: degradation effect of strain QSB-6 on phenolic acid substances

1. The test method comprises the following steps:

1.1 preparation of phlorizin culture medium (0-10 mmol/L) (phlorizin purchased from Shanghai leaf Biotech limited company, protected from light): weighing a certain amount of phlorizin, putting the phlorizin into a beaker, and adding 10 times of anhydrous ethanol for dissolving; adding a proper amount of deionized water and heating to boil so as to completely volatilize the ethanol; weighing ammonium chloride, sodium chloride, dipotassium hydrogen phosphate and magnesium sulfate according to a certain proportion, and adding the ammonium chloride, the sodium chloride, the dipotassium hydrogen phosphate and the magnesium sulfate into the phlorizin solution for dissolving; and (3) separating a part of the culture medium, adding agar powder according to the proportion of 2% as a phlorizin solid culture medium, and operating as fast as possible in a dark process.

1.2 high performance liquid chromatography for determining degradation rate

1.2.1 high pressure liquid phase sample preparation: taking 50mL of culture solution after 3d, 5d and 15d of shake culture, centrifuging at 9000r/min for 10min, removing thallus, taking equivalent dichloromethane for repeated extraction for 3 times, combining organic layers, and preparing for parallel chemical quantitative concentration.

The method for calculating the degradation rate of the high performance liquid chromatography comprises the following steps: the degradation rate is (autotoxic substance content of non-inoculated culture medium-autotoxic substance content of inoculated culture medium)/autotoxic substance content of non-inoculated culture medium x 100%.

1.2.2 concentration of the extract: the concentration and evaporation are carried out in parallel, the vacuum degree is finally reached to 63mbar on 24 concentration stations, the water bath temperature is 60 ℃, and the dissolution is carried out by 3 times by using 2mL of methanol. Then, the mixture was filtered through a 0.22 μm organic filter and placed in a 2mL centrifuge tube for further use.

1.2.3 liquid chromatography conditions: mobile phase A: acetonitrile, mobile phase B: water (pH 2.6 adjusted with acetic acid), column: acclaim 120C18(3 μm. times.150 mm. times.3 mm), column temperature 30 ℃, flow rate 0.5 mL/min.

Sample introduction mode and sample introduction volume: automatically injecting 10 mu L of sample; the detection wavelength is 280 nm.

2. And (3) test results:

2.1 High Performance Liquid Chromatography (HPLC) determination of the Strain degradation Rate

As shown in fig. 8, the contents of phlorizin, benzoic acid, ferulic acid, vanillic acid, gallic acid and phloretin were reduced with the increase of the culture time by performing high performance liquid chromatography analysis on the culture solutions of 3, 5 and 15d after shake culture of the strain QSB-6, wherein the degradation rates of

phlorizin

3, 5 and 15d were 42.89%, 62.49% and 90.20%, respectively, the content of salicylic acid was increased first and then decreased, and the degradation rate reached 44.91% on the fifth day. Phlorizin, salicylic acid and benzoic acid have been reported to be degraded into p-hydroxybenzoic acid, and benzoic acid, ferulic acid and p-hydroxybenzoic acid to be degraded into epicatechin and further catechin, leading to an increase in the catechin and p-hydroxybenzoic acid content as a result of the test.

Example 5: pot experiment

1. And (3) experimental design:

the potting test is carried out in the national apple engineering experiment center of the institute of horticultural science and engineering of Shandong agricultural university and the national key laboratory of crop biology. Potted plant test the test plants were seedlings of Malus hupehensis Rehd.

And 3, transplanting the seedlings into a nursery garden in 2017, and when the seedlings grow to a third true leaf, selecting the seedlings with the same growth vigor, transplanting the seedlings into a clay pot with the upper caliber of 38cm, the lower caliber of 28cm and the height of 26cm, and performing a potting test. Each pot contained approximately 7.543kg of soil, 3 seedlings of Malus hupehensis Rehd per pot, and 20 replicates per treatment set. When the seedlings grow to 20-30cm, two seedlings with similar growth vigor are left in each pot. The potting soil is treated as follows: old orchard soil (CK1), old orchard soil methyl bromide fumigation (CK2), bacterial manure carrier treatment (T1), and bacillus amyloliquefaciens QSB-6 bacterial manure treatment (T2); and each treatment group is uniformly managed with normal water and fertilizer, and the bacterial fertilizer carrier of the bacillus amyloliquefaciens QSB-6 are applied according to 1 percent of the soil mass.

The bacillus amyloliquefaciens QSB-6 bacterial fertilizer used for the pot culture test is manufactured by the microbial resource limited company of Chuangdi, Tex, China, and the manufacturing method comprises the following steps: firstly, QSB-6 strain is subjected to liquid fermentation (fermentation culture medium: 20.0g of sucrose, 15.0g of yeast extract and MnSO)₄ 1.0g，NaH₂PO₄·2H₂O 2.0g，Na₂HPO₄·2H₂O4.0 g and distilled water 1000mL) to obtain a liquid microbial inoculum; then mixing the liquid microbial inoculum and the sterilized bacterial fertilizer carrier according to the proportion of 1 ml: 10g of the mixture is uniformly mixed, so that the mixture of the liquid microbial inoculum and the carrier is fully wet (the humidity of 45 percent is the best), and the mixture is suitable for holding by hands without leaving watermarks; after being stirred uniformly, the mixture is put on a sunshade and covered with plastic cloth to be kept stand for 24 hours, and the temperature is kept at the best temperature of 35-38 ℃; after 24 hours, the mixture of the liquid microbial inoculum and the carrier is put into a sealed container and fermented for 15 days for use.

In the prepared bacillus amyloliquefaciens QSB-6 bacterial fertilizer, the viable count is 2 multiplied by 10⁸cfu/g。

The bacterial manure carrier is a mixture of cow dung and corn straws (cow dung: straws: 1: 3 by weight ratio) and is provided by the microbial resource company Limited of Chuangdi, Texas, China.

Sampling is carried out in 2017, 8, 15 days, three pots of seedlings with consistent growth vigor are randomly selected for each treatment, soil on the surface layer and around the pots is removed, soil impurities are filtered by a sieve with the diameter of 2mm, the soil sample is filled in a sterile plastic sealing bag and is taken back to be placed at minus 80 ℃ to extract soil DNA for further real-time fluorescent quantitative PCR analysis, the copy number of fusarium in the soil is detected, and the inhibition effect of the strain QSB-6 on fusarium in the soil is verified. The seedlings are washed clean and transported back to a laboratory for measurement of indexes such as biomass and the like.

2. Measurement indexes are as follows:

the height, ground diameter and dry fresh weight of the seedling are respectively measured by a measuring tape, a vernier caliper and an electronic scale.

And analyzing and processing the sample image by using a professional version Win RHIO (2007 edition) root system analysis system, and recording the total length of the root system, the surface area of the root system, the number of branches and the number of tips of the root system.

Determination of soil microorganisms: the quantity of bacteria, fungi and actinomycetes in the soil is measured by adopting a flat plate dilution coating method. The bacteria adopts a yeast extract peptone culture medium, the fungi adopts a potato glucose culture medium, and the actinomycetes adopts a Gao's I culture medium.

Determination of the respiration rate: the measurement was carried out by referring to the method of Maoqiquan et al and Bouma. Taking an uncolored white root system with basically consistent diameter, quickly weighing about 0.05g, cutting the root into root segments of about 2mm by using a double-sided knife, putting the root segments into a reaction cup, covering the reaction cup and starting a measuring program. The temperature of the liquid in the reaction cup was controlled at 25 ℃ by using a constant temperature bath. The apparatus used a liquid phase Oxy-Lab oxygen electrode manufactured by HANSATECH Co.

And (4) extracting the protective enzyme. 1.0g of fresh white root is taken, 8mL of phosphoric acid buffer solution is added, a small amount of quartz sand is added, the mixture is ground into homogenate and centrifuged at 12000rpm/min at 4 ℃ for 20min, the supernatant is an enzyme solution to be measured, and all measurements are carried out at 2-4 ℃ (Zhao Shijie et al, 2002).

For the measurement of the activities of superoxide dismutase (SOD), Peroxidase (POD) and Catalase (CAT), reference is made to the method of Zhaoshijie et al (2002).

The MDA content was determined by the thiobarbituric acid method in mmol/g FW (Zhao Shijie, 2002).

Taking 0.5g of fresh soil sieved by the method

The operation steps of the soil DNA extraction kit are used for extracting DNA, CFX96TMthermal Cycler (Bio-Rad) is adopted to carry out real-time fluorescence quantitative analysis on the gene copy number of the fusarium oxysporum in the soil, and the inhibition effect of QSB-6 strain on the fusarium oxysporum in the soil is verifiedThe specific primers and reaction steps are referred to the method of Lei Jia (Lei Jia, Xiang Li Feng Bing, Pan Feng Bing, Chen Zhi Sen, Shen, Yi Cheng Mi, Mao Zhi quan.2016. Ping Yi sweet tea seedling and shallot mixed together have an influence on the soil environment of continuous cropping of apple, Hi Xue Xuan, 43 (10): 1853-.

Determination of soil phenolic acid substances: accurately weighing 80g of each treatment air-dried soil sieved by a 12-mesh sieve, adding a proper amount of quartz sand, uniformly mixing in a large mortar, and repeating the treatment for 3 times. Filling 1 cellulose membrane on the bottom of a 100mL extraction pool, filling a uniformly mixed sample, and extracting according to ASE extraction conditions, namely: analytically pure absolute methanol and absolute ethanol are respectively added into two extraction bottles of ASE, the extraction temperature of an instrument is set to be 120 ℃, the pressure is set to be 10.3MPa, static extraction is carried out for 5min, the purging volume is 60%, the purging time is 90s, and the cycle is repeated for 2 times. After extraction, the extract is rotary evaporated at 45 deg.C under reduced pressure to near dryness, and 3mL methanol is added for redissolving, and the filtrate is filtered through 0.22 μm organic phase filter membrane, and after HPLC analysis determination, the method is referred to Yi Cheng Miao (2014).

Growth promoting effect of QSB-6 strain on Malus hupehensis seedling (Table 3):

table 3: influence of QSB-6 strain on biomass of Malus hupehensis seedling

Note: different lower case letters indicate significant differences at the 0.05 level (Duncan's test).

As can be seen from Table 3, the biomass of the seedlings of Malus hupehensis Rehd can be increased by sterilizing with methyl bromide, treating with a bacterial fertilizer carrier and treating with a bacterial fertilizer. The plants of the Malus hupehensis Rehd seedlings treated by CK 2T 2T 1 CK1 and T2 are high, the diameter on the ground, the weight again on the ground and the fresh weight under the ground are respectively 1.5 times, 1.32 times, 1.56 times, 1.53 times, 2.21 times and 2.27 times of the dry weight on the ground and the dry weight under the ground of T1. It can be seen that the treatment of QSB-6 strain can obviously promote the growth of Malus hupehensis seedling. The effect is slightly lower than that of methyl bromide sterilization treatment.

Effect of QSB-6 strains on soil culturable microorganisms:

as can be seen from fig. 9, the bacterial content in the soil after T2 treatment at 7, 8, and 9 months was significantly increased compared to CK1, which was 2.22 times, 3.21 times, and 4.62 times that of the control, respectively; the total content of soil fungi shows different performances in different treatments, the performance is most remarkable in September, and CK2 and T2 are respectively reduced by 75.36% and 85.51% compared with CK 1; the T1 treatment significantly increased 40.43% over July. From the number of soil actinomycetes, it can be seen that T2> T1> CK2> CK1 are all significantly higher than the control. Among these, the T2 treatment significantly increased the soil bacteria/fungi ratio, as evidenced by T2> CK2> T1> CK 1.

Inhibitory effects of QSB-6 strains on four Fusarium species:

as can be seen from fig. 10, compared with the continuous cropping soil, the gene copy number of fusarium oxysporum after the sevies and august methyl bromide fumigation and the QSB-6 bacterial manure treatment is significantly reduced by 67.12% and 68.78%, 33.96% and 60.02%, respectively, and the gene copy number of the layered fusarium oxysporum is also significantly reduced by 72.82% and 77.58%, 67.36% and 72.00%, respectively. Compared with QSB-6 bacterial manure, the gene copy number of Fusarium solani after July and August bacterial manure carrier treatment is respectively increased by 31.33% and 34.02%, and the gene copy number of Fusarium verticillum is respectively increased by 11.59% and 36.64%. The result shows that the Bacillus amyloliquefaciens QSB-6 has good inhibition effect on fusarium in soil.

Influence of QSB-6 strains on root morphology:

as shown in table 4, the total length of root system, the surface area of root system, the total volume of root system, the number of root tips and the number of branches after the treatment (T1) by the strain QSB-6 are 3.71 times, 6.13 times, 10.28 times, 2.45 times and 6.19 times of the treatment (CK1) of soil in old orchards respectively, but the effect is still not as good as that of the fumigation treatment (CK2) by methyl bromide; compared with bacterial manure carrier treatment (T2), the total length of roots, the surface area of the roots, the total volume of the roots, the number of root tips and the number of branches of the treated strain QSB-6 are respectively increased by 36.99%, 103.44%, 201.11%, 3.11% and 92.11%, and therefore, the application of the QSB-6 bacterial manure can obviously promote the growth of the roots of the seedlings of the Malus hupehensis Rehd.

Table 4: influence of QSB-6 bacterial manure on root growth of Malus hupehensis seedling

Influence of QSB-6 strains on root respiration Rate:

as shown in fig. 11, the root respiration rates of the treated seedlings of the Malus hupehensis Rehd in the measurement periods (months 7, 8 and 9) are generally in an increasing trend, and the increasing trend of the respiration rates after being treated by QSB-6 bacterial manure is obviously stronger than that of the control, and is close to that of the fumigation treatment of methyl bromide. CK2 and T2 are 1114.38 mu molO in September₂·g-1FW·min^-1And 1000.81 μmolO₂·g-1FW·min^-1All were significantly higher than the control, whereas T1 was only 9.10% of the control.

Influence of QSB-6 strains on root protective enzyme activity:

as shown in fig. 12, the SOD, POD and CAT activities were stably increased at 7, 8, 9 months for the different treatments compared to the control, with the increase being most significant with CK1 and QSB-6 bacterial manure treatments, with SOD activities at 7, 8, 9 months for T2 being 2.06, 3.53, and 3.22 times greater than the control treatment; POD activity was 1.56-fold, 1.82-fold, and 2.36-fold higher than control; CAT activity was 1.44-fold, 1.85-fold, and 2.24-fold higher than control; the MDA activity shows an opposite trend compared with the above, CK1 and QSB-6 bacterial manure treatment show a descending trend in 7, 8 and 9 months compared with a control and a bacterial manure carrier blank, QSB-6 bacterial manure treatment in 7, 8 and 9 months is respectively reduced by 0.31%, 0.45% and 0.60% compared with the control, and the descending proportion shows an increasing trend.

Influence of QSB-6 strains on soil phenolic acids:

as can be seen from Table 5, the contents of several main phenolic acid substances of the control CK1 in months 7, 8 and 9 are the highest, the total amount of phenolic acid substances in the fumigation treatment of methyl bromide is the lowest, the content change of the phenolic acid substances in the months 7, 8 and 9 is not obvious, the content of the phenolic acid substances in the soil can be reduced by the QSB-6 bacterial manure and the blank carrier treatment, and the reduction is the most obvious by the T2 treatment. Compared with T1, the content of phloretin, phlorizin, cinnamic acid, p-hydroxybenzoic acid and benzoic acid in the soil treated by QSB-6 bacterial manure in 7 months is respectively reduced by 32.00%, 36.26%, 17.95%, 57.24% and 35.21%; the contents of phlorizin, phloretin, cinnamic acid, p-hydroxybenzoic acid and benzoic acid in the soil treated by QSB-6 bacterial manure in 8 months are respectively reduced by 40.20%, 35.35%, 25.46%, 61.69% and 49.19%; the contents of phlorizin, phloretin, cinnamic acid, p-hydroxybenzoic acid and benzoic acid in the soil treated by QSB-6 bacterial manure at 9 months are respectively reduced by 49.17%, 41.26%, 25.34%, 65.89% and 49.80%.

Table 5: influence of QSB-6 bacterial manure treatment on soil phenolic acid substances

Note: the different lower case letters in the table indicate significant differences between the different treatments (P < 0.05).

Example 6: field test

1. Test materials

The apple seedlings for the test are 2-year-old grafted seedlings, the stock-spike combination is Nifu No. 3/M9T 337, purchased from Laizhou Nature horticulture science and technology Limited. The row spacing of the planted plants is 1.5m multiplied by 4 m. QSB-6 bacterial manure was prepared as in example 5.

2. Design of experiments

Each test site was set with 4 treatments, respectively: (ii) continuous cropping soil control, CK 1.② fumigating treatment by methyl bromide, CK 2.③ QSB-6 bacterial manure treatment, T1. Bacterial manure carrier treatment, T2.

And 3, treating in 2017, 20-23 days in 3 months, digging planting holes of 80cm square according to the row spacing of the seedlings, respectively uniformly mixing the bacterial manure carrier and the QSB-6 bacterial manure with soil, backfilling, digging a small hole for planting the seedlings during planting, stretching the root systems of the seedlings, strengthening, filling the soil to the original planting depth of the seedlings, compacting, and thoroughly watering. The application amount of each seedling of the young tree is controlled to be 200g, and the young tree can be applied once in the germination period, wherein the application amount is 200 g; each treated 15 trees.

3. Sampling method

And (6) collecting soil samples. Sampling at five points in a chessboard mode, taking root soil about 30cm away from apple trees, mixing the soil samples uniformly and taking the mixture back to a laboratory.

4. Measurement index

Measuring tree body indexes: the plant height is measured by using a plant height measuring ruler; the ground diameter is measured by a vernier caliper at the position 20cm away from the ground; the growth amount of new shoots is measured by selecting 3 trees per treatment, and selecting 3 branches with similar thickness for each tree and measuring the length by using a flexible meter ruler; the weight of the beancurd sheets is heavy, 5 trees are randomly extracted from each tree seed to be treated, 20 normal leaves are randomly collected from each tree, and 100 leaves are weighed as the weight of the beancurd sheets.

Extraction and activity determination of soil enzymes: soil urease, sucrase, phosphatase and catalase are measured by a micro-scale method.

5. Influence of QSB-6 bacterial manure treatment on growth indexes of field continuous cropping Fuji/M9T 337 tree bodies

As can be seen from Table 6, the QSB-6 bacterial manure treatment at each test point can promote the growth of Fuji/M9T 337 under continuous cropping conditions. The annual young shoots which are continuously used as a control are 48-65cm, and the weight of the beancurd sheets is 112-143 g. The growth vigor of the bromomethane treatment is the best, the plant height is the highest, the ground diameter is the largest, the tree body is strong, the length of the annual new shoot is 74-97cm, and the weight of the louver is 138-. The effect of treating the Dasha Ling, the Feng Mao Zi and the wan Tou village with T1 is the best, the plant height is respectively increased by 26.40%, 15.90% and 1.85% compared with the continuous control, the ground diameter is increased by 12.13%, 29.11% and 9.98%, the growth amount of young shoots is increased by 21.25%, 31.01% and 39.92%, and the weight of the beancurd sheets is increased by 5.56%, 8.54% and 1.90%.

Table 6: effect of different treatments on growth of field continuous cropping Fuji/M9T 337 Tree

6. Influence of QSB-6 bacterial manure treatment on activity of field continuous cropping Fuji/M9T 337 soil enzyme

As shown in fig. 13, the effect of T1 treatment was best in all of great sand, great village and gulitacun, in which soil urease, phosphatase, sucrase and catalase were increased by 86.15%, 100.02%, 194.22% and 89.88%, respectively, in great sand, areas of great sand, 74.17%, 95.99%, 284.02% and 84.74%, respectively, and gulitacun was increased by 54.93%, 83.76%, 278.99% and 73.14%, respectively, as compared to the control. The blank bacterial manure carrier also improves the activity of the soil enzyme, and the effect is inferior to that of QSB-6 bacterial manure treatment.

The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

SEQUENCE LISTING

<110> Shandong university of agriculture

<120> bacillus amyloliquefaciens and application thereof in prevention and treatment of apple continuous cropping obstacle

<130> 2020

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 1452

<212> DNA

<213> 16S rDNA of QSB-6 Strain

<400> 1

gccgttgcgg catgctatac atgcagtcga gcggacagat gggagcttgc tccctgatgt 60

tagcggcgga cgggtgagta acacgtgggt aacctgcctg taagactggg ataactccgg 120

gaaaccgggg ctaataccgg atggttgttt 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

caccacgaga gtttgtaaca cccgaagtcg gtgaggtaac ctttatgagc cagccgccga 1440

agtgacagat tt 1452

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence

<400> 2

agagtttgat cctggctcag 20

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence

<400> 3

ggttaccttg ttacgactt 19

<210> 4

<211> 24

<212> DNA

<213> Artificial sequence

<400> 4

cagtcaggaa atgcgtacgt cctt 24

<210> 5

<211> 24

<212> DNA

<213> Artificial sequence

<400> 5

caaggtaatg ctccaggcat tgct 24

<210> 6

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (27)..(27)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (30)..(30)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (33)..(33)

<223> n is a, c, g, or t

<400> 6

gaagtcatca tgaccgttct gcaygcnggn ggnaarttyg a 41

<210> 7

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> misc_feature

<222> (27)..(27)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (33)..(33)

<223> n is a, c, g, or t

<220>

<221> misc_feature

<222> (39)..(39)

<223> n is a, c, g, or t

<400> 7

agcagggtac ggatgtgcga gccrtcnacr tcngcrtcng tcat 44

<210> 8

<211> 23

<212> DNA

<213> Artificial sequence

<400> 8

aggtcaacta gttcagtatg gac 23

<210> 9

<211> 21

<212> DNA

<213> Artificial sequence

<400> 9

aagaaccgta accggcaact t 21

Claims

1. Bacillus amyloliquefaciens strainBacillus amyloliquefaciens) QSB-6, having a biological accession number of: CGMCC NO. 15307.

2. A Bacillus amyloliquefaciens strain of claim 1 (A), (B), (C) and C)Bacillus amyloliquefaciens) Fermentation broth, bacterial suspension and/or supernatant of QSB-6.

3. A Bacillus amyloliquefaciens strain of claim 1 (A), (B), (C) and C)Bacillus amyloliquefaciens) A Bacillus amyloliquefaciens strain (QSB-6 or claim 2)Bacillus amyloliquefaciens) Fermentation of QSB-6Use of the liquid, bacterial suspension and/or supernatant in at least one of (1) to (6) below:

(1) inhibiting phytopathogens;

(2) preparing a product for inhibiting phytopathogens;

(3) preventing and controlling diseases caused by plant pathogenic bacteria;

(5) degrading phenolic acid substances;

(6) preparing a product for degrading phenolic acid substances;

the plant pathogenic bacteria are fusarium oxysporum (F.) (Fusarium oxysporium) Fusarium verticillium (A), (B), (C)Fusarium verticillioides) Fusarium proliferatum (A), (B), (C)Fusarium proliferatum) Fusarium solani (F.), (Fusarium solani) Penicillium citrinum (II)Penicillium cyclopium) Grape anthracnose bacteria (1)Colletotrichum gloeosporioides) Alternaria zeae (B) ((B))Alternaria alternata) And rot of apple tree: (Valsa mali) One or more of;

the phenolic acid substance is one or more of phlorizin, benzoic acid, ferulic acid, vanillic acid, gallic acid and phloretin.

4. A Bacillus amyloliquefaciens strain of claim 1 (A), (B), (C) and C)Bacillus amyloliquefaciens) A Bacillus amyloliquefaciens strain (QSB-6 or claim 2)Bacillus amyloliquefaciens) Application of fermentation liquor, bacterial suspension and/or supernatant of QSB-6 in promoting growth of continuous cropping soil fruit trees.

5. A Bacillus amyloliquefaciens strain of claim 1 (A), (B), (C) and C)Bacillus amyloliquefaciens) A Bacillus amyloliquefaciens strain (QSB-6 or claim 2)Bacillus amyloliquefaciens) The use of a fermentation broth, bacterial suspension and/or supernatant of QSB-6 in any one of (1) or (2) as follows:

(1) reducing continuous cropping obstacles of apple trees;

6. A biocontrol agent for reducing continuous cropping obstacles of apple trees, which comprises Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) (B) as defined in claim 1Bacillus amyloliquefaciens) A Bacillus amyloliquefaciens strain (QSB-6 or claim 2)Bacillus amyloliquefaciens) The fermentation liquor, bacterial suspension and/or supernatant of QSB-6 are active ingredients.

7. A pharmaceutical composition containing Bacillus amyloliquefaciensBacillus amyloliquefaciens) The bacterial fertilizer of QSB-6 is characterized by being prepared by the following method:

a Bacillus amyloliquefaciens strain (B) as claimed in claim 1Bacillus amyloliquefaciens) QSB-6 is inoculated into a fermentation medium, and the composition of the fermentation medium is as follows: 20.0g of cane sugar, 15.0g of yeast extract and MnSO₄ 1.0 g，NaH₂PO₄·2H₂O 2.0 g，Na₂HPO₄·2H₂O4.0 g and 1000mL of distilled water; 30 ℃ and 200r min^-1Performing shake fermentation culture for 24h to obtain a liquid microbial inoculum;

mixing the liquid microbial inoculum and the sterilized carrier according to the ratio of 1 mL: (8-12) g, uniformly mixing, and standing for 12-24h at 35-38 ℃; then putting into a closed container, and fermenting for 14-16 days;

the carrier is formed by mixing cow dung and crop straws according to the weight ratio of 3: 1.

8. Use of the biocontrol agent of claim 6 or the bacterial manure of claim 7 in at least one of the following (1) to (4):

9. A method for alleviating continuous cropping obstacles of apple trees is characterized by comprising the following steps:

a Bacillus amyloliquefaciens strain of claim 1 (A), (B), (C) and C)Bacillus amyloliquefaciens) A Bacillus amyloliquefaciens strain (QSB-6 or claim 2)Bacillus amyloliquefaciens) The fermentation broth, bacterial suspension and/or supernatant of QSB-6 is applied to the rhizosphere soil of apple tree plants.