CN114369556A

CN114369556A - Bacillus, biocontrol microbial inoculum prepared from bacillus and application of biocontrol microbial inoculum

Info

Publication number: CN114369556A
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: 2022-04-19
Anticipated expiration: 2042-02-10
Also published as: CN114369556B

Abstract

The invention relates to bacillus, a biocontrol microbial inoculum prepared from the bacillus and application of the biocontrol microbial inoculum, and belongs to the technical field of biological microbial inoculants. The Bacillus provided by the invention is QY-1, is classified and named as Bacillus velezensis, is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms, and has the preservation addresses as follows: the No. 3 Xilu No.1 of Beijing, Chaoyang, the area of the rising of the south of the morning has the preservation number of CGMCC No.23381 and the preservation time of 2021, 9 months and 10 days. The bacillus QY-1 has good bacteriostatic action on various pathogenic bacteria whether live bacteria, supernatant or cell lysate, and has wide bactericidal spectrum. Meanwhile, the bacillus QY-1 has strong living ability and wide application range on nutrient sources, temperature, humidity and pH value. Has no pathogenicity on fruits and vegetables, better tolerance on broad-spectrum bactericides and better application prospect.

Description

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

Technical Field

The invention belongs to the technical field of biological agents, and particularly relates to bacillus, a biocontrol agent prepared from the bacillus and application of the biocontrol agent in inhibiting pathogenic bacteria in the cultivation period and the storage and transportation period of fruits and vegetables.

Background

Fruits and vegetables are easily damaged by soil-borne diseases in the planting process, and are often threatened by bacterial and fungal infections, particularly fungal diseases are the most common, and common symptoms comprise necrosis (leaf spot and leaf wither), rot (root rot and fruit rot), wilting (root, stem base and vascular tissue are invaded), and the like. At present, methods such as chemical bactericides, disease-resistant variety cultivation, intercropping and interplanting, grafting and the like are mostly adopted for preventing and treating diseases, but the prevention and treatment measures have various advantages and disadvantages.

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

At present, the main method for controlling the post-harvest infectious diseases of fruits and vegetables is to use chemical pesticides in the cultivation period, the temporary harvest period and even after the fruits and vegetables are harvested, but serious non-point source pollution and food safety problems are caused, so that biological control is a preferable scheme which gives consideration to environmental friendliness, human health and economic benefits.

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

However, the existing biocontrol strains and biocontrol microbial inoculum can not replace chemical pesticides to control diseases, and the existing microbial inoculum mainly has the following problems:

(1) biocontrol bacteria have no pertinence to the control of fruit diseases;

the existing biocontrol bacteria and microbial inoculum do not mark the planting environment from which crops the biocontrol bacteria and microbial inoculum are derived, the disease difference of different crops is huge, and the bacteriostasis spectrum of the biocontrol bacteria has limitation, so the types of pathogenic bacteria which can be inhibited by the biocontrol bacteria are not clear, and whether the biocontrol bacteria have the bacteriostasis effect or not becomes a random event.

(2) Biocontrol bacteria nutrient source, suitable habitat and undefined drug resistance;

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

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

because the existing biocontrol bacteria have no pertinence to the control of fruit diseases, a plurality of biocontrol bacteria are often used in production to be compounded to form a compound microbial inoculum for use, but the biocontrol bacteria effect needs certain biomass to be ensured, the use of the compound microbial inoculum causes nutrition competition and space competition, and even if the targeted biocontrol bacteria exist, the biomass is not enough to form strong inhibition effect on pathogenic bacteria.

Therefore, aiming at the problems of narrow antibacterial spectrum, large use limitation, incapability of fixedly planting strains and easiness in killing by various pesticides of the existing single biocontrol strain, and the problems of low biomass, large nutrition competition and space competition among the strains and poor fruit disease control effect of the composite microbial inoculum, how to further develop the high-efficiency single biocontrol strain capable of inhibiting pathogenic bacteria in the fruit and vegetable cultivation period and the storage and transportation period becomes a technical problem to be solved urgently in the field.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a biocontrol strain which can be applied to disease control in the fruit and vegetable cultivation period and the storage and transportation period, and a biocontrol bactericide prepared from the biocontrol strain and application of the biocontrol strain. The invention aims to solve the problems of narrow antibacterial spectrum, large use limitation, incapability of fixedly planting strains and easiness in killing by various pesticides of a single biocontrol bacterium, and the problems of low biomass, large nutrition competition and space competition among the strains and poor fruit disease control effect of a composite microbial inoculum.

The inventor obtains a strain of Bacillus through a large amount of experimental research and grope, wherein the Bacillus is QY-1, is classified and named as Bacillus velezensis, and the preservation unit is as follows: the China general microbiological culture Collection center has the following preservation addresses: xilu No.1 Hospital No. 3, Beijing, Chaoyang, the number of the depository center is: CGMCC No.23381, the preservation time is as follows: 9/10/2021.

The bacillus QY-1 provided by the invention has broad-spectrum bacteriostasis on fruit and vegetable pathogenic bacteria, and can inhibit the types of the pathogenic bacteria to 32 types. The bacillus QY-1 has good bacteriostatic effect on various pathogenic bacteria in the fruit and vegetable cultivation period or the storage and transportation period, has good strain planting effect, can be efficiently used for disease control and bacteriostatic preservation of fruits and vegetables, and has the advantages of easily obtained nutrient source, strong extreme temperature resistance and strong pesticide resistance. And the single strain does not have the problems of low biomass of each strain among the composite bacteria and large nutrition competition and space competition among the strains, and the application effect is extremely obvious.

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 inoculum containing the bacillus.

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

Specifically, the application is that the bio-control fungicide is adopted to inhibit pathogenic bacteria of fruits and vegetables in the cultivation period and the storage and transportation period of the fruits and vegetables so as to control diseases and realize bacteriostasis and fresh keeping.

Specifically, the fruit and vegetable pathogenic bacteria include Acremonium Sclerotigenum, Aureobasidium melanogenium, Botryosphaeria fascicularia, Botrytis cinerea, Botrytis fabae, Byssochlamys specularis, Cladosporium cladosporioides, Diaporthe ereus, Fusarium decellularea, Fusarium proliferum, Galactomyces geotrichum, Glomeella acutus, Lasiodlidia theobromae, Monilinia monorchia, Mucor circinelloides, Penicillium camemberti, Penicillium chrysogenum, Schizocium verruculosum, Rhizophyllus stolonifera, Aspergillus niger, Escherichia coli strain, Aspergillus niger strain, Aspergillus niger strain, Aspergillus niger strain, Bacillus strain, Bacillus strain, Bacillus strain, Bacillus strain.

Specifically, the fruit and vegetable diseases caused by pathogenic bacteria comprise: grape rot (Acremonium sclerotigum), mango wilt (Aureobasidium melanogenium), strawberry root rot (Borgyospora fascicularis), blueberry gray mold (Borgytis cinerea), mulberry soft rot (Borgytisisfabase), grape fruit rot (Cladosporium cladosporioides), grape rot (Diaporthe eresis), apple brown spot (Fusarium decellulare), mulberry fruit rot (Fusarium proliferatum), apple rot (Glomerella acuta), pineapple honey black rot (Lasiodipylopsia theobromae), cherry brown rot (Monilinia polystryta), citrus hairy mold (Mucor citrinovelloides), citrus soft rot (Rhizopus basicola), grape soft rot (Vitis vinifera), grape gray mold (Boragicola), and grape black rot (Penicillium nigrospora nigra), and grape black rot (Penicillium nigrosporium roseum).

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

The invention has the beneficial effects that:

(1) the bacillus QY-1 live bacteria has good bacteriostatic action on various pathogenic bacteria, wherein the bacteriostatic rate of the bacillus QY-1 live bacteria on 19 pathogenic bacteria reaches 100 percent, the growth of pathogenic fungi can be completely inhibited, and the bacteriostatic effect is extremely obvious; 6 pathogenic bacteria with the bacteriostasis rate of the live bacteria being more than 90 percent have good bacteriostasis effect; the bacteriostatic rate of the living bacteria is more than 80 percent, and the living bacteria still have good bacteriostatic effect due to 7 pathogenic bacteria.

(2) The bacillus QY-1 supernatant has certain bacteriostatic effect on different pathogenic fungi. Wherein, the bacillus QY-1 supernatant has 21 pathogenic bacteria with the bacteriostasis rate of more than 90 percent against the pathogenic bacteria, and has good bacteriostasis effect; 10 pathogenic bacteria with the bacteriostasis rate of the supernatant being more than 80 percent still have good bacteriostasis effect; only 1 kind of pathogenic bacteria with the bacteriostasis rate lower than 80% in the supernatant has slightly poor bacteriostasis effect, but still has certain bacteriostasis.

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

Drawings

FIG. 1 shows the bacteriostatic effect of viable bacteria of Bacillus QY-1 on different pathogenic bacteria, wherein A1.1 is Acremonium Sclerotigenum + QY-1 treatment; a1.2 Acremonium Sclerotigenum blank control; a2.1, Aureobasidium melanogenium + QY-1 treatment; a2.2 is Aureobasidium melanogenin blank control; a3.1, Botryosphaeria laricina + QY-1 treatment; a3.2 Botryosphaeria laricina blank control; a4.1, carrying out Botrytis cinerea + QY-1 treatment; a4.2 Botrytis cinerea blank.

FIG. 2 shows the bacteriostatic effect of the viable bacteria of Bacillus QY-1 on different pathogenic bacteria, wherein A5.1 is Botrytis fabae + QY-1 treatment; a5.2 Botrytis fabae blank control; a6.1, Byssochlamys spectabilis + QY-1 treatment; a6.2 Byssochlamys spectabilis blank control; a7.1, Cladosporium cladosporioides + QY-1 treatment; a7.2 Cladosporium cladosporioides blank control; a8.1, processing Diaporthe eres + QY-1; a8.2 Diaporthe eres blank control.

FIG. 3 shows the bacteriostatic effect of the viable bacteria of Bacillus QY-1 on different pathogenic bacteria, wherein A9.1 is Fusarium decelcellulare + QY-1 treatment; a9.2 Fusarium decelcellulare blank control; a10.1, Fusarium proliferatum + QY-1 treatment; a10.2 Fusarium proliferatum blank control; a11.1, Galactomyces geotrichum + QY-1 treatment; a11.2 Galactomyces geotrichum blank control; a12.1, glomeriella acutata + QY-1 treatment; a12.2 Glomerella acutata blank.

FIG. 4 shows the bacteriostatic effect of the live Bacillus QY-1 on different pathogenic bacteria, wherein A13.1 is Lasiodipodia theobromae + QY-1 treatment; a13.2 Lasiodipodia theobromae blank control; a14.1, Moniliniapolystroma + QY-1 treatment; a14.2 Monilinia polystroma blank; a15.1, carrying out Mucor circinelloides + QY-1 treatment; a15.2 Mucor circinelloides blank control; a16.1, Penicillium camemberti + QY-1 treatment; a16.2 Penicillium camemberti blank.

FIG. 5 shows the bacteriostatic effect of the viable bacteria of Bacillus QY-1 on different pathogenic bacteria, wherein A17.1 is Penicillium chrysogenum + QY-1 treatment; a17.2 Penicillium chrysogenum blank control; a18.1, Penicillium verruculosum + QY-1 treatment; a18.2 Penicillium verruculosum blank control; a19.1, Rhizopus stolonifer + QY-1 treatment; a19.2 Rhizopus stolonifer blank control; b1.1, Aspergillus niger + QY-1 treatment; b1.2 Aspergillus niger blank.

FIG. 6 shows the bacteriostatic effect of the viable bacteria of Bacillus QY-1 on different pathogenic bacteria, wherein B2.1 is treatment of Colletotrichum gloeosporioides + QY-1; b2.2 Colletrichum gloeosporioides blank control; b3.1, processing Alternaria alternata + QY-1; b3.2 Alternaria alternata blank control; b4.1, Penicillium expansum + QY-1 treatment; b4.2 Penicillium expansum blank control; b5.1, Penicillium multicolor + QY-1 treatment; b5.2 Penicillium multicolor blank control.

FIG. 7 shows the bacteriostatic effect of the viable bacteria of Bacillus QY-1 on different pathogenic bacteria, wherein B6.1 is Schizophyllum commune + QY-1 treatment; b6.2, a Schizophyllum commune blank control; c1.1, Arthrinium urticae + QY-1 treatment; c1.2 Arthrinium urticae blank control; c2.1, treating Aspergillus flavus and QY-1; c2.2 Aspergillus flavus blank control; c3.1, treating Talaromyces variabilis + QY-1; c3.2 Talaromyces variabilis blank control.

FIG. 8 shows the bacteriostatic effect of the viable bacteria of Bacillus QY-1 on different pathogenic bacteria, wherein C4.1 is Microdochium nivale + QY-1 treatment; c4.2 Microdochium nivale blank control; c5.1, treatment of Fusarium sp. + QY-1; c5.2 Fusarium sp. blank control; c6.1 Penicillium sp. + QY-1 treatment; c6.2 Penicillium sp. blank control; c7.1, Cladosporium bruhnei + QY-1 treatment; c7.2 Cladosporium brahnei blank control.

FIG. 9 shows the suspension of Bacillus QY-1 cultured at different pH values;

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

Detailed Description

The present invention is described in detail below by way of examples, and it should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.

Examples

First, the source of the strain

In 2019, healthy and diseased leaves, flowers and fruits of blueberries are collected for multiple times in an Qiongyuanbaoshan blueberry planting base in a Qiongyuanshan city, namely a city water gap, and the leaves, the flowers and the fruits are filled in sterile sealing bags for microbial separation and identification, wherein the purpose is to separate and obtain pathogenic microorganisms in the blueberries. However, the phenomenon that fungi cannot grow due to single-kind bacterial pollution occurs in the separation in summer and autumn, and the bacteria obtained through the two times of separation are the same strain, namely the strain provided by the invention.

II, identification of the strains

The DNA of the bacteria 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 an evolutionary tree and is preliminarily judged to be Bacillus amyloliquefaciens or Bacillus velezensis, and the gene sequence of the conserved sequence is shown as SEQ NO. 1.

After the bacteriostatic experiments were performed, the strains were subjected to full sequence sequencing and defined as Bacillus velezensis according to sequence analysis in order to further protect the strains.

The strain named as bacillus QY-1 is preserved in China general microbiological culture Collection center with the preservation address of No. 3 Xilu-Beijing province of the Korean-Yang district, and the preservation center number is: CGMCC No.23381, the preservation time is as follows: 9/10/2021.

Third, bacteriostatic test of the strains

Live bacteria bacteriostasis test of bacillus QY-1

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

1. Design of experiments

Since the main application of the biocontrol strain is to use the living bacterial strain to spray on the soil or the surface of a plant, the bacteriostatic effect of using the living bacterial strain is compared with that of using carbendazim only.

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 using a hemocytometer^7-8CFU/mL. Adding QY-1 strain cell suspension into PDA culture medium, shaking and mixing to obtain plates with QY-1 strain cell density of 1 × 10^6-7CFU/mL. The cake of Botrytis cinerea was placed in the center of a PDA plate containing the QY-1 strain using a 6mm sterile punch, and 3 replicates of each treatment were performed using a PDA plate containing 100mg/L carbendazim as a positive control and a PDA plate medium containing no QY-1 strain as a blank control. Sealing the plate containing the fungus cake with a self-sealing bag, culturing in a constant temperature incubator at 26 deg.C, and measuring the width of the antibacterial band when the plate hyphae 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 inhibited colony diameter, R1 is the colony diameter of the blank, and R2 is the colony diameter of the treated group.

2. Test results

The test results are shown in table 1 and fig. 1 to 8. As can be seen from FIGS. 1-8 and Table 1, the viable bacteria of Bacillus QY-1 have good bacteriostatic action on different pathogenic bacteria. The QY-1 live bacteria have the most remarkable bacteriostatic effect on Acremonium Sclerotigenum, Aureobasidium melanogenium, Botryosphaeria fascicularia, Botrytis cinerea, Botrytis sabae, Byssochlamys spectabilis, Cladosporium cladosporioides, Diaporthe ereus, Fusarium decellulare, Fusarium proliferum, Galactomyces geotrichum, Glomeella acuta, Lasiodiploidia theobromae, Monilinia, Mucor circinelloides, Penicillium camemberti, Penicillium chrysogenum, Penicillium verruculosum and Rhizopus stolonifer, and the bacteriostatic effect is 100% and the growth rate of fungi can be completely inhibited. Wherein, the inhibition ratio of QY-1 living bacteria to Aspergillus niger, Colletotrichum gloeosporioides, Alternaria alterna, Penicillium expansum, Penicillium multicolor and Schizophyllum commune is more than 90%, and the QY-1 living bacteria has good inhibition effect; the antibacterial effect on Arthronium 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 bacteriostatic rate of 100mg/L carbendazim, the QY-1 live bacteria have more remarkable effect on Acremonium Scrlerotigenum, Botrytis cinerea, Botrytis fabae, Byssochlamys spectabilis, Cladosporium cladosporioides, Diaporthe eres, Galactomyces geotrichum, Glomella acutata, Lasiodiptheria theobromae, Monilinia polystroma, Aspergillus niger, Penicillium expansum, Penicillium multicolor, Schizophyllum commune, Arthrothrinium urticae, Aspergillus flavus, Talaromyces variabilis, Fusarium sp. After the treatment of carbendazim of 100mg/L, the bacteriostatic effect on Colletochumgloeosporioides and Alternaria alternata is better, and the bacteriostatic rate is higher than that of the treatment of live bacillus QY-1. After subjecting Aureobasidium melanogenin, Botryosphaeria laricina, Fusarium decemcellulare, Fusarium proliferatum, Mucor circinelloides, Penicillium camemberti, Penicillium chrysogenum, Penicillium verruculosum, Rhizopus stolonifer and Microdochium nivale to QY-1 live bacteria and 100mg/L carbendazim bacteriostasis treatment, the bacteriostasis effects were similar.

TABLE 1 bacteriostatic effect of Bacillus QY-1 live thallus and carbendazim on different pathogenic bacteria

(II) bacteriostatic test of supernatant of bacillus QY-1

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

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

1. Design of experiments

And (3) removing the live bacteria of the bacillus QY-1 cultured for 12h in a repeated centrifuge filtration mode to prepare a QY-1 supernatant. 200 μ L of the supernatant was pipetted and spread evenly on a PDA plate, different pathogenic bacteria cakes were placed in the center of the surface of the PDA plate with a 6mm sterile punch, and each treatment was repeated three times with equal amounts of sterile water as control. And (3) placing the prepared flat plate in a constant-temperature incubator at 26 ℃ for culture, and measuring the width of the antibacterial band when hyphae of the flat plate of the control group are 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 a bacteriostatic effect on Acremonium Sclerotigenum, Aureobasidium melanogeninum, Botryosphaeria larcina, Botrytis cinerea, Botrytis fabae, Byssochlamys specularis, Cladosporium cladosporoides, Diaporter, Fusarium decellulare, Fusarium proliferum, Galactomyces geotrichum, Glomerella acuta, Lasioodiplodia theobromae, Monilinia strima, Mucor circiniella, Penicillium camemberti, Penicillium chrysogenum, Penicillium verruculosum, Rhizobium stolonifer, Aspergillus niger and Colyssochlia viridis more than 90%. The bacillus QY-1 supernatant has better bacteriostatic 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 bacteriostatic rate is more than 80 percent. And for Cladosporium bruhnei, the supernate of the bacillus QY-1 shows slightly poor bacteriostatic effect, and the bacteriostatic rate is lower than 80%.

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

(III) bacteriostatic test of bacillus QY-1 cell lysate

1. Design of experiments

The cell lysate bacteriostasis test is mainly used for testing whether bacteriostatic substances are contained in bacteria or not so as to prove that the extracellular bacteriostatic substances are generated by the bacteria.

Adding 10ml of sterile normal saline into the bacillus QY-1 viable bacteria, shaking up, putting the shaken solution into a 50ml centrifuge tube, and crushing the QY-1 strain cells in an ultrasonic cell crusher. Filtering the solution after cell pulverization with 0.22 μm microporous membrane filter for 1 time to obtain liquid, i.e. Bacillus QY-1 cell lysate, and storing at 4 deg.C for use.

200 μ L of cell lysate was pipetted and spread evenly on a PDA plate, different fungal cakes were placed in the center of the surface of the PDA plate with a 6mm sterile punch, and each treatment was repeated three times with equal amounts of sterile water as control. And (3) placing the prepared flat plate in a constant-temperature incubator at 26 ℃ for culture, and measuring the width of the antibacterial band when hyphae of the flat plate of the control group are full. The inhibition rate of hypha growth R formula is the same as above.

2. Test results

The test results are shown in Table 3, and it can be seen from Table 3 that the bacillus QY-1 cell lysate has certain bacteriostatic effect on different pathogenic fungi, but the bacteriostatic effect is slightly lower than that of the supernatant of the QY-1 strain. Wherein the bacillus QY-1 cell lysate has good bacteriostatic effect on Acremonium Sclerotigenum, Aureobasidium melanogeninum, Botryosphaeria larcina, Botrytis cinerea, Botrytis fabae, Byssochlamys spectabilis, Cladosporium cladosporioides, Diaporter, Fusarium decellulare, Fusarium proliferum, Galactomyces geotrichum, Glomerella acuta, Lasiodolodia theobromae, Monilinia strioma, Mucor circinelloides, Penicillium camemberti and Penicillium verruculom, and the bacteriostatic rate is more than 90%. QY-1 cell lysate has a good bacteriostatic effect 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 the bacteriostatic rate is more than 80%. The bacillus QY-1 lysate shows slightly poor antibacterial effect on Talaromyces variabilis, Penicillium sp and Cladosporium bruhnei, and the antibacterial rate is lower than 80%.

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

Fourth, life characteristics of biocontrol bacteria

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

1. Adaptability to field nutrient source

(1) Design of experiments

According to the most suitable soil environment for the growth of blueberry trees, the following soil nutrition conditions are formulated: 23.59 percent of organic carbon, 0.98 percent of total nitrogen, 0.85 percent of total phosphorus, 2.75 percent of total potassium and 70 percent of field moisture capacity under moisture condition. The C, N, P, K elemental nutrients were subjected to the following one-factor variable design, with each factor setting 4 different gradients (see table 4), according to the conditions described above:

(2) test results

The test results are shown in Table 5. As can be seen from Table 5, the Bacillus QY-1 shows stronger viability after being cultured for 60 days under different nutrient source environments. Under the condition that the nutrient sources are 23.59 percent of organic carbon, 0.98 percent of total nitrogen, 0.85 percent of total phosphorus and 2.75 percent of total potassium, the bacillus QY-1 can well survive 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, the bacillus QY-1 can survive for more than 60 days in the soil, which shows that the QY-1 strain does not depend on a specific nutrient element for survival, and the bacillus QY-1 can survive for more than 60 days under the condition of extremely low content or lack of a certain element.

TABLE 4 Bacillus QY-1 Single-factor horizon for field nutrient sources

Note: in each single factor, the non-variable factor maintains the nutrient condition formula for the optimal blueberry growth.

TABLE 560 days Bacillus QY-1 nutrient Adaptation

Note: "+" indicates the presence of viable bacteria and "-" indicates the absence of viable bacteria.

2. Adaptability to pH value

(1) Design of experiments

Adjusting pH to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 with 1mol/L HCl or 1mol/L NaOH, respectively, sterilizing, inoculating 1mL of Bacillus QY-1 seed culture solution into 100mL of LB liquid culture medium adjusted to different pH, culturing in a shaker at 37 deg.C and 150r/min, and measuring absorbance and transmittance with ultraviolet-visible spectrophotometer at 600nm wavelength after 12 h. The blank was prepared in LB liquid medium without inoculation, and each treatment was repeated 3 times. Suspended turbidity is (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 suspension degrees of the culture broth of Bacillus QY-1 were both greater than 50% after 12h at pH 5-7, indicating that Bacillus QY-1 exhibits good viability at pH 5-7. The culture suspension turbidity after 12h was 21.35% and 29.08% at

pH

4 and 8, respectively, indicating that bacillus QY-1 can grow under the conditions of meta-acid and meta-base, but slowly. When the pH is more than 9, the suspension degree of the culture solution after 12 hours is less than 10 percent, which indicates that the bacillus QY-1 is difficult to grow basically 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 growth of all fruits and vegetables.

TABLE 6 measurement of Bacillus QY-1 culture Adaptation at different pH

3. Field temperature and humidity adaptability

(1) Design of experiments

A sterile nutrient solution with the nutrient conditions of 23.59 percent of organic carbon, 0.98 percent of total nitrogen, 0.85 percent of total phosphorus and 2.75 percent of total potassium is added into a sterile sandy soil pipe, and the water content is controlled to be 30 percent, 50 percent, 70 percent and 90 percent. Inoculating 1mL of QY-1 seed culture solution, covering with sealing film, culturing at 10 deg.C, 20 deg.C, 40 deg.C, and 60 deg.C, and repeating each treatment for 3 times. Soil samples were taken at 30d, 60d and 90d, respectively, and dissolved in physiological saline, 100. mu.L of the supernatant was applied to an LB plate, and the presence or absence and the amount of viable bacteria 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 after 60 days under different field water content and temperature conditions shows that the strain has stronger survival capability at various temperatures, especially high temperature; the soil has strong survival ability under various humidity, especially under the soil moisture content of more than 30 percent. When the water content is 10%, the bacillus QY-1 cannot survive for more than 60 days, but the soil water content of 10% cannot be found in normal fruit and vegetable cultivation soil. Under the conditions of temperature of 20-60 ℃ and water content of 10-90%, the bacillus QY-1 can show better survival ability after 60 days in the soil environment.

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

TABLE 760 adaptability of Bacillus QY-1 to different field Water content

Fifthly, no pathogenicity to fruits and vegetables

Taking blueberry as an example, the bacillus QY-1 is subjected to a living body bacteriostasis test on healthy blueberry fruits. The results are shown in fig. 10, and it can be seen that the blueberry fruit has an inhibiting effect on pathogenic bacteria on the fruit surface after being treated by bacillus QY-1 live bacteria for 7 days, the bacterial strain does not cause fruit pathogenic effect, and the appearance and hardness of the fruit are normal after-ripening levels. After being placed for 7 days at normal temperature, the blueberry fruits treated by the bacillus QY-1 are not mildewed and only have slight dehydration phenomenon, while the fruits of the control group are mildewed greatly.

Sixth, tolerance to broad-spectrum fungicides

1. Design of experiments

Adding 17 chemical bactericides, including zhongshengmycin, imported agricultural streptomycin, carbendazim, pyraclostrobin, pyrimidine nucleoside antibiotic, 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 concentrations are 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 at 37 deg.C in a shaker at 150r/min, after 24h, streaking LB liquid medium with inoculating loop and observing growth of QY-1 strain, 3 times per treatment.

2. Test results

As shown in Table 8, the results of the tests show that Bacillus QY-1 has different resistance to various fungicides from Table 8. After 1% of chlorothalonil and mancozeb, 5% of zhongshenmycin, streptomycin, carbendazim, pyraclostrobin, difenoconazole, tebuconazole, tricyclazole pyraclostrobin, propiconazole, prochloraz, myclobutanil, flusilazole, epoxiconazole, azoxystrobin and metiram are co-cultured with bacillus QY-1 for 24 hours, the bacillus QY-1 can grow on an LB (lysogeny) flat plate, and the bacillus QY-1 has good drug resistance to the 16 common bactericidal drugs under the condition of low concentration and can survive in the environment of spraying the 16 drugs. Bacillus QY-1 was not viable after treatment with 0.1% pyrimidine nucleoside antibiotics.

TABLE 8 tolerance of Bacillus QY-1 to common fungicides

Note: in the table, "+" indicates survival and "-" indicates no survival.

Sequence listing

<110> Pi county space agriculture science and technology Limited

<120> bacillus, biocontrol microbial inoculum prepared from bacillus and application of biocontrol microbial inoculum

<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 QY-1, is classified and named as Bacillus velezensis, and has the preservation unit as follows: the China general microbiological culture Collection center has the following preservation addresses: xilu No.1 Hospital No. 3, Beijing, Chaoyang, the number of the depository center is: CGMCC No.23381, the preservation time is as follows: 9/10/2021.

2. The bacillus of claim 1, wherein the gene conserved sequence of the bacillus is shown in SEQ No. 1.

3. A biocontrol microbial inoculum comprising the bacillus of claim 1 or 2.

4. The application of the biocontrol microbial inoculum of claim 3 in preventing and treating fruit and vegetable diseases caused by fruit and vegetable pathogenic bacteria.

5. The application of the compound bactericide as claimed in claim 4, wherein the application is to control fruit and vegetable diseases by using a biocontrol bactericide in the cultivation period and the storage and transportation period of fruits and vegetables.

6. The use according to claim 4, wherein the fruit or vegetable pathogenic bacteria include Acremonium scleritogen, Aureobasidium melanogens, Botryosphaeria major, Botrytis cinerea, Botrytis fabae, Byssochlamys specularis, Cladosporium cladosporioides, Diaporthe species, Fusarium decemcellulare, Fusarium proliferum, Galactomyces geotrichum, Glomerella acuta, Lasiodiptheria theobromae, Monilinia, Mucor cironicella auriculata, Penicillium camemberti, Penicillium purpureum, Penicillium chrysogenum, Schizoctonium basilicium, Schizoctonium chrysogenum, Penicillium maculatum, Schizoctonium purpureum, Schizoctonium chrysosporium, Schizochytrium, Bacillus subtilis, Aspergillus, Escherichia, or Bacillus, Escherichia, or Escherichia, or Escherichia, or Escherichia, or Escherichia, or Escherichia, or Escherichia, or Escherichia, or Escherichia, or Escherichia, or Escherichia, or Escherichia, or Escherichia, or Escherichia.

7. The use of claim 4, wherein the disease of fruits and vegetables caused by pathogenic bacteria comprises grape rot, mango wilt, strawberry root rot, blueberry gray mold, mulberry soft rot, grape rot, apple brown spot, mulberry rot, apple rot, jackfruit black rot, cherry brown rot, citrus mucormycosis, citrus soft rot, grape black mold, watermelon anthracnose, pear black spot, and kiwi penicilliosis.

8. The use as claimed in claim 4, wherein the concentration of Bacillus QY-1 in the biocontrol microbial inoculum is 1 x 10^6-7CFU/mL。

9. The use of the bacillus of claim 1 or 2 in the preparation of a fruit and vegetable preservative.