CN113801804A

CN113801804A - Banana wilt biocontrol antagonistic strain and application thereof

Info

Publication number: CN113801804A
Application number: CN202110312947.6A
Authority: CN
Inventors: 何平; 杨宝明; 郑泗军; 黄玉林; 李永平; 李舒; 徐胜涛; 刘立娜
Original assignee: Institute of Agricultural Environment and Resources of Yunnan Academy of Agricultural Sciences
Current assignee: Institute of Agricultural Environment and Resources of Yunnan Academy of Agricultural Sciences
Priority date: 2021-03-24
Filing date: 2021-03-24
Publication date: 2021-12-17
Anticipated expiration: 2041-03-24
Also published as: CN113801804B

Abstract

The invention discloses a banana vascular wilt biocontrol antagonistic strain and application thereof, wherein the strain is banana vascular wilt biocontrol antagonistic strain; the collection name is Bacillus velezensis; is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation address is No. 3 of Xilu No. 1 of Beijing, Chaoyang district; the preservation date is as follows: year 2020, 11, 16; the preservation number is CGMCC NO. 21190. Two banana symbiotic bacteria are separated from a banana tissue culture seedling, and a plate confronting experiment shows that one of the two banana symbiotic bacteria has an obvious inhibition effect on No. 4 physiological race tropical type (TR4) of banana wilt bacteria, the inhibition rate is up to 79.63%, and the bacterium is identified to be a Bacillus belgii strain named YN 1282-2. Pot experiments also show that the strain has obvious effects of promoting growth of bananas and preventing and treating blight.

Description

Banana wilt biocontrol antagonistic strain and application thereof

Technical Field

The invention relates to the technical field of biofertilizers, and particularly relates to a bio-antagonistic strain for preventing banana wilt and application thereof.

Background

Bananas (Musa spp.) are important tropical and subtropical crops worldwide, not only the second largest and first traded fruit crops in the world, but also the fourth largest food crops following rice, wheat and corn. China is a big banana planting country, is mainly planted in provinces (regions) such as Hainan, Guangdong, Guangxi, Fujian, Yunnan and the like, and the scale of the banana industry is huge. Banana wilt disease caused by Fusarium oxysporum cubeba specialized type (Fusarium oxysporum f.sp. cubense, Foc) physiological races 1 (Race 1), 2 (Race 2) and 4 (Race 4), especially the 4 physiological Race Tropical type (Tropical Race 4: TR4), is the most serious destructive disease in banana producing areas in China. At present, no commercial banana varieties with high resistance and complete immunity to banana vascular wilt exist, and no ideal chemical pesticide can be used for preventing and treating banana vascular wilt. Therefore, the development of biological prevention and control of banana vascular wilt, screening of biocontrol strains with inhibition effect on banana vascular wilt and development of corresponding biocontrol microbial inoculum research are of great significance.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a biological control antagonistic strain for banana wilt with high bacteriostatic rate and application thereof.

The technical scheme of the invention is as follows: the invention provides the following technical scheme: the invention relates to a bio-control antagonistic strain for banana vascular wilt, which is a bio-control antagonistic strain for banana vascular wilt; the collection name is Bacillus velezensis; is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation address is No. 3 of Xilu No. 1 of Beijing, Chaoyang district; the preservation date is as follows: year 2020, 11, 16; the preservation number is CGMCC NO. 21190.

Further, the Bacillus velezensis strain is a biological control antagonistic strain for banana vascular wilt.

Further, the strain is subjected to streak culture on an isolated strain on an NA solid medium and cultured for 24 hours at 37 ℃, and a colony on an NA plate is circular and opaque and slightly convex in the middle; the front surface is milky white, the surface is not smooth and has wrinkles, and the back surface is milky white and is smooth and moist; gram staining reaction is positive, and the shape of the bacteria is rod-shaped and the length is about 1-2 mu m when the bacteria are observed by an electron microscope.

The banana wilt biocontrol antagonistic bacterium agent prepared by the banana wilt biocontrol antagonistic strain is provided.

The active ingredients of the biological antagonistic bacterium agent for controlling banana wilt are at least one of the following components (a), (b) and (c):

(a) the fermentation culture of the banana vascular wilt biocontrol antagonistic bacteria;

(b) the ultrasonic cracking supernatant of the banana wilt disease growth-prevention antagonistic bacteria cells;

(c) the ultrasonic cracking precipitation of the banana wilt disease growth-prevention antagonistic bacteria cells.

The preparation method of the biological anti-antagonistic bacterial agent for banana wilt comprises the following steps: inoculating the antagonistic strain into 200mL of LB liquid culture medium, carrying out shaking culture at 35-37 ℃ and 220r/min for 16-18h, taking bacterial liquid, centrifuging at 12000r/min for 5-10min, collecting the thallus, washing with sterilized water for 1-2 times, and then re-suspending the thallus with 200mL of sterilized water to obtain the biological control antagonistic bacterial agent for banana vascular wilt.

The invention relates to application of a biological control antagonistic bacterial strain for banana vascular wilt in preparation of a preparation for banana vascular wilt.

Has the advantages that: two banana symbiotic bacteria are separated from a banana tissue culture seedling, and a plate confronting experiment shows that one of the banana symbiotic bacteria has a remarkable inhibition effect on banana fusarium wilt (TR4) with the inhibition rate up to 79.63%, and the bacterium is identified to be a Bacillus beleisi strain named YN 1282-2. Pot experiments also show that the strain has obvious effects of promoting growth of bananas and preventing and treating blight.

Drawings

FIG. 1 is a morphology chart of isolated strain plates of the present invention, a-b, growth of strains in tissue culture bottles; c-d, growth of NA plate strains; e, observing the form of the scanning electron microscope;

FIG. 2 is a control diagram of the culture of TR4 alone according to the present invention; the bacteria showed significant inhibition of TR4 (FIG. 2c, d), and the hyphae of the pathogenic bacteria were lysed and expanded to a distorted state, whereas the control hyphae not antagonized by YN1282-2 did not (FIG. 2 e).

FIG. 3 is a diagram of the construction of the YN1282-2 phylogenetic tree of the present invention.

FIG. 4 shows that the result of NCBI Blast alignment of the sequencing sequence of the invention is YN1282-2 which is a strain of Bacillus velezensis.

FIG. 5 is a schematic diagram of dhbC and sboA of the present invention amplified to a desired band;

FIG. 6 is a diagram showing the control effect of YN1282-2 on banana vascular wilt potting of No. 1 (T27) banana.

FIG. 7 shows the control effect of YN1282-2 of the present invention on banana wilt potting in Brazil (Brazilian).

Detailed Description

The invention will now be described in further detail by means of the figures and the examples, which should not be construed as limiting the scope of the invention.

Example 1

The invention relates to a bio-control antagonistic strain for banana vascular wilt, which is a bio-control antagonistic strain for banana vascular wilt; the collection name is Bacillus velezensis; is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation address is No. 3 of Xilu No. 1 of Beijing, Chaoyang district; the preservation date is as follows: year 2020, 11, 16; the preservation number is CGMCC NO. 21190.

The Bacillus belgii Bacillus velezensis strain is a biological control antagonistic strain for banana vascular wilt.

The strain is subjected to streak culture on an NA solid culture medium, the strain is cultured for 24 hours at 37 ℃, and a bacterial colony on an NA flat plate is circular, non-transparent and slightly convex in the middle; the front surface is milky white, the surface is not smooth and has wrinkles, and the back surface is milky white and is smooth and moist; gram staining reaction is positive, and the shape of the bacteria is rod-shaped and the length is about 1.5 mu m when the bacteria are observed by an electron microscope.

The preparation method of the biological anti-antagonistic bacterial agent for banana wilt comprises the following steps: inoculating the antagonistic strain into 200mL of LB liquid culture medium, performing shaking culture at 37 ℃ and 200r/min for 18h, taking bacterial liquid, centrifuging at 12000r/min for 5min, collecting the thallus, washing with sterilized water for 3 times, and re-suspending the thallus with 200mL of sterilized water to obtain the bio-control antagonistic bacterial agent for banana vascular wilt.

Example 2

Example 2 differs from example 1 in that: the strain is subjected to streak culture on an NA solid culture medium, the strain is cultured for 24 hours at 37 ℃, and a bacterial colony on an NA flat plate is circular, non-transparent and slightly convex in the middle; the front surface is milky white, the surface is not smooth and has wrinkles, and the back surface is milky white and is smooth and moist; gram staining reaction is positive, and the shape of the bacteria is rod-shaped and the length is about 1 mu m when the bacteria are observed by an electron microscope.

The preparation method of the biological anti-antagonistic bacterial agent for banana wilt comprises the following steps: inoculating the antagonistic strain into 200mL of LB liquid culture medium, carrying out shaking culture at 35 ℃ and 180r/min for 16h, taking bacterial liquid, centrifuging at 12000r/min for 10min, collecting thalli, washing with sterilized water for 1 time, and suspending the thalli again with 200mL of sterilized water to obtain the bio-control antagonistic bacterial agent for banana vascular wilt.

Example 3

Example 3 differs from example 1 in that: the strain is subjected to streak culture on an NA solid culture medium, the strain is cultured for 24 hours at 37 ℃, and a bacterial colony on an NA flat plate is circular, non-transparent and slightly convex in the middle; the front surface is milky white, the surface is not smooth and has wrinkles, and the back surface is milky white and is smooth and moist; gram staining reaction is positive, and the shape of the bacteria is rod-shaped and the length is about 2 mu m when the bacteria are observed by an electron microscope.

The preparation method of the biological anti-antagonistic bacterial agent for banana wilt comprises the following steps: inoculating the antagonistic strain into 200mL of LB liquid culture medium, carrying out shaking culture at 36 ℃ and 220r/min for 17h, taking bacterial liquid, centrifuging at 12000r/min for 8min, collecting the thallus, washing with sterilized water for 2 times, and then resuspending the thallus with 200mL of sterilized water to obtain the bio-control antagonistic bacterial agent for banana vascular wilt.

Test example 1

The present invention will be described in further detail with reference to examples. The microbial fertilizer in the examples is prepared by a conventional microbial fermentation method and a conventional microbial fertilizer preparation method.

1 materials of the experiment

1.1 strains

(1) Bacillus YN1282-2 Strain: the banana endophyte separated and obtained in the laboratory is preserved in a refrigerator at the temperature of minus 80 ℃ in glycerol for later use.

(2) Banana fusarium wilt: TR4 (physiological race tropical type No. 4) (Fusarium oxysporum f.sp. cubense, Foc) strain 15-1, isolated and preserved in Xishuangbanna olive dam banana strain in 2015 this laboratory.

1.2 culture Medium

(1) LB liquid medium: dissolving 10g of tryptone, 5g of yeast extract and 10g of NaCl, adjusting the pH value to 7.0 by using 5mol/LNaOH, and fixing the volume to 1L by using deionized water.

(2) NA solid medium: 10g of peptone, 3g of beef extract, 5g of NaCl and 15-20g of agar, and metering to 1L.

(3) PDA culture medium: 200g of potato, 20g of glucose and 15-20g of agar, wherein the volume is constant to 1L, the pH value is natural, and the PDB liquid culture medium is obtained without adding the agar.

2 method of experiment

2.1 morphological identification of Strain YN1282-2

The separated strain is subjected to streak culture on an NA solid medium, is cultured for 24 hours at 37 ℃, is observed and recorded with colony characteristics such as colony morphology, transparency and colony color, and is observed under an optical microscope. And (3) scanning and observing by a cryoelectron microscope: selecting a single bacterial colony of the strain, inoculating the single bacterial colony into an LB test tube, carrying out shaking culture at 37 ℃ and 180rpm for 16h, taking out, centrifuging at 12000rpm, collecting the bacterial colony, washing the bacterial colony with sterile distilled water, then resuspending the bacterial colony, sucking 2-3 mu L of bacterial colony, placing the bacterial colony on a gold spraying plate, then rapidly placing the bacterial colony in liquid nitrogen refrigerating fluid, placing the bacterial colony in a freezing chamber for sublimation and gold spraying, and observing the shape and the form of the bacteria under a scanning electron microscope.

2.2 determination of antagonistic Activity of Strain YN1282-2

Using the point inoculation method, the ability of the isolates to inhibit TR4 on PDA plates was determined. Specific procedure a target pathogenic filament (TR4) (5 mm in diameter) was placed in the center of a PDA plate. Isolated bacteria were inoculated at four symmetrical points. The distance from the centre of the plate was 25 mm. Five replicates were performed. Plates inoculated with pathogenic bacteria alone were used as controls. The diameter of the pathogenic bacteria was measured by the cross method after culturing at 28 ℃ for 5 to 7 days. The zone of inhibition and the percentage inhibition of fungal Growth (GI) were calculated according to the formula: percent inhibition of growth ═ [ (C-T)/C ] × 100%, where C and T are the diameters of fungal hyphal growth in control and treated plates, respectively. Meanwhile, TR4 hyphae after YN1282-2 antagonism is placed in a frozen electron microscope for scanning observation, the hyphae are lightly picked and placed on a metal spraying plate, then the hyphae are rapidly placed in liquid nitrogen refrigerating fluid, the liquid nitrogen refrigerating fluid is placed in a freezing chamber for sublimation gold spraying, and the shape and the form of the fungus TR4 are observed under a scanning electron microscope.

2.3 measurement of phosphate solubilizing function of Strain YN1282-2

Inoculating Bacillus belgii YN1282-2 to NA solid culture medium by scribing method for activation, and culturing at 37 deg.C for 24 h. And (3) selecting activated Bacillus belgii YN1282-2 single colonies with autoclaved toothpicks, inoculating the single colonies on an organophosphorus culture medium plate, inoculating 3 colonies on each plate, and setting 3 groups of parallel tests. The inoculated plate was incubated at 37 ℃ for 7 days, and the size of the transparent circle was measured by cross-hatch assay. Selecting a bacterial colony with a phosphate solubilizing ring, measuring the diameter D of the bacterial colony and the diameter D of the phosphate solubilizing ring, calculating the ratio of D/D, and determining the phosphate solubilizing capacity intensity.

2.4 physiological and biochemical identification of Strain YN1282-2

The physiological and biochemical measurements of the bacteria were carried out using the Biolog microplate method according to the Biolog Gen III microplate operating manual. The specific operation is that pure cultured strains are inoculated to a BUG agar culture medium and cultured at 33 ℃; then using an Inoculatorz cotton swab to dip colonies from the flat plate, then leading the tail end of the cotton swab to penetrate into the bottom end of the inoculation tube filled with inoculation liquid, shaking the bacteria up and down to release the bacteria into the inoculation liquid, stirring uniformly to obtain cell suspension, measuring by using a turbidity meter and adjusting to the required concentration; the bacterial suspension was then added to the assay plate and capped, and after 24h and 48h, respectively, the plate was placed in a Biolog reader for assay.

2.5 molecular characterization of Strain YN1282-2

Inoculating the antagonistic strain into an NA liquid culture medium, performing shaking culture at 37 ℃ and 180r/min for 18h, centrifuging 1mL of bacterial liquid at 12000r/min for 1min, collecting thalli, and extracting DNA of the antagonistic bacterium by using an Ezup column type bacterial genome DNA extraction kit (Bio-engineering, Inc.). The PCR amplification primers were 27F/1429R (5 'AGAGAGTTTGATCCTGGCTCAG 3'/5 'CTACGGCTACCTTGTTACGA 3'), ITS1/ITS4(5 'TCCGTAGGTGAACCTGCGC 3'/5 'TCCTCCGCTTATTGATATGC 3'). The amplification conditions were 95 ℃ for 5min, 95 ℃ for 30s, 58 ℃ for 30s, 72 ℃ for 1min for 30s, 72 ℃ for 7min, 35 cycles. After the reaction, 3ul of PCR product was subjected to 1% agarose gel electrophoresis to confirm the PCR amplified fragment. The PCR product was recovered using AxyPrep DNA gel recovery kit, and the specific procedures were performed according to the kit instructions. And taking the PCR product after each strain purification, and carrying out DNA sequencing by using a sequencer ABI 3730-XL. And comparing the spliced sequence file with data in an NCBI 16S database by using an NCBI Blast program to obtain species information with the maximum similarity with the sequence of the species to be detected, and constructing a phylogenetic tree by using MEGA6.0, namely an identification result.

2.6 biocontrol gene detection of Strain YN1282-2

Taking out the strain Bacillus belgii YN1282-2 preserved on the inclined plane, activating on a NA culture medium plate, picking out a single colony by using a sterilized toothpick, uniformly mixing the single colony in 50 mu L of lysine Buffer, then placing the mixture in a water bath at 80 ℃ for cracking for 15 minutes, centrifuging at low speed for 1min, and taking the supernatant as a DNA template. YN1282-2 was PCR-amplified using 9 primers in Table 1, respectively, in an amplification system (25. mu.L): 22 mu L of Mix enzyme, 1 mu L of template, 1 mu L of upstream primer and 1 mu L of downstream primer, and the amplification condition is 98 ℃ for 2 min; 10s at 98 ℃; 15s at 54 ℃ (primers 147F/147R, FEDF1/FNDR1, bamC2F/bamC2R) or 15s at 57 ℃ (primers 110F/110R) or 15s at 51 ℃ (primers ITUCF1/ITUCR3) or 15s at 56 ℃ (primers ituD2F/ituD2R) or 15s at 58 ℃ (primers dhbCf/dhbCr, Sbo1F/Sbo1R) or 15s at 55 ℃ (primers ysnEf/ysnEr); 35 cycles; 10s at 72 ℃; the amplification products were finally detected using 1% agarose gel electrophoresis at 72 ℃ for 5min.

TABLE 1 primer Gene sequences for PCR amplification

2.7 prevention and treatment Effect of Strain YN1282-2 potting experiment

In order to further discuss the prevention and control effect and action mechanism of the Bacillus belief YN1282-2 on the banana vascular wilt and provide theoretical support for the biological prevention and control of the banana vascular wilt. The specific operation is that the antagonistic strain is inoculated in 200mL LB liquid culture medium, shaking culture and fermentation are carried out at 37 ℃ and 220r/min for 48h, bacterial liquid is taken and centrifuged at 12000r/min for 10min, fermentation liquid is collected to prepare the bio-control antagonistic bacterial agent for banana vascular wilt, 20mL of fermentation stock solution is taken and added with 180mL of sterilized water to obtain 200mL of fermented 10-time dilution. Each pot was inoculated with 40mL of fermentation broth, and the same volume of blank medium was inoculated as a blank control.

Inoculating TR4 mycelia into PDB culture medium, performing shaking culture at 28 deg.C and 180r/min for 72 hr, centrifuging bacterial liquid at 12000r/min for 10min, collecting thallus, and resuspending thallus with 200mL sterile water (1 × 10)⁸spoore/mL), and diluting the bacterial liquid by 100 times to obtain the inoculation liquid (1 × 10) of the banana fusarium wilt⁶Spore/mL). Each pot was inoculated with 40mL of the bacterial suspension, and the same volume of sterilized water was used as a blank.

This research is carried out the experiment of potting through setting up different inoculation and handling (table 2), explores YN1282-2 and prevents and control the effect to the banana wilt disease of resistant variety banana No. 1 and susceptible variety Brazil banana, YN1282-2 potted plant experimental processing is shown as table 2:

TABLE 2YN1282-2 Pot culture test treatment

3 results of the experiment

3.1 isolation and morphological characterization of Strain YN1282-2

Finding a banana symbiotic bacterium on the banana tissue culture seedlings, separating the banana symbiotic bacterium to a flat plate, and then naming the banana symbiotic bacterium as YN1282-2, wherein a bacterial colony on an NA flat plate is circular and opaque, and the middle of the bacterial colony is slightly convex; the front surface is milky white, the surface is not smooth and has wrinkles, and the back surface is milky white and is smooth and moist; gram staining reaction is positive, and the shape of the bacteria is rod-shaped and the length is about 1-2 mu m when the bacteria are observed by an electron microscope.

FIG. 1 is a morphology chart of isolated strain plates, a-b, growth of strains in a tissue culture bottle; c-d, growth of NA plate strains; e, scanning electron microscopy morphological observation, Figure 1.Plate morphology of isolated strain.a-b, the growth of isolated strain in the tissue culture lattice; c-d, the growing of isolated strain in NAplate. e, morphology under scanning electronic microscope.

3.2 determination of antagonistic Activity of Strain YN1282-2

The isolated bacteria were subjected to a TR4 challenge test, inoculated with both bacteria and pathogenic bacteria TR4, and cultured for 7 days to observe the growth of the pathogenic bacteria, and as a result, the bacteria showed significant inhibitory effects on TR4 (fig. 2a, b), and the hyphae of the pathogenic bacteria were lysed and expanded and distorted (fig. 2c, d), while the control hyphae not antagonized by YN1282-2 did not show such changes (fig. e). The bacteriostasis rate is as high as 79.63% (table 3). And enabling the isolated strain to antagonize a panel facing TR4, a, YN1282-2 and a panel facing TR 4; b, TR4 culture alone control is shown in FIG. 2, and strain YN1282-2 plate inhibition is shown in Table 3:

TABLE 3 antibacterial effect of plate confrontation of YN1282-2

3.3 measurement of phosphate solubilizing function of Strain YN1282-2

YN1282-2 is used after being cultured on an organophosphorus culture medium, and the diameters D (cm) of phosphate-solubilizing rings in3 groups of parallel experiments are respectively as follows: 1.55, 1.40, 1.43, wherein the colony diameters d (cm) are respectively: 1.08, 0.90. The D/D ratios are calculated as follows: 1.43, 1.56, 1.59 (fig. 3).

3.4 molecular characterization of YN1282-2

DNA was extracted from YN1282-2, and PCR amplification sequencing was performed to obtain a 16s rRNA gene fragment of 1420bp (GenBank accession No.: MW663765) in the strain YN 1282-2. The sequencing sequence was compared by NCBI Blast to find that YN1282-2 was a strain of Bacillus velezensis (FIG. 4). YN1282-2 phylogenetic Tree construction is shown in FIG. 3:

3.5 physiological and biochemical assays for YN1282-2

YN1282-2 assay was performed using Biolog Gen III microplates and showed that YN1282-2 was a gram positive bacterium and other biochemical assays are shown in Table 4:

TABLE 4YN1282-2 Biolog Gen III assay

3.6 identification of biocontrol gene for YN1282-2

The biocontrol/growth promotion gene detection is carried out on the strain YN1282-2, and the PCR detection result of 9 biocontrol genes detected by PCR shows that target bands of srfAB, yndJ, fenD, ituC, ituD, bamC and ysnE 7 genes are amplified in the strain YN1282-2, dhbC and sboA are amplified to the target bands, and no band exists in negative control (figure 5). Wherein, 6 genes of the amplified genes, namely srfAB, yndJ, fenD, ituC, ituD and bamC, have the functions of bacteriostasis and antagonism, and the ysnE gene has the function of growth promotion, which indicates that the strain has good biocontrol potential.

3.7YN1282-2 prevention and treatment effects on banana vascular wilt

3.7.1YN1282-2 control effect on banana wilt disease of banana No. 1

In order to further explore whether the Bacillus belgii YN1282-2 has a good prevention and control effect on banana wilt and has a growth promoting effect on bananas, a greenhouse pot experiment is carried out. The results show that: 30 days after the banana No. 1 (T27) is inoculated with TR4, the plant height of a banana plant inoculated with YN1282-2 fermentation stock solution is obviously higher than the treatment of inoculating YN1282-2 diluted 10 times fermentation solution, and is higher than a control without inoculation of biocontrol bacteria but the difference is not obvious (fig. 6a, f); whereas the pseudostem thickness of banana plants inoculated with YN1282-2 fermentation stock was significantly higher than the treatment and control diluted 10 times (FIG. 6 b); no significant change in the number of leaves (fig. 6 c); as can be seen from the disease index survey condition, the disease indexes of the banana plants inoculated with the biocontrol bacteria are all lower than those of the control, and the disease index of the banana plant inoculated with the YN1282-2 fermentation stock solution is lower than that of the banana plant inoculated with the biocontrol bacteria treated by the diluent of 10 times; the preventing and treating effect of YN1282-2 fermentation stock solution is 69.23%, and the preventing and treating effect of YN1282-2 diluted 10 times fermentation liquor is 46.15% (fig. 6d, e. table 5). In the treatment without inoculation of TR4, the plant height of the treatment with inoculated biocontrol bacteria was significantly higher than that of the control without inoculation of biocontrol bacteria (FIG. 6a, e, f); the pseudostem thickness of banana plants inoculated with YN1282-2 fermentation stock was significantly higher than the 10-fold diluted treatments and controls (FIG. 6 b); there was also no significant difference in leaf number growth (fig. 6 c). Therefore, the Bacillus belgii YN1282-2 can effectively inhibit the occurrence of canna number 1 blight and has a certain growth promoting effect on the canna number 1 plants.

FIG. 6 shows the control effect of YN1282-2 on banana vascular wilt potting of No. 1 (T27) banana. a. The height growth of each treated plant; b. (ii) pseudostem girth growth status of each treatment; c. growth of the leaves for each treatment; d. leaf disease index; e. growth of three treated plants inoculated with TR 4; f. three treated plants not inoculated with TR4 grew n-12.

3.7.2YN1282-2 control Effect on Banana banana wilt disease

The same potting experiment is carried out on the Brazil banana, and the result shows that the plant height of the banana plant inoculated with YN1282-2 fermentation stock solution is obviously higher than that of a control without inoculation of biological control bacteria 30 days after the inoculation of TR4, and the plant height is not obviously different from that of the treatment of inoculating YN1282-2 diluted 10 times fermentation solution (figures 7a and e); whereas there was no significant difference in pseudostem thickness and leaf number of banana plants (fig. 7b, c); as can be seen from the disease index survey condition, the disease indexes of the banana plants inoculated with the biocontrol bacteria are all obviously lower than those of the control, and the disease index of the banana plant inoculated with the YN1282-2 fermentation stock solution is obviously lower than that of the banana plant inoculated with the biocontrol bacteria treated by the dilution solution by 10 times; the preventing and treating effect of YN1282-2 fermentation stock solution is as high as 80.95%, and the preventing and treating effect of YN1282-2 diluted 10 times fermentation liquor is 61.90% (fig. 7d, e. table 6). In the treatment without inoculating TR4, the plant height of the banana plant inoculated with YN1282-2 fermentation stock solution is obviously higher than that of the banana plant inoculated with YN1282-2 diluted 10 times fermentation solution, and has no obvious difference with the control without inoculating biocontrol bacteria (figure 7a, e, f); there was no significant difference in pseudostem thickness and leaf number growth (fig. 7b, c). It can be seen that Bacillus bleekii YN1282-2 can effectively inhibit the occurrence of Brazil banana wilt and also has a certain growth promoting effect.

FIG. 7 shows the control effect of YN1282-2 of the present invention on banana wilt potting in Brazil (Brazilian). a, the height growth condition of each treated plant; b. (ii) pseudostem girth growth status of each treatment; c. growth of the leaves for each treatment; d. leaf disease index; e. growth and root cutting of three treated plants inoculated with TR 4; f, growth and root cutting of three treated plants not grafted with TR 4; n is 12.

4. Discussion of the related Art

The research on the biological control of banana vascular wilt has more reports, and the control of banana vascular wilt by using antagonistic bacteria is one of the main measures adopted at present, but many biocontrol bacteria are easily influenced by ambient environmental conditions and are not easy to colonize in the environment where pathogens exist, so that the control effect of the biocontrol bacteria is seriously influenced. Two banana symbiotic bacteria are separated from a banana tissue culture seedling, a plate confronting experiment shows that one of the banana symbiotic bacteria has an obvious inhibition effect on banana wilt bacteria (TR4), and the bacteria is identified as a Bacillus velezensis strain through subsequent identification. Pot experiments show that the strain has good prevention and treatment effect on banana wilt and also has obvious growth promotion effect on bananas. This is consistent with many reports on studies with banana wilt-resistant bacteria.

The experiment sets two biocontrol bacteria inoculation concentrations, the result shows that the diluted bacteria liquid has no good effect of fermentation stock solution on the control effect and the growth promotion effect of the banana vascular wilt, which may show that the biocontrol bacteria can enter the soil and can colonize and play a role in a large amount only when reaching a certain concentration, the bacteria liquid with low concentration may cause the biocontrol bacteria not to compete with the indigenous microorganisms to resist the banana vascular wilt, but the result shows that the effect of the bacteria liquid with low concentration is still obviously better than that of the control, which indicates that the growth and infection of pathogenic bacteria can be inhibited to a certain extent as long as a certain amount of biocontrol bacteria exist in the soil.

The whole attack of the banana plants in the experiment is not very serious, probably because the inoculation mode of the pathogenic bacteria is root irrigation inoculation, the roots are not injured, and the infection of the pathogenic bacteria is difficult to enter through wounds. However, the disease condition of the inoculated and non-inoculated treated plants is obviously contrasted, so that the effect of prolonging the infection time after inoculation is probably more obvious.

5. Conclusion

In the research, a banana symbiotic bacterium is separated from a banana tissue culture seedling, a plate confrontation experiment shows that one of the banana symbiotic bacteria has an obvious inhibition effect on banana wilt bacteria (TR4), and the bacterium is identified as a Bacillus velezensis strain through subsequent identification. Pot experiments show that the strain has good prevention and treatment effect on banana wilt and also has obvious growth promotion effect on bananas.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the foregoing description only for the purpose of illustrating the principles of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims, specification, and equivalents thereof.

Claims

1. A banana wilt biocontrol antagonistic strain is characterized in that: the strain is a banana vascular wilt biocontrol antagonistic strain YN 1282-2; the collection name is Bacillus velezensis; is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation address is No. 3 of Xilu No. 1 of Beijing, Chaoyang district; the preservation date is as follows: year 2020, 11, 16; the preservation number is CGMCC NO. 21190.

2. The biocontrol antagonist strain of banana vascular wilt disease according to claim 1, characterized in that: the Bacillus belgii Bacillus velezensis strain is a biological control antagonistic strain for banana vascular wilt.

3. The banana wilt biocontrol antagonist strain of claim 2, wherein: the strain is subjected to streak culture on an NA solid culture medium, the strain is cultured for 24 hours at 37 ℃, and a bacterial colony on an NA flat plate is circular, non-transparent and slightly convex in the middle; the front surface is milky white, the surface is not smooth and has wrinkles, and the back surface is milky white and is smooth and moist; gram staining reaction is positive, and the shape of the bacteria is rod-shaped and the length is about 1-2 mu m when the bacteria are observed by an electron microscope.

4. The banana vascular wilt biocontrol antagonistic bacterial agent prepared by the banana vascular wilt biocontrol antagonistic bacterial strain as described in claim 1.

5. The biocontrol antagonistic bacterial agent for banana vascular wilt according to claim 4, wherein the active ingredient is at least one of the following (a), (b) and (c):

(a) a fermentation culture of the banana vascular wilt biocontrol antagonist bacteria of claim 1;

(b) an ultrasonically lysed supernatant of the banana wilt growth prevention antagonist bacterial cells of claim 1;

(c) the ultrasonically lysed precipitate of banana wilt growth prevention antagonist bacterial cells of claim 1.

6. The method for preparing the bio-control antagonistic bacteria agent for banana vascular wilt as claimed in claim 1, which is characterized by comprising the following steps: inoculating the antagonistic strain into 200mL of LB liquid culture medium, carrying out shaking culture at 35-37 ℃ and 220r/min for 16-18h, taking bacterial liquid, centrifuging at 12000r/min for 5-10min, collecting the thallus, washing with sterilized water for 1-2 times, and then re-suspending the thallus with 200mL of sterilized water to obtain the biological control antagonistic bacterial agent for banana vascular wilt.

7. Use of the banana vascular wilt biocontrol antagonist strain of any one of claims 1 to 6 in the preparation of a banana vascular wilt formulation.