CN111876351A

CN111876351A - Bacillus belgii and application thereof in relieving apple continuous cropping obstacle

Info

Publication number: CN111876351A
Application number: CN202010714630.0A
Authority: CN
Inventors: 尹承苗; 闫助冰; 毛志泉; 陈学森; 沈向; 胡艳丽
Original assignee: Shandong Agricultural University
Current assignee: Shandong Agricultural University
Priority date: 2020-07-23
Filing date: 2020-07-23
Publication date: 2020-11-03
Anticipated expiration: 2040-07-23
Also published as: CN111876351B

Abstract

The invention discloses a Bacillus belgii strain and application thereof in reducing apple continuous cropping obstacles, wherein the strain is preserved in China general microbiological culture Collection center (CGMCC) in 6-10.2020, and the biological preservation number is as follows: CGMCC NO. 20057. The Bacillus belgii XC1 provided by the invention is a novel biocontrol bacterium with great development potential, has strong inhibition effects on Fusarium oxysporum, Fusarium moniliforme, Fusarium straticola and Fusarium solani, wherein the inhibition rate on Fusarium oxysporum is the highest and reaches 79.83%. The strain can also produce antagonistic enzymes such as protease, cellulase and the like at high yield; reducing the content of phenolic acid substances such as phlorizin, phloretin, ferulic acid, cinnamic acid and the like in the soil; promoting the growth of apple trees; has excellent effect on relieving the continuous cropping obstacle of the apple trees and provides theoretical basis for biological control of the continuous cropping obstacle of the apples.

Description

Bacillus belgii and application thereof in relieving apple continuous cropping obstacle

Technical Field

The invention relates to the technical field of agricultural microorganisms, in particular to a Bacillus belgii strain and application thereof in relieving apple continuous cropping obstacles.

Background

With the updating and upgrading of varieties and the increase of planting areas, China has large-scale old orchards which need to be updated and reformed, and the continuous cropping obstacle phenomenon faced when the old orchards are updated is very common. Researches on king marshal (2018), king xiaobao and the like (2018) show that fusarium harmful fungi are main pathogenic bacteria causing apple continuous cropping obstacles in Bohai Bay and northwest loess plateau regions; xuwenfeng (2011) and Liu Zhi (2013) are respectively separated from soil of an old apple orchard in an apple production area of Bohai Bay to obtain a large amount of Fusarium oxysporum (F.oxysporum), Fusarium moniliforme (F.moniliforme), Fusarium solani (F.solani) and Fusarium delavayi (F.prolifertum), and the Fusarium oxysporum is verified to have strong pathogenicity to seedlings of the sweet tea of the Chinese cognac, Wang Gung Shuaishui (2018) discovers that the four Fusarium oxysporum have strong pathogenicity to the sweet tea of the Chinese cognac through an inoculation test, growth retardation, yellowing and falling off of leaves occur after inoculation, and the death rate exceeds 50% after 2 months of pathogenic bacteria are inoculated. Therefore, the four fusarium species are considered to be pathogenic bacteria causing apple continuous cropping obstacle. Continuous cropping obstacles can cause the reduction of the yield and quality of apple fruits, aggravation of plant diseases and insect pests, weak tree vigor and even death, and cause serious economic loss to fruit growers. Therefore, it is of great significance to research an efficient and environment-friendly prevention and control measure to reduce the continuous cropping obstacle of the apples.

Biological control is a safe, environment-friendly, high-economic-benefit and long-acting control measure for inhibiting pathogenic bacteria in soil by using beneficial microorganisms, and has become a great hotspot of current research. The Dushu et al (2010) finds that the Bacillus belezii has higher antagonistic activity on alternaria brassicae, a pathogenic bacterium of cabbage black spot, and the disease control effect of a field test reaches 79.07%. Liu et al (2017) report that B.velezensis LS69 has strong bacteriostatic effect on carrot soft rot disease pectin bacillus and pear fire blight disease bacteria. Meanwhile, some plant rhizosphere growth-promoting bacteria can degrade autotoxic substances in soil, phenolic acid substances are main autotoxic substances causing apple continuous cropping obstacles, beneficial microorganisms are inoculated to decompose the phenolic acid substances in the continuous cropping soil, sustainable development of agriculture is facilitated, and the method is an economic and effective biocontrol measure. Qizhongzhen et al (2016) separates degradation bacteria for degrading apple autotoxic substances from rhizosphere soil, is beneficial to improving the soil environment of a continuous cropping apple orchard, Qiana et al (2019) separates and separates benzoic acid degradation bacteria, researches degradation characteristics of the benzoic acid degradation bacteria, and provides resource guarantee and scientific basis for relieving peanut continuous cropping obstacles by using the degradation bacteria. However, few studies of bacillus on controlling apple continuous cropping obstacles are currently available.

Disclosure of Invention

Aiming at the defects of the prior art, the inventor conducts long-term technical and practical exploration, and separates a Bacillus belgii XC1 strain from healthy fruit tree rhizosphere soil in a continuous cropping apple orchard, wherein the strain has antagonistic action on Fusarium oxysporum, Fusarium moniliforme, Fusarium stratiotes and Fusarium solani, can produce protease and cellulase at high yield, has excellent degradation effect on phenolic acid substances in soil, and can promote the growth of apple plants and reduce apple continuous cropping obstacles.

Specifically, the invention relates to the following technical scheme:

the first aspect of the invention provides a Bacillus velezensis XC1 strain, which is preserved in China general microbiological culture Collection center (CGMCC for short, with the address of No. 3 Siro 1. in the sunward area of Beijing) in 6.10.2020, and has the biological preservation number of: CGMCC NO. 20057.

The Bacillus belgii (Bacillus velezensis) XC1 is separated from healthy fruit tree rhizosphere soil in a continuous cropping apple orchard and has the following characteristics:

after 24 hours of culture on an LB culture medium, the bacterial colony is milky white and opaque, is initially round in shape and neat in edge, then begins to fold irregularly, spreads in a cloudy state to the periphery, has a bulge in the middle, and has a mucus-like secretion after the bacterial colony is picked up. After static culture in LB liquid culture medium, the bacteria film is formed and aerobic. The thallus is rod-shaped, has spores and is gram-positive.

In a second aspect of the invention there is provided a fermentation broth, suspension and/or supernatant of Bacillus belgii (Bacillus velezensis) XC 1.

The fermentation liquor can be prepared by the following method: inoculating Bacillus belgii (Bacillus velezensis) XC1 into LB liquid culture medium, at 37 deg.C for 180r min^-1Fermenting and culturing for 48h by a shaking table.

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 belgii (Bacillus velezensis) XC1 or Bacillus belgii (Bacillus velezensis) XC1 in at least one of the following (1) to (4):

(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.

Preferably, the phytopathogen is fusarium oxysporum, fusarium moniliforme, fusarium laminarinum or fusarium solani.

In a fourth aspect of the present invention, there is provided the use of the fermentation broth, bacterial suspension and/or supernatant of Bacillus belgii (Bacillus velezensis) XC1 or Bacillus belgii (Bacillus velezensis) XC1 in the production of protease and/or cellulase.

In a fifth aspect of the present invention, there is provided an application of the fermentation liquid, bacterial suspension and/or supernatant of Bacillus belgii (Bacillus velezensis) XC1 or Bacillus belgii (Bacillus velezensis) XC1 in reducing the content of phenolic acid substances in soil.

Preferably, the phenolic acid substances are phlorizin, phloretin, ferulic acid and cinnamic acid.

In a sixth aspect of the present invention, an application of the fermentation liquid, bacterial suspension and/or supernatant of Bacillus belgii (Bacillus velezensis) XC1 or Bacillus belgii (Bacillus velezensis) XC1 in promoting growth of fruit trees is provided.

In a seventh aspect of the present invention, there is provided a use of a fermentation broth, a bacterial suspension and/or a supernatant of Bacillus belgii (Bacillus velezensis) XC1 or Bacillus belgii (Bacillus velezensis) XC1 as described above in any one of (1) or (2) below:

(1) the continuous cropping obstacle of the apple trees is reduced;

(2) the biocontrol agent for relieving the continuous cropping obstacle of the apple trees is prepared.

In the application, the biocontrol agent takes fermentation liquor, bacterial suspension and/or supernatant of Bacillus subtilis XC1 or Bacillus subtilis XC1 as active ingredients. The biocontrol agent is in the form of wettable powder, water dispersant, water suspending agent or dispersible oil suspending agent.

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.

In an eighth aspect of the present invention, a method for alleviating continuous cropping obstacles of apple trees is provided, which comprises the following steps:

the rhizosphere soil of apple tree plants is applied with fermentation broth, bacterial suspension and/or supernatant of Bacillus belgii (Bacillus velezensis) XC1 or Bacillus belgii (Bacillus velezensis) XC 1.

The invention has the beneficial effects that:

the invention firstly separates and obtains a strain of Bacillus velezensis XC1 from healthy fruit tree rhizosphere soil in a continuous cropping apple orchard, the strain is a novel biocontrol bacterium with great development potential, and the strain has strong inhibition effects on Fusarium oxysporum, Fusarium moniliforme, Fusarium stratiotes and Fusarium solani, wherein the inhibition rate on the Fusarium oxysporum is the highest and reaches 79.83%. The strain can also produce antagonistic enzymes such as protease, cellulase and the like at high yield; reducing the content of phenolic acid substances such as phlorizin, phloretin, ferulic acid, cinnamic acid and the like in the soil; promoting the growth of apple trees; has excellent effect on relieving the continuous cropping obstacle of the apple trees and provides theoretical basis for biological control of the continuous cropping obstacle of the apples.

Drawings

FIG. 1: XC1 colony (A) and gram-stained morphology (B).

FIG. 2: XC1 strain 16S rDNA sequence phylogenetic tree (A) and gyrA gene phylogenetic tree (B).

FIG. 3: challenge test of XC1 strain with 4 Fusarium species (PDA medium); in the figure, A is Fusarium proliferatum; b, Fusarium moniliforme; c, Fusarium oxysporum; d, Fusarium solani.

FIG. 4: XC1 strain produces antagonistic enzyme activity; in the figure, a Protease production is produced; B. producing Cellulase Cellulase production.

FIG. 5: degradation condition of XC1 strain on phlorizin; in the figure, A is the growth of XC1 strain on phlorizin screening culture medium; b is the degradation rate of XC1 strain on phlorizin.

FIG. 6: influence of different treatments on the growth of the Malus hupehensis Rehd seedlings; in the figure, CK1 represents a continuous control; XC1 indicates strain XC1 treatment.

FIG. 7: the effect of different treatments on the copy number of the F.oxysporum gene; the gene copy number of the fusarium oxysporum treated by the strain XC1 is reduced by 76.3% compared with that of a continuous cropping control, which shows that XC1 has obvious bacteriostatic effect and also has good inhibitory effect on pathogenic bacteria in soil.

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, continuous cropping obstacles can cause the reduction of the yield and quality of apple fruits, aggravation of plant diseases and insect pests, weak tree vigor and even death, and cause serious economic losses to fruit growers. At present, few reports on the research of bacillus on the aspect of preventing and controlling apple continuous cropping obstacles exist.

Based on the bacteria, XC1 is obtained by separating healthy fruit tree rhizosphere soil in the continuous cropping apple orchard, and the bacteria is identified as Bacillus beleisi according to morphological, physiological and biochemical characteristic detection and phylogenetic analysis based on 16S rDNA and gyrA genes. Different from the currently reported Bacillus belgii, the screened and separated Bacillus belgii XC1 is a novel biocontrol bacterium with great development potential, and integrates multiple effects of inhibiting bacteria, producing antagonistic enzymes, degrading phenolic acid substances in continuous cropping soil, promoting the growth of fruit trees, relieving apple tree continuous cropping obstacles and the like. The inhibition effect of the bacillus subtilis on the fusarium oxysporum, the fusarium moniliforme, the fusarium stratified therein and the fusarium solani is researched by adopting a filter paper method, the bacillus XC1 shows a strong inhibition effect on four fusarium oxysporum, wherein the inhibition rate on the fusarium oxysporum is the highest and reaches 79.83%. And XC1 produces antagonistic enzymes such as protease and cellulase. After 7 days of shake flask culture, the degradation rate of Bacillus belgii XC1 on phlorizin reaches 51.55%. The pot experiment result shows that the plant height, the ground diameter, the fresh weight and the dry weight of the Malus hupehensis Rehd seedling treated by the strain XC1 are respectively improved by 39.25 percent, 43.75 percent, 148.88 percent and 151.82 percent compared with the continuous cropping control; compared with continuous cropping control treatment, the XC1 bacterial manure obviously promotes the root growth of the continuous cropping Malus hupehensis seedling, and the root length, the root surface area, the root volume and the root tip number of the continuous cropping Malus hupehensis seedling are respectively increased by 192.93%, 326.34%, 497.60% and 120.58%; compared with a continuous cropping control, the influence of methyl bromide fumigation treatment and bacterial manure carrier treatment on the content change of phenolic acid substances in soil is small, and XC1 bacterial manure treatment obviously reduces the content of the phenolic acid substances in the soil, wherein phlorizin, phloretin, ferulic acid and cinnamic acid are respectively reduced by 62.6%, 64.9%, 69.8% and 77.9%; the gene copy number of four fusarium in soil is obviously reduced. In conclusion, the bacillus beilesensis XC1 can obviously promote the growth of the seedlings of the Malus hupehensis, reduce the content of phenolic acid substances in soil and obviously inhibit the growth of four fusarium in the soil, which shows that the XC1 strain has good effect on reducing continuous cropping obstacles. At present, XC1 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO. 20057.

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.

LB liquid medium: tryptone 10.0g, yeast extract 5.0g, NaCl 10.0g, pH 7.

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.

Inorganic salt liquid culture medium: NH (NH)₄Cl 0.5g，NaCl 1g，K₂HPO₄1.3g，MgSO₄0.4g, and 1000mL of distilled water.

Phlorizin screening medium (2 mmol/L): 0.96g of phlorizin and 20g of agar were added to 1L of the inorganic salt liquid medium, and no agar was added to the liquid phlorizin screening medium.

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).

Culture medium of skimmed milk powder: 10g of skimmed milk powder, 20g of agar and 1000mL of water, and the pH value is natural.

Cellulase detection culture medium: 10g of peptone, 10g of yeast powder, 10g of sodium carboxymethyl cellulose, 5g of NaCl and KH₂PO₄1g agar 20g, 1000mL water, pH 7.0.

Example 1: isolation and characterization of strains

1. Separating and purifying the strains:

the rhizosphere bacteria are separated by adopting an improved dilution coating flat plate method, fresh soil samples are taken from rhizosphere soil of 3 continuous cropping apple orchards of 3 cities and towns (XC), Bayonet village (WT) and Feng Mao village (FM) in the tobacco pipe city, and the fresh soil samples are sieved by a sieve with the diameter of 3-4 mm, so that impurities in the soil are removed. 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, centrifuged at 10000r at 4 ℃ for 10min, the supernatant was decanted, 15% glycerol was added, and the mixture was stored at-80 ℃.

2. Screening of strains:

the initial screening of the strains adopts a plate confronting method, and the strains separated from the soil and the activated pathogenic bacteria are subjected to a confronting test on a PDA plate. Using a puncher with the diameter of 5mm to punch a bacterial block at the edge of the pathogenic bacteria, inoculating the bacterial block to the center of a PDA (personal digital assistant) plate, inoculating strains separated from soil at 3 butt joints 2cm away from the bacterial block of the pathogenic bacteria, taking another point without inoculating strains as blank control, repeating the treatment for 3 times, culturing at 28 ℃, selecting strains with inhibition zones after 7 days, and re-screening. And re-screening to obtain one antagonistic bacterium with the highest bacteriostasis rate on 4 pathogenic bacteria, and naming the antagonistic bacterium as XC 1.

Through diluting and coating the plate method, 146 strains of bacteria are detected in rhizosphere soil of 3 continuous cropping apple orchards of west cities and towns (XC), gulitacun (WT) and Pteris odorata (FM) in the tobacco pipe city, and a plate confrontation experiment is carried out after separation and purification, wherein 37 strains of bacteria have an inhibiting effect on fusarium, a bacteriostatic ring is formed around the fusarium, and XC1 which is the highest in bacteriostatic rate is finally determined as a test antagonistic strain after re-screening (figure 1A).

3. Identification of the strains:

(1) morphological and physiological biochemical identification:

the XC1 strain obtained by separation is cultured on LB culture medium at 28 ℃ for 24h, and when a single colony appears, the morphological characteristics of the colony are observed. Gram staining is carried out by adopting crystal violet, the shape and the size of bacteria are observed by utilizing a Nikon fluorescence microscope BX-51, and the preparation process of a 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, after the XC1 strain is cultured on an LB medium for 24 hours, single colony colonies are milky and opaque, are initially circular in shape and have regular edges, and then begin to fold irregularly at the edges and diffuse in a cloud form around, and have bulges in the middle of the colony, and after the colonies are picked out, mucus-like secretions exist (fig. 1A); after static culture in LB liquid culture medium, the bacteria film is formed and aerobic. When observed under a fluorescence microscope at 100X/1.30 oil microscope, the spores were short-rod-shaped and gram-stained positively (FIG. 1B).

The results of 10 physiological and biochemical index tests on XC1 strain are shown in Table 1.

Table 1: physiological and biochemical characteristics of XC1 strain

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

The results show that: v-The P test, the starch hydrolysis test, the catalase test, the nitrate reduction test, the kinetic test and the gelatin liquefaction test are positive; indole determination, methyl Red test, H₂The S test is negative; glucose can be used. According to the physiological and biochemical results of the XC1 strain, referring to Bergey's Manual of identification of bacteria and Manual of identification of common bacteria systems, it is preliminarily presumed that the XC1 strain belongs to the genus Bacillus (Bacillus spp.)

(2) Amplification, sequence analysis and phylogenetic analysis of 16S rDNA fragment:

the XC1 strain is inoculated in a liquid LB culture medium, is subjected to shaking culture at the temperature of 28 ℃ at the speed of 200r/min for 12h, and is extracted with a bacterial genome extraction kit. The amplification was performed with the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), and the gyrA conserved gene sequence was amplified with the specific primers gyrA-F (5'-CAGTCAGGAAATGCGTACGTCCTT-3') gyrA-R (5'-CAAGGTAATGCTCCAGGCATTGCT-3'). The 50 μ L PCR reaction system was: total DNA content 3. mu.L, primers 1. mu.L each, dNTP 1. mu.L, 10 XPCR buffer 5. mu.L, Taq DNA polymerase 0.6. mu.L, ddH₂O38.4. mu.L. The PCR reaction conditions are as follows: pre-denaturation at 94 deg.C for 4 min; denaturation at 94 deg.C for 1.5 min; annealing at 55 deg.C for 1 min; extending at 72 deg.C for 1.5 min; for a total of 30 cycles, the total extension was 72 ℃ for 10 min. And (3) sending the amplified product to Shanghai biological engineering technology Limited company for sequencing, carrying out BLAST comparison on a sequencing result through a nucleotide database of a National Center for Biotechnology Information (NCBI), carrying out sequence analysis on the separated antagonistic bacteria by using MEGA5 software and an adjacency N-J clustering analysis method, constructing a phylogenetic tree, and carrying out 1000 times of repeated inspection on a self-expansion value (bootstrap value).

The XC1 strain 16S rDNA gene sequence has the length of 1454bp, the GenBank accession number is MT672576, and the 16SrDNA sequence is shown as SEQ ID NO. 1. Homology alignment was performed by NCBI Blast and phylogenetic trees were established using MEGA5.0, the results are shown in FIG. 2A. It can be seen from the figure that the 16S rDNA sequence of XC1, the 16S rDNA sequence of XC1, clustered with bacillus siamensis (b.siemensis), bacillus amyloliquefaciens (b.amyloliquefaciens), and bacillus belgii (b.velezensis), could not be completely distinguished, and initially classified XC1 as the bacillus amyloliquefaciens operational group (fig. 2A). The length of the gyrA gene sequence of the XC1 strain is 950bp, the GenBank accession number is MT683381, the gyrA sequence is shown as SEQ ID NO.2, the phylogenetic tree is shown as a figure 2B, the gyrA sequence of the XC1 and the gyrA gene of the Bacillus beiensis SCGB 574(CP023431.1) are combined into one branch, and the homology is 93 percent (figure 2B). Combining the above analysis indicates that XC1 strain is bacillus belgii b.

Based on the morphological, physiological and biochemical identification and 16S rDNA sequence homology analysis results of the XC1 strain, the XC1 strain obtained by separation is identified as Bacillus velezensis. And the strain is subjected to biological preservation, and the preservation information is as follows:

the strain name is as follows: bacillus belgii

Latin name: bacillus velezensis

The strain number is as follows: XC1

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No.1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: 2020.6.10

Registration number of the preservation center: CGMCC NO. 20057.

Example 2: functional identification of Bacillus velezensis XC1

1. The bacteriostatic effect is as follows:

adopting a filter paper method to evaluate the bacteriostatic effect of XC1, inoculating XC1 strain in LB liquid culture medium, and carrying out inoculation at 37 ℃ for 180 r.min^-1Performing shake bed culture for 24h, punching a bacterial block at the edge of pathogenic bacteria by using a puncher with the diameter of 5mm, inoculating the bacterial block to the center of a PDA (personal digital assistant) flat plate, placing sterile filter paper sheets at 4 butt joints 2cm away from the bacterial block of the pathogenic bacteria, injecting 10 microliter XC1 bacterial liquid, pre-diffusing for 2h, then culturing at 28 ℃, taking pathogenic bacteria without the bacterial liquid as a contrast, observing at any time, measuring the diameter of a fungal colony by using a vernier caliper and adopting a cross method after the plate is fully grown in contrast, and expressing the size of the bacteriostatic rate by using the diameter. The above operation was repeated 3 times.

The inhibition ratio was [ (control colony diameter-treated bacteria diameter)/control colony diameter ] × 100%

Results of the confrontation test show that the XC1 strain has strong inhibition effect on the growth of 4 pathogenic fusarium hyphae (FIG. 3, Table 2). Wherein the inhibition rate to fusarium oxysporum is higher, and the inhibition rate on an improved PDA culture medium reaches 79.83%; the inhibition effect on the fusarium solani, the fusarium laminariiforme and the fusarium moniliforme is less, and the inhibition rates on the improved PDA culture medium respectively reach 68.20%, 72.95% and 70.53%.

Table 2: inhibition rate of XC1 strain on 4 kinds of fusarium

2. Producing antagonistic enzyme:

inoculating strain XC1 to skimmed milk powder culture medium, repeating the treatment for four times, culturing at 28 deg.C for 5d, and observing whether transparent ring is generated; adding XC1 point to cellulase detection culture medium, repeating for four times each treatment, culturing at 28 deg.C for 5 days, and adding appropriate amount of 1 mg/mL into the plate^-1Dyeing the Congo red solution for 1 hour, discarding the dye solution, adding an appropriate amount of 1 mol. L^-1The NaCl solution was washed for 1 hour, and the presence of the formation of a transparent circle was observed.

After 5 days of culture, XC1 forms transparent circles on a skim milk powder culture medium and a cellulase detection culture medium (figure 4), which shows that XC1 can secrete protease and cellulase, as shown in Table 3, after 5 days of culture, the diameter of a protease hydrolysis circle of a strain is more than 40mm, the diameter of a cellulase hydrolysis circle is more than 25mm, and XC1 has stronger enzyme production activity.

Table 3: antagonistic bacteria XC1 enzyme producing Activity

3. Degrading phlorizin:

XC1 is inoculated to a phlorizin screening culture medium which takes phlorizin as the only carbon source, inverted culture is carried out at 37 ℃, and XC1 is observed to grow on the phlorizin screening culture medium, so that the XC1 strain can take the phlorizin as the carbon source and has the capability of degrading the phlorizin (figure 5A). XC1 was inoculated into a basal inorganic salt culture with phlorizin as the sole carbon sourceIn nutrient medium, at 37 deg.C, 180 r.min^-1Shaking culture, periodically collecting culture solution with thallus filtered, measuring absorbance at 325nm of maximum absorption wavelength of phlorizin by using ultraviolet spectrophotometer, and finding that XC1 has degradation rate of 51.55% (2mmol/L) after 7 days (FIG. 5B). The degradation rate is (absorbance of the missed culture solution-absorbance of the missed culture solution)/absorbance of the missed culture solution x 100%.

Example 3: pot experiment

1. And (3) experimental design:

the pot culture test is carried out in 2019, 3-10 months in the national apple engineering experiment center of southern school of Shandong agricultural university and an apple continuous cropping and microorganism laboratory, the tested plant of the pot culture test is a seedling of Malus hupehensis Rehd, seeds of the Malus hupehensis Rehd are stacked at 4 ℃ for about 30 days, and the seeds are sowed in a culture bowl filled with a seedling culture medium for seedling culture after white emergence. Selecting plants which have consistent growth vigor and no plant diseases and insect pests when the seedlings grow to 6 main leaves, transplanting the plants into a clay tile pot (the upper diameter is 25cm, the lower diameter is 17cm and the height is 18cm) filled with 6.5kg of different treated soil in 1 day after 5 months, sampling in the middle ten days of 9 months, and determining related experimental indexes.

Soil for pot culture test is taken from 32-year old apple orchard in Manzhuantan Qingwan, Taian, Shandong, and is randomly taken at multiple points in an area 80.00cm from a trunk and 5.00-40.00 cm deep, and is uniformly mixed. The soil type is sandy soil, the organic matter content is 13.97g/kg, the quick-acting nitrogen is 35.76mg/kg, the quick-acting potassium is 116.64mg/kg, the quick-acting phosphorus is 20.42mg/kg, and the pH value of the soil is 5.059.

The XC1 bacterial fertilizer used for the pot culture test is manufactured by the microbial resource company Limited of Chuangdi, Tex, China, and the manufacturing method comprises the following steps: the fermentation liquid of XC1 strain is inoculated into bacterial manure carrier matrix (cow dung: straw 1: 3 by weight ratio) according to the inoculation amount of 10% (namely 10mL of XC1 fermentation liquid is inoculated into each 100g of bacterial manure carrier), and the fermentation is carried out for one week at 37 ℃.

The prepared XC1 bacterial manure is a black gray powdery solid, and the viable count is 2.10 multiplied by 10⁹cfu/g。

The experiment was set up with 4 treatments, respectively: continuous cropping soil (CK1), continuous cropping soil fumigated by methyl bromide (CK2), bacterial manure carrier treatment (CK3), XC1 bacterial manure treatment (T), wherein 2 seedlings are planted in each pot after 20 pots of treatment, and fertilizer and water management is unified. The application amount of the bacterial manure of the T treatment group and the use amount of the bacterial manure carrier of the CK3 treatment group are both 1% of the soil mass, and the continuous cropping soil and the bacterial manure are fully and uniformly mixed before planting.

The bacterial manure carrier is a mixture of cow dung and straws (cow dung: straws: 1: 3) and is provided by the microbial resource company Limited of Chundi, Tex, China.

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 volume of the root system and the number of root tips.

Taking 0.5g of fresh soil sieved by the method

The soil DNA extraction kit comprises the following operation steps of extracting DNA, carrying out real-time fluorescence quantitative analysis on gene copy number of Fusarium oxysporum in soil by using CFX96TMthermal Cycler (Bio-Rad), verifying the inhibition effect of XC1 strain on the Fusarium oxysporum in soil, and referring to the method of Lepis (Lepis family, phase, Pan-Feng, Chen schson, Shen, Yi bearing, Digitaria japonica, 2016. the influence of the mixed operation of Pingyi sweet tea seedling and shallot on the continuous cropping soil environment of apple. gardening journal, 43(10) 1853 & 1862.).

The XC1 strain has the growth promoting effect on the Malus hupehensis Rehd seedlings:

as can be seen from table 4, compared with the aged orchard soil treatment (CK1), the XC1 bacterial manure treatment (T) significantly promoted the plant height, ground diameter, fresh weight, and dry weight of the continuously cropping phyllus rubus suavissimus seedling by 39.25%, 43.75%, 148.88%, and 151.82%, respectively, and the plant height, ground diameter, fresh weight, and dry weight of the continuously cropping phyllus rubus suavissimus seedling after the XC1 bacterial manure treatment were 1.29 times, 1.43 times, 1.78 times, and 1.93 times, respectively, of the bacterial manure carrier treatment (CK 3). Therefore, XC1 bacterial manure treatment can obviously promote the growth of the Malus hupehensis Rehd seedlings.

Table 4: influence of XC1 bacterial manure on biomass of Malus hupehensis seedling

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

Influence of XC1 bacterial manure treatment on root form of Malus hupehensis Rehd seedlings

As can be seen from table 5, XC1 bacterial manure treatment (T) promoted root growth of the seedling of rubus hupehensis, which was significantly higher than that of the continuous cropping control (CK1) and bacterial manure carrier treatment (CK 2). Compared with a continuous cropping control (CK1), the root length, the root surface area, the root volume and the root tip number of the XC1 bacterial manure treatment are respectively increased by 192.93%, 326.34%, 497.60% and 120.58%. It can be seen that the bacterial manure treatment of XC1 can remarkably promote the growth of the roots of the Malus hupehensis Rehd seedlings (figure 6).

Table 5: influence of XC1 bacterial manure treatment on root growth of Malus hupehensis Rehd seedlings

5. Inhibition effect of XC1 strain on fusarium in soil:

the fusarium oxysporum in the soil is measured by a real-time fluorescent quantitative PCR technology (figure 7), and it can be seen that the copy number of the fusarium oxysporum gene which is continuously used as a control (CK1) in 4 treatments is the highest, the difference with the bacterial manure carrier treatment (CK3) is not significant, and the differences between the methyl bromide fumigation treatment (CK2) and the XC1 bacterial manure treatment (T) are respectively reduced by 83.3% and 76.3% and are significant compared with the control. It can be seen that the strain XC1 also has a good inhibition effect on Fusarium oxysporum in soil.

Effect of bacterial manure treatment of XC1 on soil phenolic acid content:

as shown in table 6, the content of the phenolic acid substances in the soil of the methyl bromide fumigation treatment (CK2) and the bacterial manure carrier treatment (CK3) is not much different from that of the continuous cropping control (CK1), which indicates that the influence of the methyl bromide fumigation treatment and the bacterial manure carrier treatment on the content of the phenolic acid substances in the soil is not obvious; compared with a continuous cropping control (CK1), the contents of phlorizin, phloretin, ferulic acid and cinnamic acid in the XC1 bacterial manure treatment (CK3) are respectively reduced by 58.33%, 64.29%, 42.67% and 50.00%, and the content of phenolic acid substances in soil can be obviously reduced by the XC1 bacterial manure treatment.

Table 6: influence of continuous cropping soil with different textures on soil phenolic acid substances

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 belgii and application thereof in relieving apple continuous cropping obstacle

<130>2020

<160>6

<170>PatentIn version 3.5

<210>1

<211>1454

<212>DNA

<213> XC1 Strain

<400>1

tgtggggcgt gcctatacat gcaagtcgag cggacagatg ggagcttgct ccctgatgtt 60

agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagactggga taactccggg 120

aaaccggggc taataccgga tggttgtttg aaccgcatgg ttcagacata aaaggtggct 180

tcggctacca cttacagatg gacccgcggc gcattagcta gttggtgagg taacggctca 240

ccaaggcaac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300

ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac 360

ggagcaacgc cgcgtgagtg atgaaggttt tcggatcgta aagctctgtt gttagggaag 420

aacaagtgcc gttcaaatag ggcggcacct tgacggtacc taaccagaaa gccacggcta 480

actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc 540

gtaaagggct cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga 600

gggtcattgg aaactgggga acttgagtgc agaagaggag agtggaattc cacgtgtagc 660

ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa 720

ctgacgctga ggagcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg 780

ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc 840

attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 900

gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 960

tcttgacatc ctctgacaat cctagagata ggacgtcccc ttcgggggca gagtgacagg 1020

tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080

caacccttga tcttagttgc cagcattcag ttgggcactc taaggtgact gccggtgaca 1140

aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca 1200

cgtgctacaa tggacagaac aaagggcagc gaaaccgcga ggttaagcca atcccacaaa 1260

tctgttctca gttcggatcg cagtctgcaa ctcgactgcg tgaagctgga atcgctagta 1320

atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380

accacgagag tttgtaacac ccgaagtcgg tgaggtaacc ttttaggagc cagccgccga 1440

aggtgaaccc agat 1454

<210>2

<211>950

<212>DNA

<213> XC1 Strain

<400>2

tgagcgttat cgtatcccgg gcgcttccgg atgtgcgtga cggtctgaag ccggttcaca 60

gacggatttt gtacgcaatg aatgatttag gcatgaccag tgacaaacca tataaaaaat 120

ctgcccgtat cgtcggtgaa gttatcggta agtaccaccc gcacggtgac tcagcggttt 180

acgaatcaat ggtcagaatg gcgcaggatt ttaactaccg ctacatgctt gttgacggac 240

acggcaactt cggttcggtt gacggcgact cagcggccgc gatgcgttac acagaagcga 300

gaatgtcaaa aatcgcaatg gaaattctgc gtgacattac gaaagacacg attgactatc 360

aagataacta tgacggttca gaaagagagc ctgccgtcat gccttcgaga tttccgaatc 420

tgctcgtaaa cggggctgcc ggtattgcgg tcggaatggc gacaaacatt cctccccatc 480

agcttggaga agtcattgaa ggcgtgcttg ccgtaagtga gaatcctgag attacaaacc 540

aggagctgat ggaatacatc ccgggcccgg attttccgac tgctggtcag attttgggcc 600

ggagcggcat ccgcaaggca tatgaatccg gacggggatc aatcacaatc cgggctaagg 660

ctgaaatcga agagacatca tcaggaaaag aaagaattat tgttacggaa cttccttatc 720

aggtgaacaa agcgagatta attgaaaaaa tcgcagatct tgtccgggac aaaaaaatcg 780

aaggaattac cgacctgcga gacgaatccg accgtaacgg aatgagaatc gtcattgaga 840

tccgccgtga cgccaatgct cacgtcattt tgaataacct gtacaaacaa acggccctgc 900

agacgtcttt cggaatcaac ctgctggcgc tcgtgacgga cagccgaagt 950

<210>3

<211>20

<212>DNA

<213> Artificial sequence

<400>3

agagtttgat cctggctcag 20

<210>4

<211>19

<212>DNA

<213> Artificial sequence

<400>4

ggttaccttg ttacgactt 19

<210>5

<211>24

<212>DNA

<213> Artificial sequence

<400>5

cagtcaggaa atgcgtacgt cctt 24

<210>6

<211>24

<212>DNA

<213> Artificial sequence

<400>6

caaggtaatg ctccaggcat tgct 24

Claims

1. A strain of Bacillus belgii (Bacillus velezensis) XC1 with the biological preservation number: CGMCC NO. 20057.

2. A fermentation broth, bacterial suspension and/or supernatant of Bacillus velezensis XC1 of claim 1.

3. Use of a fermentation broth, bacterial suspension and/or supernatant of Bacillus beijerinckii (Bacillus velezensis) XC1 according to claim 1 or Bacillus beijerinckii (Bacillus velezensis) XC1 according to claim 2 in at least one of (1) to (4) below:

(1) inhibiting phytopathogens;

(2) preparing a product for inhibiting phytopathogens;

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

4. Use according to claim 3, wherein the phytopathogen is Fusarium oxysporum, Fusarium moniliforme, Fusarium stratiotes, and Fusarium solani.

5. Use of a fermentation broth, suspension and/or supernatant of Bacillus beijerincensis (Bacillus velezensis) XC1 according to claim 1 or Bacillus beijerincensis (Bacillus velezensis) XC1 according to claim 2 for the production of a protease and/or a cellulase.

6. Use of a fermentation broth, suspension and/or supernatant of Bacillus beijerincensis (Bacillus velezensis) XC1 according to claim 1 or Bacillus beijerincensis (Bacillus velezensis) XC1 according to claim 2 for reducing the content of phenolic acids in soil.

7. The use according to claim 6, wherein the phenolic acid is phlorizin, phloretin, ferulic acid and/or cinnamic acid.

8. Use of a fermentation broth, bacterial suspension and/or supernatant of Bacillus beijerinckii (Bacillus velezensis) XC1 as defined in claim 1 or Bacillus beijerinckii (Bacillus velezensis) XC1 as defined in claim 2 for promoting the growth of fruit trees.

9. Use of a fermentation broth, bacterial suspension and/or supernatant of Bacillus beijerinckii (Bacillus velezensis) XC1 according to claim 1 or Bacillus beijerinckii (Bacillus velezensis) XC1 according to claim 2 in any one of the following (1) or (2):

(1) the continuous cropping obstacle of the apple trees is reduced;

10. A method for relieving apple tree continuous cropping obstacles is characterized by comprising the following steps:

applying a fermentation broth, a bacterial suspension and/or a supernatant of Bacillus beijerinckii (Bacillus velezensis) XC1 of claim 1 or Bacillus beijerinckii (Bacillus velezensis) XC1 of claim 2 to rhizosphere soil of apple tree plants.