CN113025522B

CN113025522B - Bacillus amyloliquefaciens, application thereof and method for preventing and/or treating banana vascular wilt

Info

Publication number: CN113025522B
Application number: CN202110281749.8A
Authority: CN
Inventors: 番华彩; 曾莉; 李舒; 徐胜涛; 郑泗军; 何平; 白亭亭; 尹可锁; 郭志祥; 李迅东; 刘立娜; 尚慧
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: 2021-03-16
Filing date: 2021-03-16
Publication date: 2022-03-22
Anticipated expiration: 2041-03-16
Also published as: CN113025522A

Abstract

The invention relates to the field of biological control and discloses a bacillus amyloliquefaciens strain, application thereof and a method for preventing and/or treating banana vascular wilt. The bacillus amyloliquefaciens provided by the invention has good inhibition effect on fusarium oxysporum f.sp.oxysporum f.sp.cubense special type 4 physiological race tropical type (Foc TR4), and shows excellent prevention and control effect on banana vascular wilt in pot experiments. Meanwhile, the strain also has good phosphate and potassium dissolving performance, and has good growth promoting effect on banana plants. The strain can be used for preventing and treating banana wilt, can solve the problem of yield reduction caused by lack of phosphorus and potassium elements in the banana planting process, and improves the economic benefit of banana planting production.

Description

Bacillus amyloliquefaciens, application thereof and method for preventing and/or treating banana vascular wilt

Technical Field

The invention relates to the field of biological control, and particularly relates to bacillus amyloliquefaciens and application thereof as well as a method for preventing and/or treating banana vascular wilt.

Background

Banana vascular wilt (Fusarium wilt of banana), also known as panama disease, yellow leaf disease and the like, is a typical destructive soil-borne fungal disease and seriously affects the planting and production of crops such as bananas and the like. Pathogenic bacteria causing banana wilt disease are Fusarium oxysporum cubeba specialization (Fusarium oxysporum f.sp. cubense), and No. 4 physiological race tropical type (Foc TR 4).

The commonly used banana vascular wilt prevention and treatment methods at present comprise chemical prevention and treatment, breeding for disease resistance, adopting proper agricultural measures (such as adjusting cultivation modes and the like) and the like, but the prevention and treatment effects of the methods are very limited and have very obvious limitations. For example, the chemical control method mainly applies chemical agents in the planting process to achieve the control purpose, but the chemical agents not only can bring environmental and soil pollution and cause food safety problems, but also can enhance the drug resistance of pathogenic bacteria after long-term large-scale application of the chemical agents, so that the effect of the chemical control method is not ideal. Although the problems caused by the application of a large amount of chemical agents can be avoided by cultivating disease-resistant banana plants and adjusting the farming mode, the breeding time of the disease-resistant banana plants is long, the early-stage scientific research investment is large, the uncertainty is high, and the adjustment of the farming mode can cause the problems of the increase of the planting cost of farmers and the like.

Biological control is a generally accepted safer and more effective agricultural disease control measure at present, mainly utilizes biological resources such as biocontrol bacteria and the like to control agricultural diseases, and the biocontrol bacteria can generate antibacterial substances and have the characteristics of inducing system resistance and the like, can effectively inhibit the invasion of pathogenic bacteria and improve the resistance of plants. And some biocontrol bacteria also have the growth promoting effect, so that the agricultural production efficiency and yield are improved while crop diseases are prevented and treated, and the agricultural economic benefit is improved. However, the prevention and control effect of the biocontrol strain developed aiming at the banana wilt at present needs to be improved, and the growth promotion effect on plants is weak, so that the biocontrol strain cannot meet the requirements of modern large-scale agricultural production and agricultural economic development.

Disclosure of Invention

The invention aims to solve the problems of poor biocontrol bacteria prevention and control effect and weak growth promotion effect on banana vascular wilt prevention and control in the prior art, and provides a bacillus amyloliquefaciens strain which has good prevention and control effect on banana vascular wilt, has good potassium and phosphorus (especially organic phosphorus) dissolving effects, and can show excellent growth promotion effect while preventing and controlling banana vascular wilt.

In order to achieve the purpose, the invention provides a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) strain, wherein the preservation number of the Bacillus amyloliquefaciens is CGMCC NO. 21189.

In a second aspect, the present invention provides the use of a strain as described above for inhibiting fungi.

In a third aspect, the invention provides the use of the strain as described above for potassium and/or phosphorus solubilization.

In a fourth aspect, the present invention provides a method for preventing and/or treating banana vascular wilt, the method comprising: applying a microbial inoculum containing bacillus amyloliquefaciens to soil and/or banana plants infected by pathogenic bacteria of banana wilt;

alternatively, the metabolites of bacillus amyloliquefaciens are applied to soil and/or banana plants infected with pathogenic bacteria of banana wilt;

wherein the Bacillus amyloliquefaciens has a preservation number of CGMCC NO. 21189.

Through the technical scheme, the invention has the following beneficial effects:

(1) the bacillus amyloliquefaciens provided by the invention can effectively inhibit fusarium oxysporum cubense special type 4 physiological race tropical type, has good prevention and treatment effects on banana wilt, has good effects of decomposing potassium and organic phosphorus, has good growth promotion effect on banana plants, and is a high-quality biocontrol strain with dual functions of prevention and treatment effects and growth promotion effects;

(2) the bacillus amyloliquefaciens provided by the invention is separated from banana plants, belongs to banana endophytes, and cannot generate adverse effects on the banana plants and pollution to soil of a banana plantation after application. Moreover, the unique and good potassium and organic phosphorus dissolving effects can also improve some soil problems caused by abuse residues of chemical fertilizers, solve the problem of yield reduction caused by lack of phosphorus and potassium elements in the planting process of bananas, and improve the stress resistance and yield of bananas;

(3) the method provided by the invention is simple and easy to implement, low in cost, good in prevention and treatment effect and suitable for large-scale popularization and application.

Drawings

FIG. 1 shows the colony morphology of strain YN0904 isolated in example 1 of the present invention;

FIG. 2 shows the morphology of strain YN0904 isolated in example 1 of the present invention under scanning electron microscope;

FIG. 3 is a 16S rDNA phylogenetic tree of the strain YN0904, which is drawn in example 1 of the present invention;

FIG. 4 shows the inhibitory effect of strain YN0904 on the tropical type of Fusarium oxysporum strain 4 on the special type of Guba in example 2 of the present invention;

FIG. 5 shows the inhibitory effect of strain YN0904 on the mycelia of Fusarium oxysporum strain Cola 4 on the tropical type of physiological race in example 2 of the present invention;

FIG. 6 is a gel electrophoresis image of PCR products obtained by amplifying 6 kinds of antagonistic genes and 1 growth promoting gene using genomic DNA of YN0904 strain as a template in example 2 of the present invention;

FIG. 7 shows the phosphate solubilizing (left side) and potassium solubilizing (right side) effects of the strain YN0904 in example 3 of the present invention;

FIG. 8 shows the control effect (leaves) of bacterial strain YN0904 on banana wilt of potted banana plants in example 4 of the present invention;

FIG. 9 shows the control effect (corms) of bacterial strain YN0904 on banana wilt of potted banana plants in example 4 of the present invention;

FIG. 10 shows the growth promoting effect of strain YN0904 on banana potted plants in example 4 of the present invention.

Biological preservation

The inventor of the invention separates a strain YN0904 from a Musa lasiocarpa plant in Meng Town of Mengla county, Xishuangna, Yunnan province, and identifies the strain YN0904 as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). Is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 Xilu No. 1 Beijing Hokko, Chaoyang, China) in 26 days 3 and 26 months 2020, and the preservation number is CGMCC NO. 21189.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The inventor of the invention separates a bacterial strain YN0904 from a banana plant which generates a mixture of bacterial soft rot and banana wilt in Menglan county Menglan of Yunnan province in the research process, and the bacterial strain YN0904 is identified as Bacillus amyloliquefaciens. Is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 Xilu No. 1 Beijing Hokko, Chaoyang, China) in 26 days 3 and 26 months 2020, and the preservation number is CGMCC NO. 21189. Through further research, the bacillus amyloliquefaciens CGMCC NO.21189 has good inhibition effect on fungi such as fusarium oxysporum and the like, can prevent and treat banana wilt, has excellent potassium and phosphorus (especially organic phosphorus) dissolving capability, and is a bifunctional strain with both bacteriostatic performance and growth promoting performance.

The first aspect of the invention provides a strain of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), wherein the preservation number of the Bacillus amyloliquefaciens is CGMCC NO. 21189.

In a second aspect, the present invention provides the use of a bacillus amyloliquefaciens as described above for inhibiting fungi.

According to a preferred embodiment of the invention, wherein said fungus is selected from the group consisting of Fusarium oxysporum f.sp.cubense No. 4 physiological race tropical form (Foc TR 4).

In a third aspect, the invention provides the use of the bacillus amyloliquefaciens as described above for potassium and/or phosphorus solubilization.

The term "potassium-solubilizing" and "phosphorus-solubilizing" as used herein means that the bacillus amyloliquefaciens is used to degrade and release potassium and phosphorus (especially organic phosphorus) in the culture environment, such as soil, so that the potassium and phosphorus can be more easily absorbed and utilized by plants (such as bananas).

The inventor of the invention finds that the bacillus amyloliquefaciens CGMCC NO.21189 and the metabolite thereof (such as fermentation liquor of the bacillus amyloliquefaciens) have the effect of inhibiting the fusarium oxysporum cubense special type 4 physiological race tropical form and can have the effect of preventing and treating banana fusarium wilt when being applied to banana plants (such as roots of the banana plants).

In a fourth aspect, the present invention provides a method for preventing and/or treating banana vascular wilt, the method comprising: applying the bacillus amyloliquefaciens and/or the fermentation broth of the bacillus amyloliquefaciens to the soil and/or the root system of the banana plant infected by the pathogenic bacteria of the banana wilt;

In the invention, the expression "preventing and/or treating banana vascular wilt" means that the method provided by the invention can produce a preventing effect on crops in a planting area which can produce banana vascular wilt, so that the crops are not easy to infect the banana vascular wilt. Or the influence of diseases on crop plants infected with the banana vascular wilt can be reduced, and the disease loss can be reduced.

According to a preferred embodiment of the invention, wherein said banana vascular wilt pathogens are selected from the fusarium oxysporum cubeba specialized type 4 physiological race tropical type.

According to a preferred embodiment of the present invention, the content of the bacillus amyloliquefaciens in the fermentation liquid of the bacillus amyloliquefaciens is 1 × 10⁷-1×10⁸CFU/mL。

According to a preferred embodiment of the present invention, when the bacillus amyloliquefaciens and/or the fermentation broth of the bacillus amyloliquefaciens is applied to soil, the bacillus amyloliquefaciens and/or the fermentation broth of the bacillus amyloliquefaciens is used in an amount such that the content of the bacillus amyloliquefaciens in the soil infected by the banana wilt pathogenic bacteria is 5 x 10⁷-5×10⁸CFU/kg, i.e. 5X 10 per kg of soil⁷-5×10⁸CFU of the above Bacillus amyloliquefaciens strain (or containing 5X 10⁷-5×10⁸CFU of said bacillus amyloliquefaciens fermentation broth of the above mentioned bacillus amyloliquefaciens).

According to a preferred embodiment of the present invention, wherein, when the bacillus amyloliquefaciens and/or the fermentation broth of the bacillus amyloliquefaciens is applied to banana plants, the amount of the bacillus amyloliquefaciens and/or the fermentation broth of the bacillus amyloliquefaciens is such that the amount of the bacillus amyloliquefaciens per each contact of banana plants is 5 x 10⁷-5×10⁸CFU, i.e. 5X 10 per banana plant⁷-5×10⁸The above amylolysis of CFUBacillus (or containing 5X 10)⁷-5×10⁸CFU of said bacillus amyloliquefaciens fermentation broth of the above mentioned bacillus amyloliquefaciens).

According to a preferred embodiment of the present invention, the method for preparing the fermentation broth of bacillus amyloliquefaciens comprises the following steps: and culturing the bacillus amyloliquefaciens under a fermentation condition to obtain a fermentation liquid containing a metabolite of the bacillus amyloliquefaciens.

Preferably, the fermentation conditions include: the temperature is 35-40 ℃, the time is 40-60h, and the shaking culture is carried out at the speed of 180-220 rpm.

Preferably, the concentration of the metabolite of Bacillus amyloliquefaciens in the fermentation broth is such that the OD of the fermentation broth is₆₀₀Values of 2 to 2.5, preferably 2.3 to 2.5.

In the method provided by the invention, the fermentation liquid can be directly applied to a plant (such as the plant root system) or soil in need, or can be prepared into a related microbial preparation (such as a liquid preparation or a solid preparation), and then the microbial preparation is applied to the plant (such as only the plant root system) or soil in need.

In the method provided by the invention, when the bacillus amyloliquefaciens metabolite is applied to prepare a related preparation, a person skilled in the art can convert the amount of the fermentation liquid and the specific content of the bacillus amyloliquefaciens metabolite in the preparation to obtain the specific amount of the bacillus amyloliquefaciens metabolite related preparation.

Any method of preparation of microbial agents known in the art may be suitable for use in the present invention. According to a particularly preferred embodiment of the present invention, wherein the microbial preparation is prepared by the following method: mixing the fermentation liquor of the bacillus amyloliquefaciens with an organic solvent. And after fully and uniformly mixing, removing the organic solvent in the obtained mixture to obtain the microbial preparation. The fermentation liquid of bacillus amyloliquefaciens is the fermentation liquid of bacillus amyloliquefaciens, and the preparation method and the characteristics thereof are not repeated.

Preferably, the organic solvent is selected from acetone, more preferably from 95 to 100 wt% aqueous acetone.

Preferably, the volume ratio of the fermentation liquid of the bacillus amyloliquefaciens to the organic solvent is 1: 1-1.2.

Preferably, the mixing conditions include: the temperature is 18-25 ℃, and the time is 10-12 h.

Preferably, the method of removing the organic solvent in the resulting liquid phase may be selected from rotary evaporation. Preferred conditions include: the temperature is 35-40 ℃, and the organic solvent is evaporated by rotary evaporation until the organic solvent is completely removed.

Preferably, the method may further comprise diluting the solution obtained after removing the organic solvent. It is preferable to dilute with deionized water, sterile water, or the like. The concentration of the diluted microbial preparation can be controlled by those skilled in the art according to actual needs, for example, it can be diluted to the same extent as the volume of the original fermentation broth.

The microbial preparation provided by the invention can be adjusted according to actual conditions, such as the concentration of the microbial preparation, the content of pathogenic bacteria of banana wilt disease in plants and/or soil to which the microbial preparation is applied, and the like when in use. For example, in the case where the microbial preparation is prepared and diluted to the volume of the original fermentation broth using the method as described above, according to a preferred embodiment of the present invention, wherein the microbial preparation is applied to the soil, the metabolite of the bacillus amyloliquefaciens is used in an amount of 0.075 to 0.1L/kg, i.e. 0.075 to 0.1L of the microbial preparation per kg of soil.

The bacillus amyloliquefaciens CGMCC NO.21189 also has excellent potassium and phosphorus dissolving functions, so the invention also provides a potassium and/or phosphorus dissolving method. The method is substantially the same as the above-described method for preventing and/or treating banana vascular wilt. The dosage of the specific bacillus amyloliquefaciens and/or the metabolites thereof can be adjusted by the skilled person according to the actual situation, and the details are not repeated.

The present invention will be described in detail below by way of examples, and it should be understood that the following detailed description is only intended to further explain and illustrate the contents and effects of the present invention, and is not intended to limit the present invention.

Example 1

This example illustrates the isolation, identification and preservation of Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) provided by the present invention.

Liquid NA medium formulation (1L): 10g of peptone, 3g of beef extract and 5g of sodium chloride, adding 1L of water, and adjusting the pH value to 7.

The solid NA culture medium is prepared by adding agar 15 wt% into a liquid culture medium.

(1) Strain isolation and purification

Taking a healthy leaf sheath of a banana plant which is collected from Meng & Wang & Town in Meng county of Xindona, Yunnan province and is generated by mixing the blight of the banana with the bacterial soft rot as a sample, washing the collected leaf sheath of the plant with sterile water, drying the leaf sheath indoors, sterilizing the surface of the leaf sheath with 75% of ethanol under the aseptic condition, cutting 2-3cm of the middle tissue of the leaf sheath with a sterile blade, soaking the leaf sheath with 75% of ethanol for 30s-1min, soaking the leaf sheath with 0.1% of mercuric chloride for 30s-1min, washing with sterile water for 3 times, and grinding the leaf sheath into homogenate. And (4) streaking the homogenate onto a solid NA plate, and separating and culturing the banana plant leaf sheath endophytic bacteria.

Placing the streaked solid NA flat plate in a30 ℃ constant temperature incubator, carrying out inverted culture for 1-2 days, and after a single colony is formed, selecting the single colony and purifying for 3 times by adopting a solid NA flat plate streaking method. The purified strain was stored in 50 wt% sterile glycerol at-80 ℃.

The strain YN0904 isolated by the method forms round opaque colonies on the surface of the NA solid medium, and the surface is dry and rough, has bulges and middle folds, and has regular wavy edges (the colony morphology of the strain YN0904 is shown in FIG. 1). The bacterium is detected to be stained in a gram positive mode, and the bacterium is observed to be rod-shaped under a scanning electron microscope (Zeiss sigma300, Germany) and can form endophytic spores which are oval and have two blunt-rounded ends (a scanning electron microscope image of a strain YN0904 is shown in figure 2). The optimal growth pH is 7, and the optimal growth temperature is 37 ℃.

(2) Strain identification

The purified strain (strain YN0904) was identified by 16S rDNA amplification and sequence analysis. The specific method comprises the following steps: extracting the genome DNA of the purified strain by using a bacterial genome DNA extraction kit (purchased from TaKaRa), and amplifying the 16S rDNA of the strain by using the following universal primers (synthesized by Shanghai Senno Biotech Co., Ltd.):

27F (upstream primer, sequence 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO:1)

1492R (downstream primer, sequence 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO:2)

The amplification systems and conditions are detailed in tables 1 and 2 below. The amplification product was sent to Shanghai Senno Biotech GmbH for sequencing.

TABLE 116S rDNA amplification System

Reagent	Dosage/. mu.L
		Template DNA
	2
		Upstream primer	2
Downstream primer	2
		Enzyme (TaKa Ra LA-Taq)	0.5
dd H₂O	30.5
		dNTP Mixture	8
PCR Buffer(10×)	5
		Total volume	50

TABLE 216S rDNA amplification conditions

The PCR product of about 1.5kb is obtained by amplification by the above method, the sequencing result is subjected to BLAST comparison on NCBI website, and a phylogenetic tree (detailed in figure 3) is constructed by using MEGA7.0 software and adopting an adjacency method, and the classification status of the separated endogenous antagonistic bacterium (strain YN0904) is determined. The classification search is referred to the following references [1] and [2 ].

[1] Chexizhu, cai miao ying, manual of systematic identification of common bacteria [ M ]. Beijing scientific Press, 2001:43-65.

[2] Plum funelanchi, cow, hosiejiang, agricultural microbiology experimental technology [ M ]. beijing: chinese agricultural press, 1996.

The result shows that the sequence similarity of the strain YN0904 and the strain Bacillus amyloliquefaciens is 99 percent, and according to the classification result of a phylogenetic tree (figure 3), the genetic relationship of the strain and the Bacillus amyloliquefaciens (MT613661) is nearest and is clustered into one branch. According to the information in GeneBank, the origin of the strain MT613661 is soil, and the strain is rhizosphere soil bacteria (soil antagonist bacteria). The strain YN0904 is separated from banana plants, belongs to plant antagonistic endophytes, and is easier to plant in a planting environment (such as soil) and plants (particularly in the plants) compared with soil antagonistic bacteria, so that the strain YN0904 can better play a role (such as banana fusarium wilt prevention and control, growth promotion, phosphate dissolving, potassium dissolving and the like) when being used as biocontrol bacteria.

According to the culture characteristics, morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis results of the strain YN0904, the strain is determined to be Bacillus amyloliquefaciens (Bacillus amyloliquefaciens). The strain is preserved in China general microbiological culture Collection center (CGMCC for short, the address is No. 3 Xilu No. 1 Beichen of the rising area in Beijing of China) within 26 days 3 months in 2020, and the preservation number is CGMCC NO. 21189.

Example 2

This example is used to illustrate the inhibitory effect of Bacillus amyloliquefaciens CGMCC NO.21189 on pathogenic bacteria of banana vascular wilt.

In this example, Fusarium oxysporum f.sp.cubense 4 # physiological race tropical type (Foc TR4) was used as a pathogenic bacterium, and a strain Foc 15-1 was specifically used (see [3] Lei, Z., et al., Identification and evaluation of resistance to Fusarium oxysporum f.sp.cubense tropical race 4in Musa acetic acid strain, Euphytoica 2018.214(7): 106.).

In this example, a PDA solid medium was used for the cultivation of pathogenic bacteria. The preparation method of the PDA solid culture medium comprises the following steps: adding 200g of potato and 20g of glucose into 1L of water, boiling, uniformly mixing, adding 15g of agar, uniformly mixing, adjusting the pH value to 7, cooling to about 60 ℃ (when the agar is not solidified while not scalding hands), adopting a 9cm culture dish, and pouring the plate according to the dosage of about 20 mL/dish.

(1) Detection of bacteriostatic Effect (bacteriostatic Rate)

The method for measuring the antibacterial rate by adopting a flat plate confrontation method comprises the following specific steps: foc 15-1 was inoculated on a PDA plate, cultured at 28 ℃ for 7 days, and then punched along the edges of the colonies with a sterilized punch to obtain a cake having a diameter of 5mm, and the cake thus obtained was inoculated in the center of a new PDA plate. The treatment group was inoculated with Bacillus amyloliquefaciens YN0904 by loop dipping, and inoculated onto PDA plate inoculated with Foc 15-1 cake by spot inoculation, wherein the specific position is 25mm away from the center of the plate edge, and each dish is inoculated with 4 spots. A control group was prepared by inoculating Foc 15-1 fungus cake only on PDA plates. Set up 3 sets of parallel experiments. The PDA plate is placed at 28 ℃ for standing culture for 7 days, then the plate is taken out for observation, the diameter of Foc 15-1 is measured by a cross method, and the bacteriostasis rate is calculated by adopting the following formula.

The bacteriostatic ratio (%) - (control pathogen colony diameter-treated pathogen colony diameter)/control diameter × 100%

The experimental results are as follows: the Foc 15-1 colonies of the treatment group were measured as: 1.8cm, 1.85cm, Foc 15-1 colonies of the control group were measured as: 8.9cm, 9cm and 9 cm. The calculated antibacterial rate of each group of experiments is as follows: 79.77%, 79.44% and 79.44%, and the average bacteriostasis rate is 79.55%.

The colony growth for one of the groups tested is shown in FIG. 4, with the left side being the treatment group and the right side being the control group. As can be seen from the figure, the growth of Foc 15-1 in the treated group is obviously inhibited, and the colony size is obviously smaller than that of the control group, which shows that the strain YN0904 has stronger inhibition effect on the tropical type of the fusarium oxysporum cubeba specialized type 4 physiological race.

(2) Effect of YN0904 on Foc 15-1 mycelium

The opposing plates (treatment groups) of the strains YN0904 and Foc 15-1 in the experiment (1) were selected and cultured for 7 days, Foc 15-1 new mycelia close to the biocontrol bacteria (YN0904 colonies) were picked up with a sterilized toothpick to prepare temporary slides, the influence of the strains YN0904 on the growth of Foc 15-1 mycelia was observed under a stereoscopic microscope (optical microscope) at a magnification of 40 times, and the new mycelia at the edges of the colonies cultured for 7 days (control groups) with PDA were used as a control.

A set of observations is shown in fig. 5. FIG. 5 shows hyphae of Foc 15-1 in the left side (YN0904+ TR4), which are swollen and deformed, enlarged and rounded at the end, and shortened in the internode between septa; the right side (CK-TR4) is the hypha of control Foc 15-1, which shows that the hypha is smooth, uniform, straight naturally, without large amount of pigment accumulation in the hypha, and the protoplasm is normal. The experimental result shows that YN0904 can effectively inhibit the growth of fusarium oxysporum cubeba special type No. 4 physiological race tropical strain.

(3) Biocontrol/growth promoting gene detection

The biocontrol/growth promoting gene detection is carried out on the strain YN0904, and the existence of 6 antagonistic genes srfAB, fenD, ituC, ituD, bamC, yndJ and 1 growth promoting gene ysnE in the strain YN0904 is detected through PCR, so that the biocontrol and growth promoting potential of the strain YN0904 can be identified. The specific method comprises the following steps: activating bacillus amyloliquefaciens YN 0904: the streaking method adopts NA solid medium to culture at 37 deg.C for 24 h. The activated single colony was picked with sterilized toothpick and mixed in 50. mu.0 lysine Buffer, then lysed in 80 homogeneous water bath for 15min, centrifuged at 8000rpm for 1min, and the supernatant (as DNA template, YN0904 genomic gene) was taken. The specific gene corresponding primers and sequences of the primers are detailed in Table 3, the amplification system is detailed in Table 4, and the amplification procedure is detailed in Table 5.

TABLE 3 detection of Gene-corresponding substance types and primers

Note: in Table 3, the last letter "F" of the primer represents the forward primer, and "R" represents the reverse primer.

Wherein, the gene for detection and its primer are obtained by referring to the following references [4] to [6 ].

[4]Joshi,R.and M.S.Gardener,Identification and Characterization of Novel Genetic Markers Associated with Biological Control Activities in Bacillus subtilis.Phytopathology,2006.96(2):p.145.

[5]Mora I,Cabrefiga J,Montesinos E.Antimicrobial peptide genes in Bacillus strains from plant environments[J].International Microbiology,2011,14(4):213-223.

[6] The bacteriostasis and growth promotion function of Zhou Xiao Jiang.2 strains of marine habitat bacillus and related control genes [ D ]. Qingdao: qingdao university of science and technology, 2015.

TABLE 4 biocontrol/growth promoting Gene amplification System

Reagent	Dosage/. mu.L
		Template DNA
	1
		Upstream primer	1
Downstream primer	1
		PCR SuperMix*	22
Total volume	25

Purchased from TsingKE, containing enzymes, dNTPs, water, PCR Buffer.

TABLE 5 biocontrol/growth promoting Gene amplification procedure

The amplification products were detected by electrophoresis on a 1 wt.% agarose gel. The results are shown in FIG. 6. Wherein, lane M is Marker,

lanes

1 and 2 are amplification products, and lane 3 is a negative control. As can be seen from FIG. 6, 6 antagonistic genes srfAB, fenD, ituC, ituD, bamC, yndJ and 1 growth promoting gene ysnE can be amplified in the YN0906 genome, which indicates that the YN0906 strain contains the genes and has good biocontrol potential.

Example 3

This example is used to illustrate the potassium and phosphorus dissolving effects of Bacillus amyloliquefaciens CGMCC NO.21189 provided by the present invention.

In this example, the phosphorus-solubilizing ability of Bacillus amyloliquefaciens YN0904 was tested by using an organophosphorus solid medium, and the potassium-solubilizing ability of Bacillus amyloliquefaciens YN0904 was tested by using a potassium-solubilizing solid medium. The specific formula and the preparation method are as follows:

organic phosphorus solid culture medium: glucose 10g, (NH)₄)₂SO₄0.5g、NaCl 0.3g、KCl 0.3g、MgSO₄·7H₂O 0.3g、FeSO₄·7H₂O 0.03g、MnSO₄·4H₂O 0.03g、Ca₃(PO₄)₂0.3g of agar and 18g of agar, adding distilled water to reach the constant volume of 1000ml, and adjusting the pH value to 7.2 +/-0.3.

The potassium-dissolving solid culture medium comprises 10g of cane sugar and Na₂HPH₄ 1g，(NH₄)₂SO₄ 0.5g，MgSO₄·7H₂O1 g, yeast extract 0.2g, FeSO₄·7H₂O 0.03g,CaCO₃2.5g, potassium feldspar powder 10g, agar powder 15g, distilled water to a constant volume of 1000mL, and pH value is adjusted to 7.2 +/-0.3.

The preparation method comprises the following steps: adding the reagents except the agar into distilled water, fixing the volume to 1000mL, then adding the agar, heating to dissolve, cooling to about 60 ℃, and pouring the solution into a plate by adopting a 9cm culture dish according to the dosage of about 20-25mL per dish.

The test method comprises the following steps: activating bacillus amyloliquefaciens YN 0904: the streaking method adopts NA solid medium to culture at 37 deg.C for 24 h. Activated bacillus amyloliquefaciens YN0904 single colonies are picked by an autoclaved toothpick and inoculated on an organophosphorus plate and a potassium desolvation medium plate, 1 colony is inoculated on each plate, and 3 groups of parallel tests are set. The inoculated plate was incubated at 30 ℃ for 7 days, and the size of the transparent circle was measured by a cross-hatch method.

The experimental results are as follows:

and (3) detecting the phosphate solubilizing capability: 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. Through measurement, in 3 groups of parallel tests, the diameters (cm) of the phosphate solubilizing rings are respectively as follows: 2.55, 2.75, 2.55, wherein the colony diameters (cm) are respectively: 1.85, 2.0, 1.95. The D/D ratios are calculated as follows: 1.38, 1.31.

And (3) detecting the potassium dissolving capacity: selecting a colony with a potassium-resolving ring, measuring the diameter D of the colony and the diameter D of the potassium-resolving ring, calculating the ratio of D/D, and determining the strength of the potassium-resolving capability. Through measurement, in 3 groups of parallel tests, the diameters (cm) of potassium-dissolving rings are respectively as follows: 3.45, 3.75, 3.8, wherein the colony diameters (cm) are respectively as follows: 2.05, 1.95 and 1.9. The D/D ratios are calculated as follows: 1.68, 1.92 and 2.

Figure 7 shows the results of one plate in the phosphate (left) and potassium (right) dissolution tests, respectively.

Example 4

This example is used to illustrate the prevention and cure effect and growth promotion effect of Bacillus amyloliquefaciens CGMCC NO.21189 on banana wilt potted plants.

Taking tissue-cultured Brazil banana seedlings, cleaning a root culture medium, transplanting the Brazil banana seedlings into a seedling bag, and transplanting the Brazil banana seedlings into a plastic pot which takes vermiculite as a substrate and has the diameter of 11cm and the height of 12cm after the banana seedlings grow 3-4 leaves (about one month). After transplanting, the seedlings are frequently drenched for moisture preservation and fertilized once a week. The specific fertilization mode is as follows: dissolving compound fertilizer in water according to the dosage of 2g per banana seedling for application, wherein the content of N in the compound fertilizer is 15 wt%, and P in the compound fertilizer₂O₅In an amount of 15 wt.%, K₂The content of O was 15% by weight. After the banana seedlings grow 5-6 leaves (about one month), the banana seedlings are used as banana pot seedlings to perform banana wilt prevention and control and growth promotion experiments.

Preparing a bacillus amyloliquefaciens fermentation liquid: activating bacillus amyloliquefaciens YN 0904: the streaking method adopts NA solid culture medium to culture at 30 deg.C for 24 h. Then, a single colony is picked by using a sterile inoculating loop and inoculated in an NA liquid culture medium (200mL), and shake culture is carried out at 37 ℃ and 220rpm for 2 days to obtain a strain fermentation liquid. Preparing the strain fermentation liquor into 1 × 10 by adopting sterile water⁸CFU/mL of Bacillus amyloliquefaciens fermentation liquid for later use.

The banana wilt spore liquid is adopted to treat banana potted seedlings to simulate the occurrence of banana wilt. Banana cakeThe preparation method of the wilt spore liquid comprises the following steps: inoculating the separated and purified banana vascular wilt disease 4 # microspecies (Foc 15-1) cake into a liquid PDA culture medium, shaking and culturing for 2 days at 28 ℃ and 220rpm, and filtering the culture solution by 4 layers of sterile gauze to obtain a pathogen spore suspension. Prepared with sterile water to a concentration of 1 × 10⁶CFU/mL of banana vascular wilt spore liquid for later use.

The banana potted seedlings were divided into four groups: (1) independently irrigating NA liquid culture medium; (2) separately irrigating the bacillus amyloliquefaciens fermentation liquor; (3) irrigating NA liquid culture medium and banana fusarium wilt spore liquid; (4) pouring the bacillus amyloliquefaciens fermentation liquid and the banana fusarium wilt spore liquid. Each group was tested with 4 banana potted seedlings, and three groups of parallel tests were set up. The specific irrigation method comprises the following steps: in the test, the plant is watered once, and the total dosage of the poured solution (fermentation liquor and culture medium) is 40mL per plant. After pouring the bacillus amyloliquefaciens fermentation liquid for 7 days, inoculating banana fusarium wilt bacteria (each plant is poured with 40mL of banana fusarium wilt spore liquid).

On the day of treatment, biological indexes such as plant height, pseudostem diameter and the like of each treated banana plant are measured, then the plants are placed under the same conditions for pot culture, and after 40 days, the measurement is carried out again. The disease incidence of each treatment is investigated, the disease index of each treatment (obtained by calculation according to a formula according to the disease grading standard in the table 6) is counted, and the prevention and treatment effect is calculated. The results are detailed in tables 7-12.

In tables 9 to 11, the superscript letters indicate the difference between the measurement results at day 0 and day 40, and if the data in the same row are superscripted with different letters, the difference is significant, and if the data are the same letters, the difference is not significant.

TABLE 6 Classification Standard of disease of Banana wilt

Measuring method

Plant height: measuring the distance from the ground to the top of the intersection point of two petioles

Pseudostem diameter is measured by measuring the diameter of the base of the pseudostem about 1cm from the ground surface with a vernier caliper

The control effect is calculated by adopting the following formula:

the preventing and treating effect (%) is [ (disease index of control group-disease index of treatment group)/disease index of control group ] × 100%

Growth rate (end time of measurement-start time of measurement)/day

TABLE 7 disease index comparison

TABLE 8 comparison of leaf and bulb (mean) disease indices

Group of	Corm	Blade
			(3)	56.25±3.61	70.83±2.08
(4)	14.58±2.08	12.5±3.61
			Preventive effect/%)	74.26±2.27	82.58±4.61

TABLE 9 comparison of plant height growth promoting effects

Group of	Plant height/cm at 0 day	Plant height/cm of 40 days	Growth rate
				(1)	21.06±1.69^a	30.58±1.81^b	0.24
(2)	21.74±2.07^a	36.17±2.86^a	0.36
				(3)	21.65±1.44^a	22.6±1.48^c	0.02
(4)	20.10±1.27^a	29.55±1.65^b	0.22

TABLE 10 comparison of growth promoting effect of pseudostem diameter

Group of	Pseudostem diameter/mm at day 0	Pseudostem diameter/mm for 40 days
			(1)	7.18±0.29^a	9.88±0.51^ab
(2)	7.26±0.48^a	11.64±0.71^a
			(3)	7.21±0.18^a	8.69±0.44^c
(4)	7.20±0.47^a	10.93±0.38^b

TABLE 11 comparison of the growth promoting effects of the leaf number of plants

Group of	Leaf number of 0 day	Leaf number of 40 days
			(1)	5.33±0.31^a	6.75±0.41^b
(2)	5.17±0.22^a	7.17±0.31^a
			(3)	5±0.32^a	5.67±0.24^c
(4)	5.08±0.37^a	6.58±0.35^ab

TABLE 12 comparison of root growth promoting effects

Group of	Root mean coefficient of 40 balance
		(1)	6.58
(2)	7.75
		(3)	5.75
(4)	6.2

In addition, the growth of banana potted plants of group (3) (left) and group (4) (right) is shown in fig. 8 after 40 days, and it can be seen that banana plants not treated with YN0904 had poor plant development after infection with Foc 15-1, with significantly smaller leaves and yellowing.

The root-cut results of banana pot plants of group (3) (left) and group (4) (right) after 40 days are shown in fig. 9, and it can be seen that the color of the bulbs of banana pot plants treated with YN0904 is normal, while the color of the bulbs of banana pot plants not treated with YN0904 is red to dark brown, showing significant symptoms of banana wilt.

The results of fig. 8 and 9 demonstrate that YN0904 has good control effect on banana vascular wilt.

The growth of the banana plants after 40 days in group (1) (left) and group (2) (right) banana pot plants is shown in fig. 10, from which it can be seen that the banana plants treated with YN0904 grew better and the leaves were significantly larger. It is proved that YN0904 has growth promoting effect on banana plants.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

SEQUENCE LISTING

<110> research institute of agricultural environmental resources of agricultural academy of sciences of Yunnan province

<120> Bacillus amyloliquefaciens, application thereof and method for preventing and/or treating banana vascular wilt

<130> I68282YNN

<160> 16

<170> PatentIn version 3.5

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Claims

1. Bacillus amyloliquefaciens strainBacillus amyloliquefaciens) The bacillus amyloliquefaciens is characterized in that the preservation number of the bacillus amyloliquefaciens is CGMCC NO. 21189.

2. Use of the bacillus amyloliquefaciens of claim 1 for inhibiting fungi.

3. Use according to claim 2, wherein the fungus is selected from the group consisting of Fusarium oxysporum cubeba speciality type (C) (Fusarium oxysporum f. sp. cubense) No. 4 physiological race tropical type.

4. Use of the bacillus amyloliquefaciens according to claim 1 for potassium and/or phosphorus solubilization.

5. A method for preventing and/or treating banana vascular wilt, comprising: applying bacillus amyloliquefaciens and/or a fermentation broth of the bacillus amyloliquefaciens to soil and/or a root system of a banana plant infected by pathogenic bacteria of banana wilt;

wherein the preservation number of the bacillus amyloliquefaciens is CGMCC NO. 21189.

6. The method according to claim 5, wherein the content of Bacillus amyloliquefaciens in the fermentation broth of Bacillus amyloliquefaciens is 1 x 10⁷-1×10⁸CFU/mL。

7. The method of claim 5, wherein the fermentation broth of Bacillus amyloliquefaciens is prepared by a method comprising: and culturing the bacillus amyloliquefaciens under a fermentation condition to obtain a fermentation liquid containing a metabolite of the bacillus amyloliquefaciens.

8. The method of claim 7, wherein the fermentation conditions comprise: the temperature is 35-40 ℃, the time is 40-60h, and the shaking culture is carried out at the speed of 180-220 rpm.

9. The method of claim 7, wherein the concentration of the metabolite of Bacillus amyloliquefaciens in the fermentation broth is such that the OD of the fermentation broth₆₀₀The value is 2-2.5.

10. The method of claim 9, wherein the concentration of the metabolite of bacillus amyloliquefaciens in the fermentation broth is such that the OD of the fermentation broth₆₀₀The value is 2.3-2.5.

11. The method of claim 5, wherein said banana vascular wilt pathogen is selected from the Fusarium oxysporum cubeba specialized type 4 physiological race tropical form.

12. The method according to claim 5 or 6, wherein the amount of the Bacillus amyloliquefaciens and/or the Bacillus amyloliquefaciens fermentation broth is such that the content of the Bacillus amyloliquefaciens in the soil infected with the pathogenic bacteria of banana vascular wilt is 5 x 10 when the Bacillus amyloliquefaciens and/or the Bacillus amyloliquefaciens fermentation broth is applied to the soil⁷-5×10⁸CFU/kg。

13. The method according to claim 5, wherein when the Bacillus amyloliquefaciens and/or the fermentation broth of the Bacillus amyloliquefaciens is applied to the roots of banana plants, the amount of the Bacillus amyloliquefaciens is such that the amount of the Bacillus amyloliquefaciens per contact of each banana plant is 5 x 10⁷-5×10⁸CFU。