CN114369556B

CN114369556B - Bacillus, biocontrol microbial agent prepared from bacillus and application of biocontrol microbial agent

Info

Publication number: CN114369556B
Application number: CN202210125856.6A
Authority: CN
Inventors: 刘继
Original assignee: Chengdu Academy of Agriculture and Forestry Sciences
Current assignee: Chengdu Academy of Agriculture and Forestry Sciences
Priority date: 2022-02-10
Filing date: 2022-02-10
Publication date: 2023-07-04
Anticipated expiration: 2042-02-10
Also published as: CN114369556A

Abstract

The invention relates to bacillus, a biocontrol microbial inoculum prepared from the bacillus and application thereof, and belongs to the technical field of biological microbial inoculum. The bacillus provided by the invention is QY-1, is classified and named as bacillus bailii Bacillus velezensis, and is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with the preservation addresses: the preservation number is CGMCC No.23381, and the preservation time is 2021, 9 months and 10 days. The bacillus QY-1 of the invention has better antibacterial effect on various pathogenic bacteria no matter whether the bacillus QY-1 is a living thallus or a supernatant fluid or a cell lysate, and has wide bactericidal spectrum. Meanwhile, the bacillus QY-1 has strong living ability and wide application range for nutrient sources, humiture and pH value. The bactericide has no pathogenicity to fruits and vegetables, and has better tolerance to broad-spectrum bactericides and better application prospect.

Description

Bacillus, biocontrol microbial agent prepared from bacillus and application of biocontrol microbial agent

Technical Field

The invention belongs to the technical field of biological bactericides, and particularly relates to bacillus, a biocontrol bactericides prepared from the bacillus and application of the biocontrol bactericides in the aspects of inhibiting pathogenic bacteria in fruit and vegetable cultivation period and storage and transportation period.

Background

Fruits and vegetables are easy to be affected by soil-borne germs in the planting process, are often threatened by bacterial and fungal infection, and particularly, common symptoms such as necrosis (leaf spots and leaf withering), rot (root rot and fruit rot), wilting (root, stem base and vascular bundle tissue are affected) and the like are most common in fungal diseases. At present, chemical bactericides, breeding disease-resistant varieties, intercropping, grafting and other methods are mostly adopted for disease control, but the control measures have advantages and disadvantages.

Fresh fruits and vegetables are subjected to different degrees of infectious diseases (pathogenic microorganism infection) due to the change of physiological and biochemical characteristics of the fresh fruits and vegetables after picking and during long-term storage. The infectious diseases of the picked fruits and vegetables are caused by the infection of pathogenic bacteria on the fruits and vegetables in the cultivation period, the initial processing period and the storage and transportation period. Wherein, part of diseases in the cultivation period show symptoms and seriously affect the yield in the fruit ripening process, and part of diseases in the cultivation period do not show macroscopic signs before the fruits are fully ripe, but cause great loss in the post-harvest storage and transportation process.

At present, the main method for controlling the postharvest infectious diseases of fruits and vegetables is to use chemical pesticides in the cultivation period, the temporary harvest period and even after harvest, but the chemical pesticides cause serious non-point source pollution and food safety problems, so biological control is a preferred scheme taking environmental protection, human health and economic benefits into account.

Biological control is a technology for controlling disease occurrence by using living organism biocontrol bacteria and metabolic active substances thereof, wherein the biocontrol bacteria can colonize and grow on crop plants and rhizosphere to form biological barriers for protecting crops from being invaded by bacteria, and the metabolic active substances thereof inhibit and kill pathogenic fungi on one hand and induce plants to improve disease resistance on the other hand.

However, the existing biocontrol strain and biocontrol microbial inoculum can not replace chemical pesticides for disease control, and the existing microbial inoculum mainly has the following problems:

(1) The biocontrol bacteria have no pertinence on fruit disease control;

the existing biocontrol bacteria and microbial inoculum are not marked with the planting environment from which crops are sourced, and the disease difference of different crops is huge, and the bacteriostasis spectrum of the biocontrol bacteria is limited, so that the types of pathogenic bacteria which can be inhibited by the biocontrol bacteria are unclear, and whether the biocontrol bacteria have bacteriostasis effect becomes a random event.

(2) The biocontrol bacteria have undefined nutrient source, suitable habitat and drug resistance;

the existing biocontrol bacteria and microbial inoculum are not marked with optimal and extreme nutrition sources, optimal and extreme habitats and drug resistance to common chemical bactericides, pesticides and herbicides. The problem causes the situation that the strain cannot be planted fixedly or the strain is quickly killed by various pesticides after the biocontrol bacteria or the microbial inoculum is applied, and the survival of the biocontrol bacteria also becomes a random event.

(3) Too many mixed types of biocontrol bacteria cannot quickly form competitive advantages;

because the existing biocontrol bacteria have no pertinence to fruit disease control, a plurality of biocontrol bacteria are often used for compounding to form a composite microbial inoculum for use in production, but the effect of the biocontrol bacteria needs a certain biomass as a guarantee, the use of the composite microbial inoculum can cause nutrition competition and space competition, and even if the biocontrol bacteria with pertinence exist in the situation, the biomass is insufficient for forming a stronger inhibition effect on pathogenic bacteria.

Therefore, aiming at the problems that the existing single biocontrol bacterium has narrower bacteriostasis spectrum, large use limitation, incapability of field planting of strains and easiness of killing by various pesticides, the compound microbial inoculum has low biomass, large nutrition competition and space competition among the strains, and poor fruit disease control effect, how to further develop more efficient single biocontrol bacterium capable of inhibiting pathogenic bacteria in the fruit and vegetable cultivation period and storage and transportation period is a technical problem to be solved in the field.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a biocontrol strain capable of being applied to disease control in the fruit and vegetable cultivation period and the storage and transportation period, and a biocontrol microbial agent prepared from the biocontrol strain and application of the biocontrol strain. The invention aims to solve the problems that a single biological bacteria-proofing agent has narrow bacteriostasis spectrum, large use limitation, incapability of field planting strains and easiness of killing by various pesticides, and the compound bacteria agent has low biomass, large nutrition competition and space competition among the strains and poor fruit disease-proofing effect.

Through a great deal of experimental study and fumbling, the inventor of the invention obtains a bacillus, which is QY-1 and classified and named as bacillus bailii Bacillus velezensis, and the preservation unit is: the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) has a preservation address of: beijing, chaoyang, north Chen Xili No.1, 3, accession number is: CGMCC No.23381, the preservation time is: 2021, 9 and 10.

The bacillus QY-1 provided by the invention has broad-spectrum antibacterial property on pathogenic bacteria of fruits and vegetables, and can inhibit the types of the pathogenic bacteria to 32 types. The bacillus QY-1 has good antibacterial effect on various pathogenic bacteria in both the cultivation period and the storage period of fruits and vegetables, has good strain planting effect, can be effectively used for disease control and antibacterial fresh-keeping of fruits and vegetables, and has easily obtained nutrition source, strong extreme temperature resistance and strong pesticide resistance. The single strain has no problems of low biomass of each strain among the composite bacteria and large nutrition competition and space competition among the strains, and has extremely obvious application effect.

The conserved sequence of the bacillus provided by the invention is shown as SEQ NO.1 in a sequence table.

The invention further provides a biocontrol microbial agent containing the bacillus.

The invention further provides application of the biocontrol microbial inoculum in preventing and controlling fruit and vegetable diseases caused by fruit and vegetable pathogenic bacteria and application of the bacillus in preparing fruit and vegetable fresh-keeping agents.

Specifically, the application is to inhibit pathogenic bacteria of fruits and vegetables by adopting a biocontrol microbial inoculum in the cultivation period and the storage and transportation period of the fruits and vegetables so as to prevent and treat diseases and realize bacteriostasis and fresh-keeping.

Specifically, the fruit and vegetable pathogenic bacteria include Acremonium Sclerotigenum, aureobasidium melanogenum, botryosphaeria laricina, botrytis cinerea, botrytis isfabae, byssochlamys spectabilis, cladosporium cladosporioides, diaporthe eres, fusarium decemcellulare, fusarium proliferatum, galactomyces geotrichum, glomerella acutata, lasiodiplodia theobromae, moniliapolystroma, mucor circinelloides, penicillium camemberti, penicillium chrysogenum, penicillium verruculosum, rhizopus stolonifer, aspergillus niger, colletotrichum gloeosporioides, alternaria alternata, penicillium expansum, penicillium multicolor, schizophyllum commune, arthrinium urticae, fusarium sp.

Specifically, the fruit and vegetable diseases which can be caused by the pathogenic bacteria include: grape rot (Acremonium Sclerotigenum), mango blight (Aureobasidium melanogenum), strawberry root rot (Botryosphaeria laricina), blueberry gray mold (Botrytis cinerea), mulberry soft rot (Botrytis cinerea), grape fruit rot (Cladosporium cladosporioides), grape rot (Diaporthe eres), apple brown spot (Fusarium decemcellulare), mulberry fruit rot (Fusarium proliferatum), apple rot (Glomerella acutata), jackfruit black rot (Lasiodiplodia theobromae), cherry brown rot (monilinia polyspora), citrus aspergillosis (Mucor circinelloides), citrus soft rot (Rhizopus stolonifer), grape black mold (Aspergillus niger), watermelon anthracnose (Colletotrichum gloeosporioides), pear black spot (Alternaria alternata), kiwi fruit green rot (Penicillium expansum).

The action concentration of the bacillus QY-1 in the biocontrol microbial inoculum is 10 ^6-7 CFU/mL。

The invention has the beneficial effects that:

(1) The bacillus QY-1 viable bacteria has good antibacterial effect on the various pathogenic bacteria, wherein the antibacterial rate of the bacillus QY-1 viable bacteria against 19 pathogenic bacteria reaches 100%, the growth of the pathogenic bacteria can be completely inhibited, and the antibacterial effect is extremely remarkable; the bacteria inhibition rate of the living bacteria is more than 90 percent, and 6 pathogenic bacteria exist, so that the bacteria inhibition effect is good; 7 pathogenic bacteria exist in the living bacteria with the antibacterial rate of more than 80%, and the living bacteria still have a good antibacterial effect.

(2) The bacillus QY-1 supernatant has certain antibacterial effect on different pathogenic fungi. Wherein 21 pathogenic bacteria exist in the bacillus QY-1 supernatant with the bacteriostasis rate of more than 90 percent against the pathogenic bacteria, and the bacillus QY-1 has good bacteriostasis effect; the supernatant has more than 80 percent of antibacterial rate and 10 pathogenic bacteria, and still has better antibacterial effect; the supernatant has a bacteriostasis rate of less than 80% and only 1 pathogenic bacteria, and the bacteriostasis effect is slightly poor, but still has certain bacteriostasis.

(3) The bacillus QY-1 cell lysate provided by the invention has a certain antibacterial effect on different pathogenic fungi, but the antibacterial effect is slightly lower than that of the supernatant of the QY-1 strain. Wherein, 17 pathogenic bacteria exist in the bacillus QY-1 cell lysate with the bacteriostasis rate of more than 90 percent, and the bacteriostasis effect is good; the cell lysate has 12 pathogenic bacteria with a bacteriostasis rate of more than 80 percent, and has good bacteriostasis effect; 3 pathogenic bacteria exist in the cell lysate with the antibacterial rate lower than 80%, and the antibacterial effect is slightly poor.

Drawings

FIG. 1 shows the bacteriostatic effect of live bacillus QY-1 on different pathogenic bacteria, wherein A1.1: acremonium Sclerotigenum +QY-1 is treated; a1.2: acremonium Sclerotigenum blank; a2.1, aureobasidium melanogenum +QY-1 treatment; a2.2: aureobasidium melanogenum blank; a3.1, botryosphaeria laricina +QY-1 treatment; a3.2: botryosphaeria laricina blank; a4.1 Botrytis cinerea+QY-1 treatment; a4.2 Botrytis cinerea blank.

FIG. 2 shows the bacteriostatic effect of live Bacillus QY-1 on different pathogenic bacteria, wherein A5.1 is Botrytis fabae+QY-1 treatment; a5.2 Botrytis fabae blank; a6.1, byssochlamys spectabilis +QY-1 treatment; a6.2: byssochlamys spectabilis blank; a7.1, cladosporium cladosporioides +QY-1 treatment; a7.2: cladosporium cladosporioides blank; a8.1 Diaphorthe eres+QY-1 treatment; a8.2 Diadorthe eres blank.

FIG. 3 shows the bacteriostatic effect of live bacillus QY-1 on different pathogenic bacteria, wherein A9.1: fusarium decemcellulare +QY-1 is treated; a9.2: fusarium decemcellulare blank; a10.1: fusarium proliferatum +QY-1 treatment; a10.2: fusarium proliferatum blank; a11.1, galactomyces geotrichum +QY-1 treatment; a11.2: galactomyces geotrichum blank; a12.1, glomerella acutata +QY-1 treatment; a12.2: glomerella acutata blank.

FIG. 4 shows the bacteriostatic effect of live bacillus QY-1 on different pathogenic bacteria, wherein A13.1: lasiodiplodia theobromae +QY-1 is treated; a13.2: lasiodiplodia theobromae blank; a14.1 Moniliariapolystroma+QY-1 treatment; a14.2: monilinia polystroma blank; a15.1: mucor circinelloides +QY-1 treatment; a15.2: mucor circinelloides blank; a16.1, penicillium camemberti +QY-1 treatment; a16.2: penicillium camemberti blank.

FIG. 5 shows the bacteriostatic effect of live Bacillus QY-1 on different pathogenic bacteria, wherein A17.1: penicillium chrysogenum +QY-1 is treated; a17.2: penicillium chrysogenum blank; a18.1, penicillium verruculosum +QY-1 treatment; a18.2: penicillium verruculosum blank; a19.1: rhizopus stolonifer +QY-1 treatment; a19.2: rhizopus stolonifer blank; b1.1: aspergillus niger +QY-1 treatment; b1.2: aspergillus niger blank.

FIG. 6 shows the bacteriostatic effect of live Bacillus QY-1 on different pathogenic bacteria, wherein B2.1: colletotrichum gloeosporioides +QY-1 is treated; b2.2: colletotrichum gloeosporioides blank; b3.1: alternaria alternata +QY-1 treatment; b3.2: alternaria alternata blank; b4.1. Penicillium expansum +QY-1 treatment; b4.2: penicillium expansum blank; b5.1: penicillium multicolor +QY-1 treatment; b5.2: penicillium multicolor blank.

FIG. 7 shows the bacteriostatic effect of live Bacillus QY-1 on different pathogenic bacteria, wherein B6.1 is Schizophyllum commune +QY-1; b6.2: schizophyllum commune blank; c1.1: arthrinium urticae +QY-1 treatment; c1.2: arthrinium urticae blank; c2.1: aspergillus flavus +QY-1 treatment; c2.2: aspergillus flavus blank; c3.1: talaromyces variabilis +QY-1 treatment; c3.2: talaromyces variabilis blank.

FIG. 8 shows the bacteriostatic effect of live Bacillus QY-1 on different pathogens, with C4.1: microdochium nivale +QY-1 treatment; c4.2: microdochium nivale blank; c5.1 Fusarium sp. + QY-1 treatment; c5.2. Fusarium sp. C6.1 Penicillium sp. + QY-1 treatment; c6.2. Penicillium sp. C7.1 Cladosporium ruhnei+QY-1 treatment; c7.2 Cladosporium ruhnei blank.

FIG. 9 shows the turbidity of the culture of Bacillus QY-1 at different pH;

FIG. 10 shows the results of in vivo bacteriostasis test of Bacillus QY-1 on blueberry fruit, wherein the left panel shows blank control and the right panel shows QY-1 bacterial liquid treatment.

Detailed Description

The present invention is described in detail below by way of examples, which are necessary to be pointed out herein for further illustration of the invention and are not to be construed as limiting the scope of the invention, since numerous insubstantial modifications and adaptations of the invention will occur to those skilled in the art in light of the foregoing disclosure.

Examples

1. Bacterial strain origin

The method is characterized in that the urban green house in 2019 is a urban green house, healthy leaves, flowers and fruits of blueberries are collected for multiple times, and the healthy leaves, flowers and fruits of the blueberries are placed in a sterile sealing bag for microbial separation and identification, so that pathogenic microorganisms in the blueberries are isolated and obtained initially. However, the phenomenon that fungi cannot grow due to single bacterial pollution occurs in the separation in summer and autumn respectively, and the bacteria obtained by the two separation are the same strain, namely the strain provided by the invention.

2. Identification of strains

The bacterial DNA was extracted and the 16S rDNA sequence of the strain was sequenced with the following primers:

27F：GAGAGTTTGATCCTGGCTCAG

1492R：TACGGCTACCTTGTTACGAC

the conserved sequence obtained by sequencing is analyzed by a evolutionary tree, and is preliminarily judged to be Bacillus amyloliquefaciens or Bacillus velezensis, and the gene sequence is shown as SEQ NO. 1.

After the bacteriostasis experiment, to further protect the strain, the strain was subjected to full sequence sequencing and defined as Bacillus velezensis according to sequence analysis.

The strain named as bacillus QY-1 is preserved in China general microbiological culture Collection center (China Committee for culture Collection), the preservation address is North Chen Xili No.1, 3 in the Korean area of Beijing, and the preservation center is numbered: CGMCC No.23381, the preservation time is: 2021, 9 and 10.

3. Bacteriostasis test of strains

Bacterial inhibition test of live bacteria of bacillus QY-1

The strain has rich antibacterial types and has good control effect on pathogenic bacteria on strawberries, blueberries and grapes.

1. Test design

Because the main application mode of the biocontrol strain is to use the living strain to spray on the soil or the plant surface, the antibacterial effect of the living strain is only compared with that of carbendazim.

Single colonies of the QY-1 strain were inoculated into LB medium, cultured with constant shaking at 28℃at 150rpm for 24 hours, and the cell suspension density was determined to be 1X 10 by using a hemocytometer ^7-8 CFU/mL. Adding QY-1 strain cell suspension into PDA culture medium, shaking and mixing to obtain plates, wherein the cell density of each plate QY-1 strain is 1×10 ^6-7 CFU/mL. A6 mm sterile punch was used to take the Botrytis cinerea bacterial cake and place it in the center of the PDA plate containing QY-1 strain, with PDA plate containing 100mg/L carbendazim as positive control and PDA plate medium without QY-1 strain as blank control, each treatment was repeated 3 times. Sealing the flat plate with the bacterial cake by using a self-sealing bag, culturing in a constant temperature incubator at 26 ℃, and measuring the width of the antibacterial zone when the hypha of the flat plate of the control group grows full.

Calculating the inhibition rate R of hypha growth:

R(％)＝(R1-R2)/R1×100％

where R is the percentage of inhibition colony diameter, R1 is the colony diameter of the blank control, and R2 is the colony diameter of the treatment group.

2. Test results

The test results are shown in Table 1 and FIGS. 1-8. As can be seen from FIGS. 1 to 8 and Table 1, the live bacteria of Bacillus QY-1 have excellent antibacterial effect on different pathogenic bacteria. The antibacterial effect of QY-1 living bacteria on Acremonium Sclerotigenum, aureobasidium melanogenum, botryosphaeria laricina, botrytis cinerea, botrytis fabae, byssochlamys spectabilis, cladosporium cladosporioides, diaporthe eres, fusarium decemcellulare, fusarium proliferatum, galactomyces geotrichum, glomerella acutata, lasiodiplodia theobromae, moniliapolystroma, mucor circinelloides, penicillium camemberti, penicillium chrysogenum, penicillium verruculosum and Rhizopus stolonifer is most obvious, the antibacterial rate is 100%, and the growth of pathogenic fungi can be completely inhibited. Wherein, the antibacterial rate of QY-1 living bacteria on Aspergillus niger, colletotrichum gloeosporioides, alternaria alternata, penicillium expansum, penicillium multicolor and schizophyllum commune is more than 90%, and the bacterial inhibition effect is very good; the antibacterial effect on Arthrinium urticae, aspergillus flavus, talaromyces variabilis, microdochium nivale, fusarium sp, penicillium sp and Cladosporium bruhnei is still better, and the antibacterial rate is more than 80%.

Compared with the bacteriostasis rate of 100mg/L carbendazim, the bacteriostasis effects of QY-1 living bacteria on Acremonium Sclerotigenum, botrytis cinerea, botrytis fabae, byssochlamys spectabilis, cladosporium cladosporioides, diadorthe eres, galactomyces geotrichum, glomerella acutata, lasiodiplodia theobromae, monilinia polystroma, aspergillus niger, penicillium expansum, penicillium multicolor, schizophyllum commune, arthrinium urticae, aspergillus flavus, talaromyces variabilis, fusarium sp, penicillium sp and Cladosporium bruhnei are more prominent, and the bacteriostasis rate is obviously greater than that of 100mg/L carbendazim. After 100mg/L carbendazim treatment, the antibacterial effect on colletotrichumgloosporides and Alternaria alternata is better, and the antibacterial rate is greater than that of bacillus QY-1 living bacteria treatment. After QY-1 viable cells and 100mg/L carbendazim were subjected to bacteriostasis treatments of Aureobasidium globosum, botryosphaeria laricina, fusarium decemcellulare, fusarium proliferatum, mucor circinelloides, penicillium camemberti, penicillium chrysogenum, penicillium verruculosum, rhizopus stolonifer and Microdochium nivale, the bacteriostasis effects were similar.

TABLE 1 antibacterial effect of Bacillus QY-1 viable cells and carbendazim on different pathogenic bacteria

Supernatant bacteriostasis test of bacillus QY-1

The antibacterial test of the supernatant is mainly used for judging whether the inhibition effect of the biocontrol bacteria on pathogenic bacteria is caused by nutrition competition, and when the supernatant has a wider antibacterial effect, the strain can secrete substances for inhibiting the pathogenic bacteria into the living environment of the strain.

The supernatant bacteriostasis test mainly tests the bacteriostasis effect of extracellular bacteriostasis substances, and the method mainly eliminates nutrition competition factors and is an important index for evaluating the biocontrol effect of biocontrol bacteria.

1. Test design

And (3) culturing bacillus QY-1 for 12 hours, and removing living bacteria by a repeated centrifugal machine filtering mode to prepare a QY-1 supernatant. 200. Mu.L of supernatant was pipetted and spread evenly on PDA plates, and the different pathogenic bacterial cakes were placed in the center of the PDA plate surface with a 6mm sterile punch, and each treatment was repeated three times with equal amounts of sterile water as a control. And (3) placing the prepared flat plate in a constant temperature incubator at 26 ℃ for culturing, and measuring the width of the antibacterial zone when the hypha of the flat plate of the control group grows full.

Calculating the inhibition rate R of hypha growth

R(％)＝(R1-R2)/R1×100％

2. Test results

The bacteriostatic effect of the bacillus QY-1 supernatant on different pathogenic fungi is shown in table 2. Wherein, the QY-1 supernatant has the best bacteriostasis effects on Acremonium Sclerotigenum, aureobasidium melanogenum, botryosphaeria laricina, botrytis cinerea, botrytis fabae, byssochlamys spectabilis, cladosporium cladosporioides, diaporthe eres, fusarium decemcellulare, fusarium proliferatum, galactomyces geotrichum, glomerella acutata, lasiodiplodia theobromae, monilinia polystroma, mucor circinelloides, penicillium camemberti, penicillium chrysogenum, penicillium verruculosum, rhizopus stolonifer, aspergillus niger and Colletotrichum gloeosporioides, and the bacteriostasis rate is more than 90%. The bacillus QY-1 supernatant has better antibacterial effects on Alternaria alternata, penicillium expansum, penicillium multicolor, schizophyllum commune, arthrinium urticae, aspergillus flavus, talaromyces variabilis, microdochium nivale, fusarium sp and Penicillium sp, and the antibacterial rate is more than 80%. And for Cladosporium bruhnei, the bacillus QY-1 supernatant shows slightly poorer antibacterial effect, and the antibacterial rate is lower than 80%.

TABLE 2 bacteriostatic Effect of Bacillus QY-1 supernatant on different pathogenic fungi

Bacterial inhibition test of bacillus QY-1 cell lysate

1. Test design

The cell lysate bacteriostasis test mainly tests whether the bacteria body contains bacteriostasis substances or not, so as to prove that extracellular bacteriostasis substances are produced by the bacteria body.

10ml of sterile physiological saline is added into bacillus QY-1 viable cells, the mixture is uniformly shaken, the uniformly shaken solution is put into a 50ml centrifuge tube, and QY-1 bacterial strain cells are crushed in an ultrasonic cell crusher. Filtering the solution after cell pulverization with a microporous filter membrane filter with the aperture of 0.22 μm for 1 time to obtain liquid, namely bacillus QY-1 cell lysate, and preserving at 4 ℃ for later use.

200 mu L of cell lysate is sucked and uniformly coated on a PDA plate, mold cakes of different pathogenic fungi are taken by a 6mm sterile puncher and placed in the center of the surface of the PDA plate, and each treatment is repeated three times by taking the same amount of sterile water as a control. And (3) placing the prepared flat plate in a constant temperature incubator at 26 ℃ for culturing, and measuring the width of the antibacterial zone when the hypha of the flat plate of the control group grows full. The inhibition rate R formula of hypha growth is the same as above.

2. Test results

The test results are shown in Table 3, and from Table 3, it can be seen that the bacillus QY-1 cell lysate has certain antibacterial effect on different pathogenic fungi, but the antibacterial effect is slightly lower than that of the supernatant of the QY-1 strain. Wherein, the bacillus QY-1 cell lysate has good antibacterial effects on Acremonium Sclerotigenum, aureobasidium melanogenum, botryosphaeria laricina, botrytis cinerea, botrytis fabae, byssochlamys spectabilis, cladosporium cladosporioides, diaporthe eres, fusarium decemcellulare, fusarium proliferatum, galactomyces geotrichum, glomerella acutata, lasiodiplodia theobromae, monilinism polystremia, mucor circinelloides, penicillium camemberti and Penicillium verruculosum, and the antibacterial rate is more than 90%. The QY-1 cell lysate has good antibacterial effects on Penicillium chrysogenum, rhizopus stolonifer, aspergillus niger, colletotrichum gloeosporioides, alternaria alternata, penicillium expansum, penicillium multicolor, schizophyllum commune, arthrinium urticae, aspergillus flavus, microdochium nivale and Fusarium sp. And for Talaromyces variabilis, penicillium sp.and Cladosporium bruhnei, the bacteriostasis effect of the bacillus QY-1 lysate is slightly bad, and the bacteriostasis rate is lower than 80%.

TABLE 3 bacteriostatic Effect of Bacillus QY-1 cell lysate on different pathogenic fungi

4. Living characteristics of biocontrol bacteria

The bacillus QY-1 has strong living ability and is mainly characterized by wide applicability of nutrient sources, temperature and humidity and pH value.

1. Adaptation of nutrient source in field

(1) Test design

According to the most suitable soil environment for the growth of blueberry fruit trees, the following soil nutrition conditions are set: 23.59% of organic carbon, 0.98% of total nitrogen, 0.85% of total phosphorus, 2.75% of total potassium and 70% of water-holding capacity in the field under the condition of moisture. According to the conditions described above, the following one-factor variable design was performed on the C, N, P, K element nutrient, with 4 different gradients per factor set (see table 4):

(2) Test results

The test results are shown in Table 5. As can be seen from Table 5, bacillus QY-1 shows a strong viability after 60 days of culture under different nutrient source environments. Under the conditions that the nutrition source is 23.59% of organic carbon, 0.98% of total nitrogen, 0.85% of total phosphorus and 2.75% of total potassium, the bacillus QY-1 can survive well for more than 60 days. The QY-1 strain can survive under the condition of low content of carbon, nitrogen, phosphorus and potassium elements. Under the condition of respectively lacking carbon, nitrogen, phosphorus and potassium elements, bacillus QY-1 can survive for more than 60 days in soil, which shows that the strain QY-1 does not depend on a specific nutrient element to survive, and under the condition that the content of a certain element is extremely low or is in a missing state, bacillus QY-1 can still survive for more than 60 days.

TABLE 4 Bacillus QY-1 Single-factor level Table for nutrient source in field

Note that: among the single factors, the non-variable factors maintain the nutritional condition formula optimal for blueberry growth.

TABLE 5 60 Bacillus QY-1 nutrient source adaptation cases

Note that: "+" indicates a living bacterium and "-" indicates a sterile bacterium.

2. pH adaptability

(1) Test design

The pH values are respectively adjusted to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 by using 1mol/L HCl or 1mol/LNaOH, 1mL of bacillus QY-1 seed culture solution is respectively inoculated in 100mL of LB liquid culture medium which is adjusted to different pH values after sterilization, the culture is carried out in a shaking table at 37 ℃ and 150r/min, and the absorbance and the transmittance of the culture solution are measured at 600nm by using an ultraviolet-visible spectrophotometer after 12 hours. The LB liquid medium without bacteria was used as a blank, and 3 replicates were treated. Turbidity= (100-transmittance) ×100%.

(2) Test results

The test results are shown in Table 6 and FIG. 9. As can be seen from Table 5 and FIG. 9, the turbidity of the culture solution after 12 hours was greater than 50% at pH=5-7 for Bacillus QY-1, indicating that Bacillus QY-1 exhibited better viability at pH=5-7. At ph=4 and ph=8, the culture turbidity after 12h was 21.35% and 29.08%, respectively, indicating that bacillus QY-1 can grow under meta-acid and meta-base conditions, but more slowly. When the pH is more than 9, the turbidity of the culture solution is less than 10% after 12 hours, which indicates that bacillus QY-1 is basically difficult to grow in a strong alkaline environment. The pH value suitable for growth is 5-8, and the pH value range basically comprises the pH value range of soil suitable for all fruits and vegetables to grow.

TABLE 6 determination of Bacillus QY-1 culture adaptability at different pH values

3. Field temperature and humidity adaptability

(1) Test design

In a sterile sandy soil pipe, a sterile nutrient solution with the nutrition conditions of 23.59% of organic carbon, 0.98% of total nitrogen, 0.85% of total phosphorus and 2.75% of total potassium is added, and the moisture is controlled to be 30%, 50%, 70% and 90%. 1mL of QY-1 seed culture solution was inoculated, and the sealing film was covered, and the culture was carried out at 10℃and 20℃and 40℃and 60℃respectively, and each treatment was repeated 3 times. Soil samples were taken at 30d, 60d and 90d, respectively, dissolved in physiological saline, 100. Mu.L of supernatant was taken and spread on LB plates, and the presence or absence of viable bacteria and the number thereof were observed.

(2) Test results

The test results are shown in Table 7. As can be seen from Table 7, the survival condition of the bacillus QY-1 under different field water content and temperature conditions after 60 days shows that the strain has stronger survival capability at various temperatures, especially at high temperature; the survival ability is strong under various humidity, especially at the water content of soil above 30%. At a moisture content of 10%, bacillus QY-1 cannot survive for more than 60 days, but the soil moisture content of 10% is unlikely to occur in normal fruit and vegetable cultivation soil. And under the conditions of 20-60 ℃ and 10-90% of water content, bacillus QY-1 can show better survival ability after 60 days in a soil environment.

The strain has strong tolerance to high temperature and is not suitable for low temperature and extremely low temperature environments, but the soil water content condition below 10% cannot appear in normal crop production areas, so the viability of the strain is suitable for most crop soil. When the air humidity is high, the strain can be mixed with inorganic nutrient solution and sprayed on the surface of crops to serve as foliar fertilizer and protective agent.

TABLE 7 Water content adaptability of Bacillus QY-1 to different fields for 60 days

5. No pathogenicity to fruit and vegetable

Taking blueberry as an example, bacillus QY-1 was subjected to an in vivo bacteriostasis test on healthy blueberry fruits. As shown in fig. 10, the blueberry fruit has an inhibiting effect on pathogenic bacteria on the surface of the fruit after being treated by bacillus QY-1 viable bacteria for 7 days, the strain does not cause the pathogenic effect of the fruit, and the appearance and hardness of the fruit are both at the normal post-ripening level. After being placed for 7 days at normal temperature, the blueberry fruits treated by the bacillus QY-1 are not mildewed, only have slight water loss phenomenon, and the fruits of the control group are mildewed in a large amount.

6. Tolerance to broad spectrum bactericides

1. Test design

Adding 17 chemical bactericides including mesomycin, imported agricultural streptomycin, carbendazim, pyraclostrobin, azoxystrobin antibiotics, difenoconazole, tebuconazole, tricyclazole pyraclostrobin, propiconazole, prochloraz, myclobutanil, flusilazole, epoxiconazole, azoxystrobin, metiram, chlorothalonil and mancozeb into an LB liquid culture medium to ensure that the pesticide concentration is 0.1%, 1% and 5% respectively; the same concentration of QY-1 strain was inoculated with LB liquid medium without bactericide as a control. Culturing in a shaking table at 37 ℃ and 150r/min, dipping LB liquid medium with an inoculating loop after 24 hours, streaking on an LB plate, and observing the growth condition of the QY-1 strain, wherein each treatment is repeated for 3 times.

2. Test results

As shown in Table 8, the test results are shown in Table 8, and the resistance of Bacillus QY-1 to various bactericides is different. Wherein, 1% of chlorothalonil, mancozeb, 5% of mesomycin, streptomycin, carbendazim, pyraclostrobin, difenoconazole, tebuconazole, tricyclazole pyraclostrobin, propiconazole, prochloraz, myclobutanil, flusilazole, epoxiconazole, azoxystrobin and metiram are jointly cultured for 24 hours with bacillus QY-1, bacillus QY-1 can grow out on LB plates, which indicates that bacillus QY-1 has better drug resistance to the 16 common bactericidal drugs under the condition of low concentration and can survive under the environment of spraying the 16 drugs. And bacillus QY-1 is not viable after 0.1% treatment with azoxystrobin antibiotics.

TABLE 8 tolerance of Bacillus QY-1 to general bactericides

Note that: in the table "+" indicates survival and "-" indicates non-survival.

Sequence listing

<110> Pi county Yi agricultural science and technology Co., ltd

<120> Bacillus, biocontrol agent prepared from the same and application thereof

<141> 2022-02-10

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1425

<212> DNA

<213> Bacillus velezensis

<400> 1

cgatccttct tggatccaac gggcaccgct ccgacttcgg gtgttacaaa ctctcgtggt 60

gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct gatccgcgat 120

tactagcgat tccagcttca cgcagtcgag ttgcagactg cgatccgaac tgagaacaga 180

tttgtgggat tggcttaacc tcgcggtttc gctgcccttt gttctgtcca ttgtagcacg 240

tgtgtagccc aggtcataag gggcatgatg atttgacgtc atccccacct tcctccggtt 300

tgtcaccggc agtcacctta gagtgcccaa ctgaatgctg gcaactaaga tcaagggttg 360

cgctcgttgc gggacttaac ccaacatctc acgacacgag ctgacgacaa ccatgcacca 420

cctgtcactc tgcccccgaa ggggacgtcc tatctctagg attgtcagag gatgtcaaga 480

cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat gctccaccgc ttgtgcgggc 540

ccccgtcaat tcctttgagt ttcagtcttg cgaccgtact cccccagggc ggagtgctta 600

atgcgttagc tgcagcacta aggggccgga aaccccctaa ccacttagca cttcatcgtt 660

tacggcgtgg actaccaggg gtatctaatc ctgttcgctc cccacgcttt cgcttcctca 720

gcgtcagtta cagaccagag agtcgccttc gccactggtg ttcctccaca tctctacgca 780

tttcaccgct acacgtggaa ttccactctc ctcttctgca ctcaagttcc ccagtttcca 840

atgaccctcc ccggttgagc cgggggcttt cacatcagac ttaagaaacc gcctgcgagc 900

cctttacgcc caataattcc ggacaacgct tgccacctac gtattaccgc ggctgctggc 960

acgtagttag ccgtggcttt ctggttaggt accgtcaagg tgccgcccta tttgaacggc 1020

acttgttctt ccctaacaac agagctttac gatccgaaaa ccttcatcac tcacgcggcg 1080

ttgctccgtc agactttcgt ccattgcgga agattcccta ctgctgcctc ccgtaggagt 1140

ctgggccgtg tctcagtccc agtgtggccg atcaccctct caggtcggct acgcatcgtc 1200

gccttggtga gccgttacct caccaactag ctaatgcgcc gcgggtccat ctgtaagtgg 1260

tagccgaagc caccttttat gtctgaacca tgcggttcag acaaccatcc ggtattagcc 1320

ccggtttccc ggagttatcc cagtcttaca ggcaggttac ccacgtgtta ctcacccgtc 1380

cgccgctaac atcagggagc aagctcccat ctgtccgctt gagga 1425

Claims

1. The bacillus is characterized in that the bacillus is QY-1, and is classified and named as bacillus belicus Bacillus velezensis, and the preservation unit is as follows: the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) has a preservation address of: beijing, chaoyang, north Chen Xili No.1, 3, accession number is: CGMCC No.23381, the preservation time is: 2021, 9 and 10.

2. A biocontrol microbial agent comprising the bacillus of claim 1.

3. Use of the biocontrol microbial inoculum according to claim 2 for controlling fruit and vegetable diseases caused by fruit and vegetable pathogens, characterized in that the fruit and vegetable pathogens comprise Acremonium Sclerotigenum, aureobasidium melanogenum, botryosphaeria laricina, botrytis cinerea, botrytis fabae, byssochlamys spectabilis, cladospora cladosporarioides, diaporthe eres, fusarium decemcellulare, fusarium proliferatum, galactomyces geotrichum, glomerella acutata, lasiodiplodia theobromae, monilinia polystroma, mucor circinelloides, penicillium camemberti, penicillium chrysogenum, penicillium verruculosum, rhizopus stolonifer, aspergillus niger, colletotrichum gloeosporioides, alternaria alternata, penicillium expansum, penicillium multicolor, schizophyllum commune, arthrinium urticae, aspergillus flavus, talaromyces variabilis, microdochium nivale, fusarium sp.

4. The use according to claim 3, wherein the use is to control fruit and vegetable diseases with biocontrol agents during the cultivation and storage periods of fruit and vegetable.

5. The use according to claim 3, wherein the fruit and vegetable diseases caused by pathogenic bacteria include grape rot, mango wilt, strawberry root rot, blueberry gray mold, mulberry soft rot, grape fruit rot, grape rot, apple brown rot, mulberry fruit rot, apple rot, jackfruit black rot, cherry brown rot, citrus mucor, citrus soft rot, grape black mold, watermelon anthracnose, pear black spot, kiwi green mold.

6. The use according to claim 4, wherein the concentration of bacillus QY-1 in the biocontrol agent is 1 x 10 ^6-7 CFU/mL。

7. The use of the bacillus of claim 1 in preparing fruit and vegetable fresh-keeping agent.