CN117568199A

CN117568199A - Biocontrol bacterium and application thereof in pepper epidemic disease prevention and control

Info

Publication number: CN117568199A
Application number: CN202311045101.6A
Authority: CN
Inventors: 钱新; 陈亚丽; 李美霞; 王晓飞; 王宏宝; 周长勇; 曹凯歌; 毛佳; 陈香华; 刘伟中
Original assignee: JIANGSU XUHUAI DISTRICT HUAIYIN AGRICULTURAL SCIENCE RESEARCH INSTITUTE
Current assignee: JIANGSU XUHUAI DISTRICT HUAIYIN AGRICULTURAL SCIENCE RESEARCH INSTITUTE
Priority date: 2023-08-18
Filing date: 2023-08-18
Publication date: 2024-02-20

Abstract

The invention relates to a novel biocontrol bacterium and application thereof in the aspect of controlling pepper epidemic disease; the biocontrol strain bacillus amyloliquefaciens 1LN2 can effectively prevent and control pepper epidemic diseases and has growth promoting effect on peppers; particularly in the aspect of pepper planting application, the pepper epidemic disease prevention and control effect is remarkable, and the pepper growth and the yield can be promoted; furthermore, the biological control bacteria has wide antibacterial spectrum, can effectively inhibit the growth of nine plant pathogenic bacteria including phytophthora, and has wide application range. In addition, the biocontrol bacterium 1LN2 is obtained from the rhizosphere soil of the capsicum, is nontoxic and environment-friendly compared with chemical reagents, and has good development and application prospects.

Description

Biocontrol bacterium and application thereof in pepper epidemic disease prevention and control

Technical Field

The invention belongs to the technical field of biological control of crops, and particularly relates to a biocontrol bacterium and application thereof in the aspect of controlling pepper epidemic disease.

Background

Peppers are used as important cooking materials, and have been planted for over 300 years in China. At present, the yield of the capsicum in China is first in the world, is as high as 1.3 hundred million tons, and accounts for about 25% of the total yield of the capsicum worldwide. The pepper phytophthora blight is a soil-borne disease with destructive damage caused by phytophthora capsici (Phytophthora capsici), which causes yield reduction and even sterilization on peppers, and causes serious economic loss. Usually, the disease area reaches 20% -30%, and more than 80% and even hundred percent in severe cases, so that large-area dead plants are caused, and the healthy development of agricultural production is hindered. After the first discovery of the epidemic disease of the capsicum in Jiangsu province of China in the 20 th century, the capsicum is gradually spread to a plurality of areas, so that the epidemic disease of the capsicum producing area occurs in a large area. In recent years, the rapid development of the pepper industry in China is accompanied with specialized and large-scale production of planting, and the incidence trend and the hazard degree of pepper epidemic diseases are all in an annual rising trend.

At present, the main measures for preventing and treating the pepper epidemic disease are the application of chemical agents and the selection of resistant varieties. However, the long-term use of chemical pesticides induces phytophthora capsici to generate drug resistance to bactericides, thereby leading to continuous weakening of the sterilizing effect. Meanwhile, the long-term use of chemical pesticides aggravates water pollution, damages ecological environment and endangers human health. In addition, the selective breeding of the resistant variety is used as another means for preventing and controlling phytophthora capsici, however, so far, the resistance level of all varieties in production to epidemic diseases is low, so that an efficient and economic preventing and controlling method is still lacking at the present stage. With the enhancement of green prevention and control consciousness, the advantages of biological control in recent years are remarkable, and the effect of controlling vegetable diseases is remarkable.

Therefore, the search for new control measures is becoming a great importance in the control of epidemic diseases. The biological control measure is used as a safe pollution-free control measure, has the advantages of safety, stability, greenness, innocuity and the like, and simultaneously avoids a series of problems caused by chemical control. The biocontrol agent has good disease control effect, is nontoxic to human and livestock, does not pollute the environment and has no residue; the killing specificity to the plant diseases and insect pests is strong, natural enemies are not damaged, beneficial organisms are avoided, and ecological balance can be maintained; the production raw materials and the active ingredients are natural products which are easy to degrade, can return to the nature, promote the green development of agricultural rural areas and promote the industrial ecology.

Biological control of phytophthora blight is becoming a growing point of research, and it is desirable in the art to screen microorganisms capable of controlling phytophthora blight.

Disclosure of Invention

In view of the above-mentioned problems and/or other problems of the related art, the present invention provides a biocontrol bacterium, wherein the biocontrol bacterium is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) 1LN2, which has been preserved in the general microbiological center of the China general microbiological culture collection center, called CGMCC for short, with the address of the north chen west road No.1 No. 3 in the korean region of beijing, and the preservation number of the biocontrol bacterium is CGMCC No.27381, at 5 months 18 of 2023.

The invention provides an application of the biocontrol bacteria in preventing and controlling phytophthora root rot of crops, an application in promoting growth of crops or an application in pepper planting.

The biocontrol bacterium CGMCC No.27381 provided by the invention can effectively prevent and treat crop epidemic diseases and has growth promoting effect on crops; particularly in the aspect of pepper planting application, the pepper epidemic disease prevention and control effect is remarkable, and the pepper growth and the yield can be promoted; in addition, the biocontrol bacterium 1LN2 is obtained from the rhizosphere soil of the capsicum, is nontoxic and environment-friendly compared with chemical reagents, and has good development and application prospects.

The specific technical scheme is as follows:

the bacillus amyloliquefaciens strain is 1LN2 and is preserved in China general microbiological culture Collection center with a preservation number of CGMCC No.27381.

A biological microbial inoculum comprises active ingredients of bacillus amyloliquefaciens 1LN2 or fermentation broth of bacillus amyloliquefaciens 1LN2. Preferably, the viable bacteria concentration of the bacterial liquid in the bacillus amyloliquefaciens 1LN2 fermentation broth is 1 multiplied by 10 ⁵ ～1×10 ¹⁰ CFU/ml。

The use of said bacillus amyloliquefaciens or said biological agent for inhibiting phytophthora, fusarium graminearum, rhizoctonia cerealis, sclerotinia sclerotiorum, verticillium capsici, bipolaris zeae or phoma asparagi. In particular for inhibiting phytophthora.

The bacillus amyloliquefaciens or the biological microbial inoculum is used for preventing and treating pepper epidemic diseases and promoting pepper growth. Preferably for promoting an increase in stem thickness, leaf number, plant height and bud number.

Compared with the prior art, the beneficial technical effects are achieved:

the biocontrol bacterium 1LN2 can effectively prevent and control phytophthora capsici and has growth promoting effect on peppers; especially in the aspect of pepper planting application, the pepper epidemic disease prevention and control effect is remarkable, and the pepper growth can be promoted and the yield can be improved.

Drawings

Fig. 1: is a evolutionary tree constructed from the sequenced sequences, wherein the sequences in the sequence library are all from the NCBI gene library;

fig. 2: photographs of the results of a plate antagonism assay against phytophthora;

fig. 3: flat panel antagonism of 1LN2 against other pathogens;

fig. 4:1LN2 in the presence of a sterile broth with an inhibitory effect on hyphal growth;

fig. 5: growth promotion effect of 1LN2 on pepper;

Detailed Description

1. The following describes the operation process of separating and screening the biocontrol strain (bacillus amyloliquefaciens Bacillus amyloliquefaciens) CGMCC No.27381 obtained by the inventor of the application from natural environment.

(1) Isolation of potential biocontrol bacteria

Isolation of potential biocontrol bacteria present outside the plant body:

cutting 3g of roots, stems and leaves of capsicum (picked from Huaian vegetable greenhouse) respectively, weighing 3g of root surrounding soil, and adding the collected samples into 30mL of sterilized water (containing sterilized glass beads) respectively;

the sterilized water containing the sample was placed in a shaker for shaking at a rotational speed of 170rmp for 30min. Standing for 5min after oscillation is finished. And respectively sucking the supernatant of the obtained samples, and carrying out gradient dilution. Multiple of sequential dilution of 10 ⁴ 、10 ⁵ And 10 ⁶ Then, 0.1mL of three gradient dilutions were taken on LB plates, respectively, and placed in a 28℃incubator. After incubation for 24-48 hours, plates were picked up with colony counts between 50-300, and all colonies were picked up simultaneously. After purification, the strain is inoculated into a test tube containing 5mL of LB liquid medium, and is cultivated for 24-48 hours at 28 ℃ under 200rpm, and the bacterial suspension is taken and evenly mixed with 80% glycerol solution with equal volume and stored at-70 ℃ for standby.

Isolation of potential biocontrol bacteria present in plants:

firstly, respectively taking 3g of roots, stems and leaves of capsicum (picked from Huaian vegetable greenhouse) as samples, and then sterilizing the surfaces of the taken samples, wherein the specific operation process is as follows: firstly, 1mL of 1% sodium hypochlorite is used for sterilization for 5min, secondly 70% alcohol is used for sterilization for 2min, and finally sterile water is used for cleaning for 3 times.

The surface sterilized samples were ground with sterilized mortar, 3mL of sterilized water was added thereto, the sterilized water was replenished after filtration through sterilized cotton cloth, and the final sample was diluted 10 times. 0.1mL of the slurry was applied to LB plate, and each spot was repeated 3 times and incubated at 28℃for 48 hours at 200 rpm. Counting and picking all the colonies on the plate, separating and purifying, inoculating to a liquid culture medium, culturing at 28 ℃ for 24-48h by shaking, and preserving 40% glycerol at-70 ℃ for later use.

(2) Determination of enzyme production and metabolite Activity Primary screening of biocontrol Strain

Whether the separated strain has better antagonistic activity is identified, and the production potential of protease, chitinase, glucanase, cellulase and the like of the separated strain is evaluated, so that whether the strain can degrade the cell wall of fungi (plant pathogenic fungi cell wall components comprise protein, chitin, glucan, cellulose and the like) can be estimated preliminarily, and the strain can be used as a standard for primary screening biocontrol bacteria. Bacterial strain colonies in a vigorous growth period are picked up by toothpicks and placed on a protein culture medium, a culture medium (Chi-Ayers) taking colloidal chitin as a unique carbon source, a beta-1, 3-glucanase enzyme activity measurement culture medium and a cellulase enzyme activity measurement culture medium, the bacterial strains are placed in a 28 ℃ incubator after inoculation, the transparent rings are observed after 3d, and the outer diameter and the inner diameter of the transparent rings are recorded respectively. Strains with one or more of the above enzyme-producing and secondary metabolite activities remain for further screening.

(3) Plate antagonism test with phytophthora to further screen phytophthora-resistant biocontrol strain

Phytophthora fungi are cultivated in an incubator at 28 ℃ for 5 days, a sterilized puncher is used for punching a fungus dish with the diameter of 0.6cm, and the fungus dish is inoculated in the center of the V8 culture medium. 2.5. Mu.l of the bacterial liquid of the biocontrol bacterium 1LN2 of the invention was sampled and inoculated (live bacterial liquid concentration of 5X 10) by a pipette at a position equidistant from the inoculation site and relatively everywhere ⁹ ～1×10 ¹⁰ CFU/ml), 2.5. Mu.l ddH was removed by a pipette on another V8 medium at an equidistant relative four places from the inoculation site ₂ O was used as a control and incubated in a 28 ℃ incubator, and 3 replicates were run simultaneously for each of the above treatments. After 4 days of culture, the existence and the size of the inhibition zone are observed, so that the existence and the intensity of antagonism of the potential biocontrol strain to phytophthora can be judged.

A strain is separated from root soil of a pepper healthy strain, and is confirmed to be a potential biocontrol strain by enzyme activity detection and a flat plate antagonism test, and is named as biocontrol strain 1LN2.

2. The identification process of the biocontrol bacterium 1LN2 according to the invention is as follows:

molecular biological identification

And (3) carrying out molecular biology identification on the biocontrol bacteria 1LN2 obtained by screening by adopting phylogenetic analysis according to an identification method of the bacterial 16S rDNA gene fragment sequence.

Extracting 1LN2 bacterial genome DNA by using a genome DNA rapid extraction kit of Shanghai Saighur gene technology Co., ltd, and amplifying the 16S rRNA sequence and gyrB gene fragment of the biocontrol bacterium 1LN2 by using a PCR amplification kit of Tiangen biochemical technology Co., ltd with the extracted DNA product as a template, wherein the specific operation is carried out according to the instruction of the kit.

The forward primer sequence adopted for amplifying the 16S rRNA sequence is as follows: 5'-AGAGTTTGATCACTGGCTCAG-3', the reverse primer sequence adopted for amplification is as follows: 5'-CTACGGAGTACCTTGTTACGAC-3'.

The forward primer sequence adopted for amplifying the gyrB gene fragment is as follows: 5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3', the reverse primer sequence adopted in the amplification is as follows: 5'-AGCAGGATACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3'.

The amplified bands were cut and recovered using gel recovery kit from Tiangen Biochemical technologies Co. The recovered samples were sent to general biosystems (Anhui) Inc. for sequencing.

The sequencing results were subjected to homology comparison (similarity alignment with 16SrDNA sequences and gyrB gene fragments of known bacteria) by NCBI BLAST software, and the biocontrol bacterium 1LN2 of the present invention was identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).

FIG. 1 is a evolutionary tree constructed from sequenced sequences, wherein sequences in the sequence library are all from the NCBI gene library.

3. Preservation of strains

The biocontrol bacterium 1LN2 for preventing and treating crop epidemic diseases belongs to bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and is preserved in China general microbiological culture collection center (CGMCC) for short in the year 2023, 5 and 18, and has the address of Hospital No.1 and No. 3 in the North Chen West road of the Korean region of Beijing city and the preservation number of CGMCC No.27381.

4. The invention relates to a method for culturing biocontrol bacteria and application thereof

The biocontrol bacterium 1LN2 is streaked on an LB plate, placed at 28 ℃ for culturing for 24 hours, and after single colony of the strain grows out, single colony of the strain is selected to be cultured in a test tube containing 5ml of LB culture solution at 28 ℃ for 12 hours at 200rpm to be used as seed culture solution. Inoculating the seed bacterial liquid into 500mL LB culture liquid at a ratio of 1:100, culturing at 28deg.C and 200rpm for 24 hr until the total concentration of viable bacteria is 5×10 ⁹ ～1×10 ¹⁰ CFU/ml。

Regarding the application of the biocontrol bacterium 1LN2 of the invention, generally, after the pepper is transplanted into the soil, the bacterial liquid obtained by the culture is diluted by 100-200 times immediately, the transplanted pepper is root irrigated, and the pepper is diluted by 500-1000 times simultaneously for foliage spraying.

5. Flat plate antagonism test result of biocontrol bacterium 1LN2 of the invention on phytophthora

The specific test method comprises the following steps: phytophthora fungi are cultivated in an incubator at 28 ℃ for 5 days, a sterilized puncher is used for punching a fungus dish with the diameter of 0.6cm, and the fungus dish is inoculated in the center of the V8 culture medium. 2.5. Mu.l of the bacterial liquid of the biocontrol bacterium 1LN2 of the invention was sampled and inoculated (live bacterial liquid concentration of 5X 10) by a pipette at a position equidistant from the inoculation site and relatively everywhere ⁹ ～1×10 ¹⁰ CFU/ml), 2.5 μl of LB solution was taken as control on another V8 medium at opposite four positions equidistant from the inoculation site with a pipette, and placed in a 28 ℃ incubator for culture, and 3 replicates were performed simultaneously for each of the above treatments.

See FIG. 1 for photographs of plate antagonism test against Phytophthora, wherein the plate inoculated with 1LN2 plaque is a plate experimental group, and the plate inoculated with LB solution at the same relative position is a control group.

As shown in table 1: the average diameter of the experimental group is 4.11cm (the average distance between the upper, lower, left and right biocontrol bacteria inoculation points is taken as the diameter) measured by a vertical crossing method, the average diameter of the control group is 6.96cm, and the average hypha growth inhibition rate is 40.95%. The junction of the biocontrol bacterium 1LN2 and phytophthora forms a clear transparent ring, while the control group does not form a transparent ring.

Thus, from the above plate antagonism test results, it can be seen that: 1LN2 has obvious antagonistic activity against Phytophthora.

TABLE 1 Phytophthora diameter and hypha growth inhibition ratio of control and experimental groups

6. Flat plate antagonism test result of biocontrol bacterium 1LN2 of the invention on other pathogenic fungi

Taking fungus discs of Fusarium graminearum, rhizoctonia graminearum, sclerotinia sclerotiorum and the like by using a puncher, respectively inoculating the fungus discs on a PDA flat plate, and respectively inoculating 2.5 mu l of the fungus solution of biocontrol bacterium 1LN2 of the invention on four points by taking the fungus discs as a center through a vertical crossing method, wherein the fungus solution is an experimental group; 2.5. Mu.l of LB solution was used as a control on another PDA medium at equal distances from the inoculation site and placed in a 28℃incubator for incubation, and 3 replicates were performed simultaneously for each of the above treatments.

As shown in FIG. 3, the biocontrol bacterium 1LN2 of the invention has obvious antagonism to a plurality of pathogenic bacteria such as Fusarium graminearum, rhizoctonia cerealis and the like.

7. Flat plate antagonism test result of biocontrol bacterium 1LN2 sterile fermentation liquid of the invention on phytophthora

Mixing the sterile fermentation broth of the biocontrol bacterium 1LN2 of the invention with a culture medium according to different proportions (10%, 20%, 30%, 40%, 50%) and inoculating phytophthora; the control group was set up by mixing LB with medium in different proportions (10%, 20%, 30%, 40%, 50%) and inoculating Phytophthora. Culturing in a 28 ℃ incubator, and simultaneously performing 3 groups of parallel repeated experiments on the treatments. And measuring the diameter of phytophthora capsici falling after 4d, and calculating the phytophthora capsici silk inhibition rate.

The results are shown in FIG. 4, which shows that the higher the concentration of 1LN2 in the sterile broth, the higher the hyphal inhibition of phytophthora. When the sterile fermentation liquid of 1LN2 is diluted by one time, the hypha inhibition rate is as high as eighty percent, and the hypha growth inhibition rate of other dilution times is about forty or more, which shows that the sterile fermentation liquid of 1LN2 has better plate inhibition effect on phytophthora.

TABLE 2 results of inhibition of hyphal growth by sterile fermentation broth of LN2

8. Greenhouse test result of biocontrol bacterium 1LN2 for promoting growth of capsicum

Transplanting the pepper seedlings growing to 3-4 leaf periods into disposable plastic cups, wherein each cup is one plant. After the seedlings were relaxed, the biocontrol bacterium 1LN2 was inoculated with a bacterial solution (bacterial concentration 5X 10) ⁷ CFU/mL) for root irrigation treatment, and each seedling is irrigated with 20mL; this is the experimental group (experimental group of pepper growth promotion test).

Meanwhile, in the same transplanting mode, 20mL of LB solution is irrigated for each seedling to serve as a control group (a control group of a pepper growth promotion test).

After 45 days of treatment, the growth vigor of the pepper seedlings is observed, and the biomass of each plant is counted: the results of the stem thickness, leaf number, plant height and flower bud number are shown in the following table 3.

As shown in FIG. 5 and Table 3, the biomass (stem thickness, leaf number, plant height, flower bud number) of the biocontrol bacterium 1LN2 treated experimental group was significantly increased as compared with the control group. Therefore, the result shows that the biocontrol bacterium 1LN2 has growth promoting effect on pepper planting.

TABLE 3 growth promoting results of LN2 on Capsici fructus

9. The field disease prevention test result of the biocontrol bacterium 1LN2 on pepper epidemic disease

The field test is carried out in continuous cropping area of Huaian yin region of Huaian city of Jiangsu province, and is divided into 5 treatment areas, 2m wide protection row is set in the middle, and the field test is carried out according to conventional cultivation management technologyLine field management, each treatment area is 20m ² The length is 10m, the width is 2m, irrigation and drainage ditches are formed around each treatment area, energy and irrigation can be drained, and irrigation and drainage are convenient.

On the day of seedling transplanting, the biocontrol strain solution (living strain concentration 5×10) ⁷ CFU/mL) for root irrigation treatment, and each seedling is irrigated with 20mL; this is the experimental group and the positive control group.

Meanwhile, in the same transplanting mode, 20mL of LB solution is irrigated to each seedling as a blank control group.

After the control onset, the onset was observed.

Regarding statistics of disease conditions, the classification criteria for phytophthora disease are presented as follows:

level 0: no disorder;

stage 1: the root and stem parts of the seedlings are slightly blackened, and the leaves are not withered;

3 stages: blackening root and stem of seedling to 1-2cm, and withering irrecoverable leaf to obtain sporadic leaf

Falling off;

5 stages: the root and stem parts of the seedlings are blackened by more than 2cm, and the leaves are obviously withered, so that the leaves are obviously withered or fallen leaves are obviously removed;

7 stages: blackening the rhizome parts of the seedlings, shrinking, and falling off the outer parts of growing points or withering the whole plant;

stage 9: the whole plant dies.

The calculation formulas for the "disease severity" and "control effect" are as follows:

disease severity= { Σ (leaf number of each stage×relative stage number) }/(total number of investigation×9);

control effect= (control disease severity-treatment disease severity)/control disease severity;

see table 3 below, which is a field test result for controlling pepper epidemic disease.

The results in Table 4 show that the field control effect of the biocontrol bacterium 1LN2 of the invention on phytophthora capsici reaches 86.63%, which shows that the biocontrol bacterium has better control effect on phytophthora capsici.

TABLE 4 field test results of 1LN2 control of pepper epidemic disease

Remarks: positive control groups were used: bacillus amyloliquefaciens IBFCBF-1 with a preservation number of CGMCC No.11230; bacillus amyloliquefaciens strain YB1701 with a preservation number of CGMCC No.16003; bacillus amyloliquefaciens Kc-5 with a preservation number of CGMCC No.17654.

In conclusion, the biocontrol bacterium 1LN2 can effectively prevent and control phytophthora capsici and has growth promoting effect on peppers; especially in the aspect of pepper planting application, the pepper phytophthora capsici control effect is remarkable, and the pepper growth can be promoted and the yield can be improved. In addition, the biocontrol bacterium 1LN2 has wide bacteriostasis spectrum, and can effectively inhibit the growth of nine plant pathogenic fungi including phytophthora.

Claims

1. The bacillus amyloliquefaciens is characterized in that the bacillus amyloliquefaciens strain is 1LN2 and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.27381.

2. A biological microbial agent, which is characterized in that the active ingredient of the microbial agent is bacillus amyloliquefaciens 1LN2 or a fermentation broth of bacillus amyloliquefaciens 1LN2 according to claim 1.

3. The biological agent according to claim 2, wherein the viable bacterial concentration of the bacterial liquid in the fermentation broth of Bacillus amyloliquefaciens 1LN2 is 1X 10 ⁵ ～1×10 ¹⁰ CFU/ml。

4. Use of bacillus amyloliquefaciens according to claim 1 or the biological agent according to any one of claims 2-3 for inhibiting phytophthora, fusarium graminearum, sclerotinia capsici, maize bipolaris or phoma asparagi.

5. The use according to claim 4 for the inhibition of phytophthora.

6. Use of bacillus amyloliquefaciens according to claim 1 or the biological agent according to any one of claims 2-3 for controlling pepper epidemic disease and promoting pepper growth.

7. The use according to claim 6, for promoting an increase in stem thickness, leaf number, plant height and bud number.