CN112899201B

CN112899201B - Bacillus belgii, application thereof and method for preventing and treating banana wilt

Info

Publication number: CN112899201B
Application number: CN202110281761.9A
Authority: CN
Inventors: 番华彩; 徐胜涛; 曾莉; 李舒; 郑泗军; 何平; 白亭亭; 尹可锁; 李迅东; 刘立娜; 郭志祥; 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: 2021-12-07
Anticipated expiration: 2041-03-16
Also published as: CN112899201A

Abstract

The invention relates to the field of biological control and discloses a Bacillus belgii strain, application thereof and a method for preventing and/or treating banana vascular wilt. The Bacillus belgii provided by the invention has a strong inhibition effect on Fusarium oxysporum Guba specialization type No. 4 physiological race tropical type (Foc TR4), and can well prevent and treat banana wilt. In addition, the Bacillus belgii also has a good growth promoting effect on banana plants and can help the banana plants to grow faster and better. Moreover, the Bacillus belgii is a banana rhizosphere antagonist, does not generate side effects or pollution problems when in use, and is safer and more environment-friendly.

Description

Bacillus belgii, application thereof and method for preventing and treating banana wilt

Technical Field

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

Background

Bananas are perennial herbaceous plants and are one of important commercial crops in tropical and subtropical regions. The banana planting in China is mainly distributed in areas such as Guangdong, Hainan, Guangxi, Fujian, Yunnan and Taiwan, the yield of bananas in China is huge, and the bananas are the fourth most fruits after apples, grapes and oranges in China at present. However, banana cultivation has been frequently plagued in recent years by banana wilt (Fusarium wilt of banana), the pathogenic bacterium of which is Fusarium oxysporum cubeba specialized (Fusarium oxysporum f.sp. cubense, FOC) tropical type No. 4 physiological race (TR 4). The banana wilt does not show obvious symptoms in the early stage, but the young bananas carry pathogenic bacteria after infection until entering the growth period, particularly the bud drawing period, infected plants rapidly attack and finally die, so that the yield of the bananas is reduced, and the income of banana farmers is seriously damaged. Moreover, if the incidence rate of banana vascular wilt in the banana plantation reaches more than 20%, the area is not suitable for planting bananas any more, so that the large-scale banana vascular wilt outbreak severely limits banana production, and immeasurable economic loss is caused to the banana planting industry.

At present, the methods for preventing and treating banana vascular wilt mainly comprise four methods: chemical prevention method, disease-resistant breeding, farming mode adjustment and biological prevention method. Among them, the biological control method has been receiving attention in recent years as a control method having the best control effect, the minimum side effect on the environment and plants, the low cost, and the convenient operation. The core of the biological control method is to develop and utilize biocontrol bacteria, and the biocontrol bacteria can inhibit pathogenic bacteria, so that the purpose of preventing and treating plant diseases is achieved. In addition, some biocontrol bacteria can also have the growth promoting effect on crops, and the yield is improved while the diseases are prevented and treated. However, the current development work of biocontrol bacteria aiming at banana vascular wilt is not deep, the prevention and control effect of the available biocontrol bacteria is limited, the growth promotion effect is not obvious, and the advantages of biological prevention and control cannot be fully exerted. Therefore, the development of a novel biocontrol bacterium for controlling banana vascular wilt, which has good control effect and good crop growth promoting effect, is urgently needed to meet the requirement of biological control of banana vascular wilt.

Disclosure of Invention

The invention aims to solve the problems that the biocontrol bacteria control effect for controlling banana vascular wilt is not ideal, the growth promotion effect is not available or is not obvious and the like in the prior art, and provides a strain of Bacillus belezii, which has the characteristics of good control effect on banana vascular wilt and obvious growth promotion effect on banana plants.

In order to achieve the purpose, the invention provides a Bacillus subtilis (Bacillus velezensis) on one hand, which is characterized in that the preservation number of the Bacillus subtilis is CGMCC NO. 21872.

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

In a third aspect, the present invention provides the use of a bacillus beiesei as defined above for promoting plant growth.

The fourth aspect of the invention provides a method for preventing and treating banana vascular wilt, which is characterized in that the Bacillus belgii and/or the fermentation liquor of the Bacillus belgii are applied to soil infected by pathogenic bacteria of banana vascular wilt and/or roots of banana plants;

wherein the Bacillus belgii has a preservation number of CGMCC NO. 21872.

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

(1) the Bacillus belgii provided by the invention has strong inhibition effect on Fusarium oxysporum Guba specialization type No. 4 physiological race tropical type (Foc TR4), and has good prevention and treatment effect on banana vascular wilt;

(2) the Bacillus belgii provided by the invention has good growth promoting effect;

(3) the Bacillus belgii provided by the invention is separated from rhizosphere soil of banana plants, the microbial environment in the banana planting environment cannot be damaged by using the Bacillus belgii, the soil is not polluted, the cost is low, and the using method is simple.

Drawings

FIG. 1 shows the colony morphology of the strain YN1910 in example 1;

FIG. 2 is a scanning electron micrograph of the strain YN1910 in example 1;

FIG. 3 is a phylogenetic tree prepared according to the 16S rDNA sequencing result of strain YN1910 in example 1;

FIG. 4 is a graph showing the inhibitory effect of Bacillus belgii on the tropical type of the fusarium oxysporum cubense No. 4 physiological race in example 2;

FIG. 5 shows the inhibitory effect of strain YN1910 on the mycelia of Fusarium oxysporum cubeba specialization type No. 4 physiological race tropical type in example 2 of the present invention;

FIG. 6 is a gel electrophoresis image of a PCR product obtained by amplifying 6 kinds of antagonistic genes and 1 growth promoting gene using genomic DNA of YN1910 as a template in example 2 of the present invention;

FIG. 7 shows the control effect of bacterial strain YN1910 on banana wilt of potted banana plants (leaves) in example 4 of the present invention;

FIG. 8 shows the control effect of bacterial strain YN1910 on banana wilt of potted banana plants (bulbs) in example 4 of the present invention;

FIG. 9 shows the growth promoting effect of strain YN1910 on banana potted plants in example 4 of the present invention.

Biological preservation

The rhizosphere soil bacterium YN1910 obtained by separating rhizosphere soil of healthy banana plants in Yuxi city Yuanjiang county of Yuxi, Yunnan in 2019 by the inventor is identified as Bacillus velezensis. Is preserved in China general microbiological culture Collection center (CGMCC, address: No. 3 Xilu No. 1 Xinchen Yang district, Beijing, China) at 3 months and 5 days in 2021, with the preservation number of CGMCC NO. 21872.

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 rhizosphere soil bacterium YN1910 obtained by separating rhizosphere soil of healthy banana plants in the plots with banana wilt disease in Yuxi county of Yuanjiang province in Yunnan province in the research process is identified as Bacillus velezensis. Is preserved in China general microbiological culture Collection center (CGMCC, address: No. 3 Xilu No. 1 Beijing, Chaoyang district, Beijing, China) at 3 months and 5 days in 2021, with the preservation number of CGMCC NO. 21872. Through further research, the Bacillus beilisi CGMCC NO.21872 has a good inhibition effect on fungi such as fusarium oxysporum and the like, can prevent and treat banana wilt, has excellent capability of promoting the growth of banana plants, and is a bifunctional strain with both antibacterial performance and growth promoting performance.

The first aspect of the invention provides a Bacillus belgii (Bacillus velezensis), and the preservation number of the Bacillus belgii is CGMCC NO. 21872.

According to a preferred embodiment of the invention, wherein the fungus is selected from the group of fungi capable of causing banana vascular wilt to occur.

Preferably, the fungus is selected from the group consisting of Fusarium oxysporum cubeba specialization type No. 4 physiological race tropical type (Foc TR 4).

Preferably, the plant is selected from bananas.

The expression "promoting plant growth" in the invention means that the Bacillus belgii is adopted to promote banana growth, i.e. the growth state of banana plants is better; promoting the growth process of bananas, namely enabling the bananas to grow more quickly; promoting banana yield, namely enabling the banana yield to be more and the fruits to be larger, and the like.

The inventor of the invention discovers in the research process that the Bacillus belgii CGMCC NO.21872 and the metabolite (such as fermentation liquor) thereof have the effect of inhibiting fusarium oxysporum f.sp.cubense No. 4 physiological race tropical type (Foc TR4) and other fungi, and can effectively prevent and control the occurrence and development of banana wilt when applied to banana plants.

In a fourth aspect, the present invention provides a method for controlling banana vascular wilt, comprising: applying the Bacillus belgii and/or the Bacillus belgii fermentation broth to soil and/or banana plant roots infected with pathogenic bacteria of banana wilt;

wherein the Bacillus belgii has a preservation number of CGMCC NO. 21872.

In the invention, the 'control of banana vascular wilt' means that the method provided by the invention can produce a prevention effect on crops in a planting area which is likely to produce banana vascular wilt, so that the crops are not easily infected with 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 (Foc TR 4).

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

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

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 belgii in the fermentation broth is such that the OD of the fermentation broth₆₀₀Values of 2 to 2.5, preferably 2.3 to 2.5.

In the method provided by the invention, the fermentation liquor can be directly applied to a needed plant or soil, and the metabolite of the Bacillus belgii can be extracted according to the method in the prior art to be prepared into a related microbial preparation (such as a liquid preparation or a solid preparation) for use. Any method for extracting active ingredients from fermentation liquor in the field of the method can be applied to the method.

In the method provided by the invention, when the Bacillus bleedensis metabolite is prepared into a related microbial preparation for application, a person skilled in the art can convert the dosage of the fermentation liquor and the specific content of the Bacillus bleedensis metabolite in the preparation to obtain the specific dosage of the Bacillus bleedensis 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 broth of Bacillus belgii 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 the Bacillus belgii is the fermentation liquid of the Bacillus belgii, and the preparation method and the characteristics of the fermentation liquid are not repeated.

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

Preferably, the volume ratio of the fermentation liquor of the bacillus belgii 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 removed by rotary evaporation.

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, where the microbial preparation is prepared and diluted to the volume of the original fermentation broth using the method described above, according to a preferred embodiment of the invention, wherein the microbial preparation, when applied to soil, is used in an amount of 0.075 to 0.1L/kg/time of the metabolite of bacillus belgii, i.e. 0.075 to 0.1L/time of the microbial preparation per kg of soil.

According to a preferred embodiment of the present invention, wherein, when the Bacillus belgii and/or the Bacillus belgii fermentation broth is applied to soil, the Bacillus belgii and/or the Bacillus belgii fermentation broth is used in an amount such that the content of the Bacillus belgii in the soil infected with pathogenic bacteria of banana vascular wilt is 5 × 10⁷-5×10⁸CFU/kg, i.e. 5X 10 per kg of soil⁷-5×10⁸CFU of Bacillus belgii (or containing 5 × 10)⁷-5×10⁸CFU of said Bacillus belgii broth of said Bacillus belgii).

According to a preferred embodiment of the invention, wherein when the Bacillus bleekerii and ^ is/areOr the fermentation broth of Bacillus belgii is applied to banana plants, the Bacillus belgii and/or the fermentation broth of Bacillus belgii is used in an amount such that the amount of Bacillus belgii per banana plant contact is 5 x 10 per time⁷-5×10⁸CFU, i.e. 5X 10 per banana plant⁷-5× 10⁸CFU of Bacillus belgii (or containing 5 × 10)⁷-5×10⁸CFU of said Bacillus belgii broth of said Bacillus belgii).

The Bacillus belgii CGMCC NO.21872 provided by the invention also has an excellent growth promoting effect on plants, so the invention further provides a method for promoting plant growth. The method is basically the same as the method for preventing and treating the banana vascular wilt. For the specific Bacillus belgii and/or the amount of the Bacillus belgii fermentation broth, those skilled in the art can adjust the dosage according to actual conditions, and details are not repeated herein.

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 velezensis CGMCC NO.21872 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. The preparation method comprises dissolving the components except agar in water, boiling, adding agar, dissolving, mixing, sterilizing with high temperature steam, taking out, cooling to about 60 deg.C (without scalding hands and the agar not solidifying), and pouring into flat plate. And pouring about 20mL of the solid NA culture medium into each 9m culture dish, and cooling and solidifying to obtain the NA flat plate.

(1) Strain isolation and purification

Healthy banana plant rhizosphere soil collected from banana wilt disease plots in Yuxi City, Yuanjiang county, Yunnan province is taken as a sample. The specific sampling method comprises the following steps: sampling points are randomly selected from the plot by adopting a five-point sampling method, 0-10cm of soil is collected by a soil collector, and each treatment is repeated for 3 times. Collecting soil attached to the root system of banana as rhizosphere soil sample, removing impurities such as root hair, pebbles and the like, uniformly mixing the collected soil sample, sealing the mixture by using a polyethylene sterile bag, placing the mixture in an ice box, bringing the mixture back to a laboratory, and storing the mixture at-20 ℃ for later use.

Weighing 2g of uniformly mixed soil sample, adding the uniformly mixed soil sample into a centrifugal tube filled with 18mL of sterile water, oscillating and uniformly mixing the uniformly mixed soil sample by using a vortex oscillator to prepare a soil suspension, and sequentially diluting the soil suspension by a 10-time gradient until the dilution multiple is 10^-7. Respectively diluting by 10 times by using a micropipette^-4、10^-5、10^-6、10^-7The suspension was pipetted 100. mu.L each, and each was spread on NA plates and cultured in an inverted state at 28 ℃ for 24 hours. Single colonies of different morphologies were picked and placed in 50 wt% sterile glycerol and stored at-80 ℃ until use.

In the rhizobacteria of soil obtained by the method, the bacterial strain YN1910 forms a circular milky opaque colony on the surface of a NA solid medium, the surface is not smooth, the middle is slightly convex and bright, the bacterial membrane is formed, and the edge is regular (the colony form of the bacterial strain YN1910 is shown in figure 1). The bacteria were detected to be gram-positive stained, and were rod-shaped with blunt ends under an optical microscope under a scanning electron microscope (zeiss sigma300, germany) (scanning electron microscope image of strain YN1910 is shown in fig. 2). The optimal growth pH is 7, and the optimal growth temperature is 37 ℃.

(2) Strain identification

The purified strain (strain YN1910) was identified by 16S rDNA amplification and sequence analysis. The specific method comprises the following steps: extracting the genome DNA of the purified strain YN1910 by using a bacterial genome DNA extraction kit (TaKaRa), and amplifying the 16S RNA of the strain by using the following universal primers (synthesized by the granted injury Parsonino Biotech Co., Ltd.): 27F (forward primer, sequence 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO:1) and 1492R (reverse primer, sequence 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 2). The amplification system and conditions are detailed in tables 1-2 below. The amplification product was sent to Shanghai Senno Biotech GmbH for sequencing.

TABLE 116S rDNA amplification System

Reagent	Dosage/. mu.L
		Template DNA (Strain 1910 genomic 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 method, the sequencing result is subjected to BLAST comparison on NCBI website, MEGA7.0 software is used, the adjacency method is adopted to construct a phylogenetic tree, and the classification status of the separated strain YN1910 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 results showed that the sequence similarity of strain YN1910 with strain Bacillus velezensis was 99%. According to the classification result of the phylogenetic tree (see figure 3 in detail), the strain has the closest genetic relationship with Bacillus belgii and is clustered into one branch.

According to the culture characteristics, morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis results of the strain YN1910, the strain is determined to be Bacillus velezensis. The strain is preserved in China general microbiological culture Collection center (CGMCC for short, the address: No. 3 Xilu No. 1 Xinchen of the sunward area in Beijing, China) at 2021 year, 3 month and 5 days, and the preservation number is CGMCC NO. 21872.

Example 2

This example illustrates the inhibitory effect of Bacillus belgii CGMCC NO.21872 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 and the preparation method of the PDA flat plate comprise 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, sterilizing by using high-pressure steam, cooling to about 60 ℃ (when the agar is not too hot and is not solidified), adopting a 9cm culture dish, and pouring the plate according to the dosage of about 20 mL/dish to obtain the PDA plate.

(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 dipped the Bacillus beiLeisi YN1910 by an inoculating ring, inoculated on a PDA plate just inoculated with Foc 15-1 fungus cake in a spot inoculation mode, specifically positioned at the position 25mm away from the center of the plate edge, and inoculated at 4 spots per dish. 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: 1.65cm, 1.6cm, 1.65cm, 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: 81.46%, 82.22% and 81.67%, and the average bacteriostasis rate is 81.78%.

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, the colony size is obviously smaller than that of the control group, and the bacterial strain YN1910 has stronger inhibition effect on fusarium oxysporum cubeba specialized type 4 physiological races.

(2) Effect of YN1910 on Foc 15-1 mycelium

A facing plate (treatment group) of the strain YN1910 and Foc-1 in the experiment (1) is selected to be opposite to each other for culturing for 7 days, Foc-15-1 new hyphae close to the biocontrol bacteria (YN1910 colony) are picked by a sterilization toothpick to prepare a temporary slide, the influence of the strain YN1910 on the growth of the Foc-1 hyphae is observed under a body type microscope at a magnification of 40 times (an optical microscope), and the new hyphae at the edge of the colony of the PDA normally cultured for 7 days (the control group) are 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 (YN1910+ TR4), which are swollen and deformed, enlarged and rounded at the ends, shortened hyphal internodes between diaphragms, and concentrated and aggregated endoplasmic reticulum; 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 YN1910 can effectively inhibit the growth of fusarium oxysporum cubeba specialized No. 4 physiological race tropical strain.

(3) Biocontrol/growth promoting gene detection

And performing biocontrol/growth promoting gene detection on the strain YN1910, and detecting whether 6 antagonistic genes srfAB, fenD, ituC, ituD, bamC, yndJ and 1 growth promoting gene ysnE exist in the strain YN1910 by PCR (polymerase chain reaction), thereby identifying the biocontrol and growth promoting potentials of the strain YN 1910. The specific method comprises the following steps: activating bacillus belgii YN 1910: 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 ℃ water bath for 15min, centrifuged at 8000rpm for 1min, and the supernatant was taken as DNA template (i.e., genomic DNA of strain YN1910 as DNA template). 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 1-2 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 YN1910 genome, which shows that the YN1910 strain contains the genes and has good biocontrol potential.

Example 3

This example illustrates the prevention and treatment effect and growth promotion effect of Bacillus belgii CGMCC NO.21872 on banana vascular 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.

B, preparing Bacillus beiLeisi fermentation liquor: bacillus belgii preserved with ultralow temperature (-80 deg.C) glycerolActivation of Bacillus YN 1910: 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 Bacillus belgii fermentation broth for use.

The banana wilt spore liquid is adopted to treat banana potted seedlings to simulate the occurrence of banana wilt. The preparation method of the banana vascular 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 Bacillus belgii fermentation liquor; (3) irrigating NA liquid culture medium and banana fusarium wilt spore liquid; and (4) irrigating the Bacillus belgii fermentation liquor and the banana fusarium wilt spore liquid. Wherein, the (1) group and the (3) group are control groups, and the (2) group and the (4) group are experimental groups (treatment groups). Each group was tested with 4 banana potted seedlings, and 3 groups of parallel tests were set up. The specific irrigation method comprises the following steps: the test period is 1 time of irrigation, and the amount of each irrigation is 40 mL. Wherein the roots of the potted banana plants in the groups (1) and (2) are respectively watered with NA liquid culture medium and Bacillus beiLeisi fermentation liquor on the 0 th day, and then potted plant culture is carried out for 40 days; and (3) respectively irrigating the roots of the potted banana plants with NA liquid culture medium and Bacillus beiLeisi fermentation liquid on the 0 th day, and inoculating banana fusarium wilt bacteria (namely respectively irrigating 40mL of banana fusarium wilt spore liquid per plant on the roots of the banana plants) after the potted banana plants are cultured to the 7 th day.

On the day of treatment (day 0), biological indicators such as plant height, pseudostem diameter and the like of each treated banana plant were measured, and then potted under the same conditions for culture, and after 40 days, measurement was performed again. The disease occurrence of each treatment group is investigated, the disease index of each treatment group (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 the following two formulas:

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 (leaf) 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)	10.42±2.08	6.25±3.61
			Preventive effect/%)	81.76±2.94	91.41±4.82

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)	21.21±2.07^a	37.15±2.86^a	0.4
				(3)	21.65±1.44^a	22.6±1.48^d	0.02
(4)	20.28±1.27^a	28.88±1.65^c	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^b
(2)	7.28±0.48^a	11.36±0.71^a
			(3)	7.21±0.18^a	8.69±0.44^c
(4)	7.13±0.47^a	10.74±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±0.21^a	6.75±0.31^ab
(2)	5.25±0.32^a	7.67±0.51^a
			(3)	5±0.22^a	5.67±0.24^a
(4)	5.08±0.27^a	7.17±0.48^b

TABLE 12 comparison of root growth promoting effects

Group of	Root mean coefficient of 40 balance
		(1)	6.58
(2)	8
		(3)	5.75
(4)	6.5

In addition, the growth conditions of the plants of the group (1) (left side) and the group (2) (right side) banana potted plants after being cultured for 40 days are compared in the graph 7, and the graph shows that the banana potted plants watered by YN1910 fermentation liquid have obviously larger leaves and better growth conditions, so that the Bacillus belgii YN1910 provided by the invention has better growth promoting effect.

A comparison of the growth of the plants after 40 days of culture is shown in FIG. 8 for the banana potted plants of group (3) (left) and group (4) (right), where it can be seen that the banana plants infected with Foc 15-1 grew better after being watered with YN1910 fermentation broth. The banana plants which are not watered by the YN1910 fermentation liquor on the left side are short and have small and yellow leaves due to the influence of banana wilt, and the banana plants which are watered by the YN1910 fermentation liquor on the right side have good growth state, large leaves and few yellow leaves and are less influenced by banana wilt.

The root-cut results of banana pot plants of group (3) (left) and group (4) (right) are shown in fig. 9 after 40 days, and it can be seen that the color of the bulbs of banana pot plants treated with YN1910 is normal, while the color of the bulbs of banana pot plants not treated with YN1910 is red to dark brown, showing obvious symptoms of banana wilt.

The results of FIGS. 8 and 9 demonstrate that Bacillus belgii YN1910 used in the present invention has a good effect of controlling banana vascular wilt.

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 belgii, application thereof and method for preventing and treating banana wilt

<130> I68283YNN

<160> 16

<170> PatentIn version 3.5

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Claims

1. Bacillus belgiiBacillus velezensis) The bacillus beleisi has a preservation number of CGMCC NO. 21872.

2. Use of bacillus belgii according to claim 1 for inhibiting fungi.

3. Use according to claim 2, wherein the fungus is capable of causing banana vascular wilt to occur.

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

5. Use of the bacillus belgii of claim 1 for promoting plant growth.

6. A method of controlling banana vascular wilt, the method comprising: applying the Bacillus belgii and/or the Bacillus belgii fermentation broth to soil and/or banana plant roots infected with pathogenic bacteria of banana wilt;

wherein the Bacillus belgii has a preservation number of CGMCC NO. 21872.

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

8. The method of claim 6, wherein the Bacillus belgii fermentation broth is prepared by a process comprising: culturing the Bacillus belgii under a fermentation condition to obtain a fermentation liquid containing a metabolite of the Bacillus belgii.

9. The method of claim 8, 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.

10. The method of claim 8, wherein the concentration of the metabolite of Bacillus belgii in the fermentation broth is such that the OD of the fermentation broth₆₀₀The value is 2-2.5.

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

12. The method of claim 6, wherein said banana vascular wilt pathogenic bacteria is selected from the Fusarium oxysporum cubeba specialized type 4 physiological race tropical form.

13. The method according to claim 6 or 7, wherein the Bacillus belgii and/or the Bacillus belgii broth is used in an amount such that the content of Bacillus belgii in the soil infected with banana vascular wilt pathogens is 5 x 10 when applied to the soil⁷-5×10⁸CFU/kg。

14. A method according to claim 6 or 7, wherein the Bacillus belgii and/or Bacillus belgii broth is used in an amount such that the amount of Bacillus belgii per contact per banana plant is 5 x 10 when the Bacillus belgii and/or Bacillus belgii broth is applied to the banana plant root system⁷-5×10⁸CFU。