CN116515691A

CN116515691A - Bacillus bailii LG001 and application thereof

Info

Publication number: CN116515691A
Application number: CN202310434429.0A
Authority: CN
Inventors: 雷高; 杨文玲; 权淑静; 王佰涛; 刘德海; 刁文涛; 李珊珊; 李亮亮; 胡虹; 巩涛; 李磊; 甄静; 马焕; 王继雯; 冯菲; 解复红; 张志龙; 陈国参
Original assignee: Henan Academy Of Sciences Institute Of Biology LLC; Henan Academy of Sciences
Current assignee: Henan Academy Of Sciences Institute Of Biology LLC; Henan Academy of Sciences
Priority date: 2023-04-21
Filing date: 2023-04-21
Publication date: 2023-08-01

Abstract

Bacillus bailii LG001, classified and named as Bacillus bailii @Bacillus velezensis) The microbial strain is preserved in China general microbiological culture collection center (CGMCC) at 9/6 of 2021, with the preservation number of CGMCC No.23366 and the preservation address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city. The bacillus belicus LG001 is applied to a medicament for preventing and treating plant pathogenic fungi. The bacillus bailii LG001 can generate various antibacterial substances, has obvious inhibition effect on the growth of various plant pathogenic fungi, has good antibacterial effect, and is a novel biocontrol microorganism which is safe, efficient and broad-spectrum for preventing and controlling plant fungal diseases. The provided biocontrol microbial agent has good control effect and no toxicityCan cause environmental pollution, can replace pesticides to prevent and treat gray mold of cucumber, and has remarkable social and economic benefits.

Description

Bacillus bailii LG001 and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to bacillus beleiensis LG001 with remarkable antagonism on various plant pathogenic bacteria and application thereof.

Background

Botryiscinerea belongs to Botryiscinerea genus, and is a pathogenic bacterium causing gray mold of cucumber. The botrytis cinerea infects cucumber plants in a growing period, so that the yield and the quality of the cucumber plants are seriously reduced, and the serious economic loss is caused.

At present, the control of Botrytis cinerea mainly adopts chemical control, such as bactericides of benzimidazoles, anilinopyrimidines, dimethyl imides, succinic acid dehydrogenase inhibitors and the like. However, the frequent use of chemical agents causes the emergence of a large number of resistant gray mold bacteria. In order to ensure the prevention effect, diseases are often prevented and treated by increasing the dosage and the application frequency, so that the pesticide residue exceeds the standard. In addition, the pollution of pesticide residues to soil and water is also gradually aggravated to destroy the ecological balance of agriculture.

Compared with chemical control, biological control has the advantages of low pollution, no toxic residue and good specificity. Bacillus bailii (Bacillus velezensis) is a recent research hot spot for bacterial control, and reports on bacillus bailii are increasing at home and abroad, and besides basic researches on antagonizing pathogenic bacteria, promoting growth of animals and plants, identifying antibacterial substances and gene clusters thereof, antagonizing action mechanisms and the like, the application of bacillus bailii in agricultural production is increasing. There are several patents currently reporting the use of bacillus beljalis in controlling plant diseases. Therefore, the bacillus subtilis has good application prospect in biological control and agricultural production, can provide a bacillus beijerinckii strain, has remarkable antagonism to various plant pathogenic bacteria, but has not been reported so far.

Disclosure of Invention

Aiming at the situation, in order to overcome the defects of the prior art, the invention aims to provide bacillus bailii LG001 and application thereof, which can effectively solve the problems of less types of existing biocontrol bacteria, limited selection, narrow antibacterial spectrum and poor control effect.

In order to achieve the above purpose, the technical scheme of the invention is that bacillus beleiensis LG001, classified and named bacillus beleiensis (Bacillus velezensis) is preserved in China general microbiological culture collection center (CGMCC) No.23366 and the preservation address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

The bacillus belicus LG001 is applied to a medicament for preventing and treating plant pathogenic fungi.

The effective component of the biocontrol microbial inoculum is bacillus bailii LG001, and the biocontrol microbial inoculum is culture bacterial suspension, fermentation liquor or metabolic products and volatile substances generated by fermentation of bacillus bailii LG001.

The application of the biocontrol microbial inoculum in the medicine for preventing and controlling the gray mold of cucumber.

The bacillus bailii LG001 disclosed by the invention can generate various antibacterial substances, has an obvious inhibition effect on the growth of various plant pathogenic fungi, is wide in antibacterial spectrum and good in antibacterial effect, and has a better biological control effect. Is a novel biocontrol microorganism which is safe, efficient and broad-spectrum for preventing and controlling plant fungal diseases. The biocontrol microbial inoculum provided by the invention has the advantages of simple preparation, low cost, easy industrial production, good control effect, no environmental pollution, capability of replacing pesticides to control gray mold of cucumber and the like, great significance in guaranteeing food safety and remarkable social and economic benefits.

Drawings

FIG. 1 is a colony morphology of Bacillus belicus LG001 of the present invention on NA medium; wherein A is colony morphology, and B is cell morphology under a microscope.

FIG. 2 is a 16SrDNA sequence evolutionary phylogenetic tree of Bacillus bailii LG001 of the present invention.

FIG. 3 is a graph showing the inhibition of various plant pathogenic fungi by Bacillus bailii LG001 fermentation broth of the present invention.

FIG. 4 is a graph comparing the inhibition of Botrytis cinerea by different strains of the present invention, wherein a: CK (NB liquid medium); b, bacillus bailii LG001; b, bacillus bailii LG001 fermentation broth (0.2 um filter membrane filtration); 50% wettable powder carbendazim (d 4:10 ug/mL).

Detailed Description

The following detailed description of specific embodiments of the invention refers to the accompanying drawings and the detailed description.

The strain Bacillus bailii (Bacillus velezensis) LG001 is obtained by separating rhizosphere soil of long-term cucumber planting in Zhengyang county of a standing-horse store. The bacillus is classified and named as bacillus belicus (Bacillus velezensis) and is preserved in China general microbiological culture Collection center (CGMCC No. 2366) in the year 9 and 6 of 2021, and the preservation number is CGMCC No. 2366 and the preservation address is: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

The strain is applied to a medicament for preventing and treating plant pathogenic fungi, wherein the plant pathogenic fungi are one or more of botrytis cinerea, fusarium aphanidermatum, peanut southern blight, kiwi fruit black spot, watermelon fusarium wilt, corn stem rot, potato late blight and fusarium graminearum.

The invention also provides a biocontrol microbial inoculum, the active ingredient of which is bacillus bailii LG001, and the biocontrol microbial inoculum is fermentation broth of bacillus bailii LG001, which is prepared by the following steps: inoculating the activated bacillus belicus LG001 into a sterile LB liquid medium, culturing at 180r/min and 30 ℃ for 48 hours to obtain a fermentation broth, namely the biocontrol microbial agent.

The bacillus belicus LG001 can generate various antibacterial substances, has obvious inhibition effect on various plant pathogenic fungi, has wide antibacterial spectrum and good antibacterial effect, has better biological control effect, and the biocontrol microbial inoculum obtained by fermenting the strain has the advantages of simple preparation, low cost, easy industrial production, good control effect, no environmental pollution and quite good beneficial technical effect through experiments, and the related data are as follows:

1. screening and identification of strains

(1) Taking 2.00g of rhizosphere soil sample of cucumber planted in Zhengyang county of a standing horse, adding the rhizosphere soil sample into a sterile conical flask with glass beads, adding 20mL of sterile physiological saline, putting into a shaking table, oscillating for 20min at 30 ℃ at 180r/min, and standing for 5min. Taking supernatant and adopting ten times dilution method to dilute to 10 in turn ^-2 、10 ^-3 、10 ^-4 And 10 ^-5 And (3) respectively sucking 100 mu L of each concentration, coating on an LB culture medium, uniformly coating, repeating each concentration for three times, reversely placing a flat plate, culturing in a 30 ℃ incubator, and selecting different bacteria according to the different forms and colors of bacterial colonies.

(2) Antagonistic bacteria were screened by plate counter method. Solid culture media of different plant pathogenic fungi with the diameter of 5mm and hyphae are selected, inoculated to the center of a PDA flat plate, dipped with bacterial colonies to two sides by using a sterile inoculating loop, placed in a 28 ℃ incubator for 4d, and observed for bacteriostasis diameter, and repeated three times for each group. Transferring bacteria with antibacterial activity screened on the flat plate to NA culture medium, purifying, and culturing to obtain purified active strain with strongest antibacterial effect, namely Bacillus bailii (Bacillus velezensis) LG001.

2. Morphological identification of strains

Strain LG001 was inoculated on NA plate medium and cultured at 30 ℃ for 16h, and the colony morphology was observed as round, milky white, smooth in surface, irregular in edge, sticky in texture and wrinkled (see fig. 1A). The bacterial cells of strain LG001 were short bar-shaped under a microscope (as shown in FIG. 1B), and gram staining was positive.

The physiological and biochemical characteristics of strain LG001 were identified using a VITEK bacillus identification card, and the results are shown in Table 1.

TABLE 1 Biochemical reactions for identification LG001

3. Molecular biological identification of strains

Physiological and biochemical identification refers to methods of Dongxiu beads et al (2001). The strain LG001 was amplified by colony PCR to 16 SrDNA. Primers were used as universal primers 27F (5 'AGAGTTTGATCTCTMTGGCTCA 3') and 1492R (5'TACGGYTACCTTGTTACGACTT 3'). After gel electrophoresis, the 16S rDNA gene was found to be between 1000 and 2500bp in length. And (3) comparing the nucleotide SEQUENCE with DNA maker, then sending the nucleotide SEQUENCE to a qinghao organism for sequencing, and splicing the obtained base SEQUENCE by using Biokit software to obtain the 16SrDNA with the effective SEQUENCE length of 1455bp, wherein the specific SEQUENCE is shown as SEQUENCE Listing. The amplified sequences were subjected to BLAST homologous sequence searches at NCBI (https:// www.ncbi.nlm.nih.gov) and phylogenetic tree patterns were constructed using MEGA6.06 software, as shown in FIG. 2. The results showed that the 16S rDNA sequence of the strain was 99.31% similar to Bacillus bailii, and the strain was identified as Bacillus bailii by combining morphological, physiological and biochemical features and 16S rDNA (Bacillus Velezensis). Bacillus belicus (Bacillus Velezensis) LG001 is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No.23366, a preservation date of 2021, 9 months and 6 days, and a preservation address of North Chen Lu No. 1 and 3 in the Qiangyang area of Beijing city.

The bacillus beleiensis LG001 16S rDNA sequence is as follows:

GGGGGGGGTGCCAATTCATGCAAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATG

TTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCC

GGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGT

GGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACG

GCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACT

GAAACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGA

AAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTT

GTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGA

AAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCC

GGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCC

GGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGT

GGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAG

GCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGAT

TAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGC

CCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACT

GAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCG

AAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGAC

GTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAG

ATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTG

GGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATC

ATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGC

GAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCA

ACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATA

CGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGT

CGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGGTGAACCAGAAGG

4. fermentation liquor application of bacillus bailii LG001

Preparation of fermentation liquor: picking single colony of LG001 thallus on solid culture medium with aseptic inoculating loop, inoculating into 100mL sterilized LB culture medium, shaking culturing at 30deg.C for 48 hr at 180r/min to obtain 10-containing strain ^6～9 CFU/mL fermentation broth was used.

Antagonism of 4.1LG001 fermentation broth against pathogenic fungi.

Inoculating different plant pathogenic fungi respectively in the center of a sterile PDA culture medium: the preparation method comprises the steps of (1) carrying out spot grafting on botrytis cinerea, aphis aphanidermatum, southern blight of peanut, black spot of kiwi fruit, fusarium wilt of watermelon, corn stalk rot, potato late blight and fusarium graminearum on two sides of each germ by respectively sucking 10 mu L of LG001 fermentation liquor, and carrying out dark culture at 30 ℃ for 3d to serve as an experimental group; the control group replaced the bacterial fermentation broth with sterile water, and the other steps were the same as those of the experimental group. Each set is provided with 3 parallels. And observing the antibacterial effect, and calculating the antibacterial rate. The bacteriostatic effect is shown in figure 3 and table 2. The calculating method of the bacteriostasis rate comprises the following steps:

antibacterial ratio (%) = (control colony diameter-treated colony diameter)/control colony diameter×100% (1)

As can be seen from the graph in FIG. 3, the LG001 fermentation broth has antagonism to different degrees on plant pathogenic fungi such as botrytis cinerea, fusarium aphanidermatum, southern blight germ, kiwi fruit black spot germ, fusarium oxysporum, corn stalk rot germ, potato late blight germ and fusarium graminearum.

TABLE 2 antagonism test of Bacillus bailii LG001 against several plant pathogenic fungi

The results are shown in Table 2, the strain LG001 shows obvious antibacterial activity on 8 plant pathogenic fungi, the inhibition rate is more than 35.88%, and the highest inhibition rate reaches 69.41%, so that the bacterial residue LG001 fermentation liquor has good biocontrol potential.

Antagonism of the plant pathogenic fungus Botrytis cinerea by 4.2LG001 sterile fermentation broth.

The LG001 cells were removed from the broth with a 0.22 μm bacterial filter to give a sterile broth for use. The central part of the PDA culture medium is inoculated with botrytis cinerea which is a plant pathogenic fungus. Around Botrytis cinerea, 4 sterile oxford cups were placed, each 100 μl of sample was added, and the sample was added as shown in FIG. 4. The plate was placed in an incubator at 28℃and incubated for 3d, and the antibacterial effect was observed, and the results are shown in FIG. 4.

The result shows that the inhibition strength of the LG001 sterile fermentation liquid on the growth of the botrytis cinerea is equivalent to that of carbendazim with the final concentration of 10 mug/mL, and the antibacterial effect is obvious.

5. Experiment for preventing and controlling gray mold of cucumber in pot culture by fermentation broth

The LG001 fermentation broth test for preventing and controlling the gray mold of the cucumber is carried out outdoors in a potting mode. The soil is taken from farmland planted with cucumber in Zhengyang county of standing horse shop, 2.0kg of the soil is filled in each pot, 4.0g of the fourth element 17-6-22 compound fertilizer (Shidanli) is applied, and 1 plant of 5 leaf-age cucumber seedlings with consistent implantation state and size is planted. The experiments were set up 6 groups of 10 pots each, 3 replicates were set up, 6 groups were:

(1) Blank group: spraying sterile water on the whole plant leaves to wet the leaves and drip water until the leaves are wet, wherein the following treatment groups are the same;

(2) Control group: only the pathogenic bacteria group is inoculated, and the foliar spray is 1 multiplied by 10 ⁶ CFU/mL of botrytis cinerea spore suspension;

(3) 50% carbendazim wettable powder+pathogen group: 50% carbendazim wettable powder with 500 times of liquid sprayed on leaf surface is sprayed for 24 hours to 1X 10 ⁶ CFU/mL of botrytis cinerea spore suspension;

(4) Pathogenic bacteria +50% carbendazim wettable powder group: foliar spray 1×10 ⁶ CFU/mL of botrytis cinerea spore suspension, and spraying 50% carbendazim wettable powder of 500 times of liquid after 24 hours;

(5) LG001 broth + pathogen group: spraying LG001 fermentation liquid diluted 5 times on leaf surface, and spraying 1×10 after 24 hr ⁶ CFU/mL of botrytis cinerea spore suspension;

(6) Pathogen + LG001 broth group: foliar spray 1×10 ⁶ CFU/mL of botrytis cinerea spore suspension, and after 24 hours, LG001 fermentation broth diluted 5 times is sprayed.

When the incidence rate of disease treatment approaches 40%, the leaf numbers and the disease degree of all cucumber plants are counted, and the indexes are as follows:

level 0: the whole leaves are free from diseases;

stage 1: the total area of the disease spots is less than 1% of the leaf area;

3 stages: the area of the disease spots accounts for 1% -5% of the area of the leaf blades;

5 stages: the total area of the disease spots accounts for 6-10% of the total leaf area;

7 stages: the total area of the lesion accounts for 11% -20% of the total leaf area;

stage 9: the total area of the disease spots accounts for more than 21% of the total leaf area;

and calculating the morbidity, the disease index and the prevention and treatment effect according to the formulas (2), (3) and (4).

Incidence (%) = number of treated plants/total number of treated plants×100% (2)

Control effect (%) = (control disease index-treatment disease index)/control disease index×100% (4)

TABLE 3 Gray mold statistics of potted cucumber

TABLE 4 control effect of LG001 fermentation broth on gray mold of potted cucumber

Note that: the lower case letters in the same column show significant differences (P is less than or equal to 0.05)

The results are shown in table 4 above, with the data indicating: gray mold disease does not occur in the blank group; the incidence rate and the disease index of the control group inoculated with only Botrytis cinerea are 40% and 4.07 respectively; compared with a control group, the 50% carbendazim wettable powder treatment group is applied, which shows that the chemical pesticide can obviously reduce the morbidity and the disease index by preventing and treating gray mold; the results of the LG001 fermentation liquor treatment group show that the spraying of the LG001 fermentation liquor can obviously prevent and treat the occurrence of cucumber gray mold, and the effect difference of the LG001 fermentation liquor treatment group and 50% carbendazim wettable powder is obvious, and the control effect is 83.29% and 62.16%.

6. LG001 fermentation liquor field test for controlling cucumber gray mold

The field biocontrol test is carried out in the 3 months of cucumber planting season at the town age Zhuang Cun after the wine in the Yichuan county of Luoyang city. The test packets are set to:

(1) Blank group: spraying tap water on the leaf surfaces to wet the leaf surfaces (the same applies below);

(2) Treatment group 1: spraying 500 times of 50% carbendazim wettable powder;

(3) Treatment group 2: spraying LG001 fermentation liquor on leaf surfaces;

and (3) randomly selecting the ridges, wherein each test group is 4 ridges, and 50 strains of cucumbers are planted in each ridge. After 14 days, 100 cucumber leaves in each group are randomly selected to count the number of disease spots and disease indexes, and the morbidity, the disease indexes and the prevention and treatment effects are calculated, and the specific results are shown in Table 5.

TABLE 5 control of cucumber gray mold results with LG001 fermentation broth

Table 5 the results show that: the morbidity and the disease index of the treatment group 1 and the treatment group 2 are lower than those of the blank group, which shows that the 50% carbendazim wettable powder sprayed with 500 times of liquid and the LG001 fermentation liquor have remarkable effect on preventing the gray mold of the cucumber, the prevention effect of the LG001 fermentation liquor on the gray mold of the cucumber is obviously higher than that of the 50% carbendazim wettable powder, and the pollution caused by using chemical pesticides is reduced.

In conclusion, the bacterial colony and the fermentation liquor of the bacillus belicus LG001 can obviously inhibit various plant pathogenic fungi, and the fermentation liquor has good effect on preventing the gray mold of cucumber, is environment-friendly, can reduce the use of chemical pesticides, has good market application potential, and has the following advantages compared with the prior art:

1. the bacillus belicus LG001 obtained by screening of the invention not only has the capacity of producing indoleacetic acid, protease, amylase and beta-mannase, but also can secrete antibacterial active substances, can inhibit the growth of various pathogenic bacteria, and is suitable for various plant pathogenic fungi: the growth of the botrytis cinerea, the fusarium solani of melons and fruits, the southern blight of peanuts, the black spot of kiwi fruits, the fusarium wilt of watermelons, the corn stem basal rot of potatoes, the late blight of potatoes and the fusarium graminearum all have obvious inhibition effect, broad antibacterial spectrum and good antibacterial effect, and have better biological control effect. The strain is derived from field soil, is harmless to the ecological environment, is not easy to cause the drug resistance of pathogens, is easy to reproduce and prepare into biopesticide, and is a novel biocontrol microorganism which is safe, efficient and broad-spectrum for preventing and controlling plant fungal diseases;

2. the biocontrol microbial inoculum provided by the invention has the advantages of simple preparation, low cost, easiness in industrial production, good control effect, no environmental pollution, environmental protection and no pollution, can replace pesticides to control gray mold of cucumbers, reduce application of chemical pesticides, reduce environmental pollution, improve cucumber yield, ensure food safety, and is significant, beneficial to long-term, healthy and sustainable development of agricultural production, and significant in social and economic benefits, and is a great innovation in biological control of plant fungal diseases.

Claims

1. Bacillus bailii LG001, classified and named as Bacillus bailii @Bacillus velezensis) The microbial strain is preserved in China general microbiological culture collection center (CGMCC) at 9/6 of 2021, with the preservation number of CGMCC No.23366 and the preservation address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

2. Use of bacillus belgium LG001 according to claim 1 in a medicament for combating phytopathogenic fungi.

3. The use according to claim 2, wherein the phytopathogenic fungi are one or more of the group consisting of botrytis cinerea, fusarium aphanidermatum, fusarium solani, fusarium wilt, fusarium solani, fusarium graminearum, and fusarium graminearum.

4. The biocontrol microbial inoculum is characterized in that the effective component of the biocontrol microbial inoculum is bacillus bailii LG001, and the biocontrol microbial inoculum is culture bacterial suspension, fermentation liquor or metabolic products and volatile substances generated by fermentation of bacillus bailii LG001.

5. The biocontrol microbial agent according to claim 4, wherein the fermentation broth is prepared by activating bacillus belicus LG001, inoculating to a sterile LB liquid medium, culturing at 180r/min at 30 ℃ for 48 h.

6. The use of the biocontrol microbial inoculum according to claim 4 in medicaments for controlling cucumber gray mold, which is sprayed on plants with pathogenic bacteria like a stripe.