CN107858315B

CN107858315B - Bacillus amyloliquefaciens for preventing and treating lotus seed rot disease and application thereof

Info

Publication number: CN107858315B
Application number: CN201711372342.6A
Authority: CN
Inventors: 肖炎农; 张臻; 孟俊峰; 王高峰; 肖雪琼
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2017-12-14
Filing date: 2017-12-14
Publication date: 2020-09-11
Anticipated expiration: 2037-12-14
Also published as: CN107858315A

Abstract

The invention discloses bacillus amyloliquefaciens for preventing and treating lotus seed rot disease and application thereof, wherein the bacillus amyloliquefaciens is: (Bacillus amyloliquefaciens) B-36, accession number: CCTCC NO: m2017652. In an indoor flat plate confronting test, the growth inhibition rate of the B-36 strain to Fusarium reaches 74.07%; the growth inhibition rate of the B-36 fermentation liquor with the concentration of 20 percent on fusarium is as high as 90.46 percent; indoor pot culture test results show that the leaf diameter and leaf height of the lotus seedpod in the B-36 bacterial suspension treatment group are obviously improved relative to blank control, and the effect of preventing and treating the lotus seedpod rot in the later growth stage of the lotus seedpod can reach 46.20 percent, so that the strain is a biocontrol bacterium with great development potential for preventing and treating the lotus seedpod rot.

Description

Bacillus amyloliquefaciens for preventing and treating lotus seed rot disease and application thereof

Technical Field

The invention belongs to the technical field of biological control of plant diseases, and particularly relates to bacillus amyloliquefaciens for controlling a lotus seed rot disease and application thereof.

Background

The lotus seed is a common aquatic vegetable, is fresh, sweet and delicious, can be prepared into various dishes, and is a common food material. In addition, the lotus seeds, lotus plumule, lotus leaves, lotus flowers and the like have various health-care and medicinal functions. Therefore, the lotus seed industry has been rapidly developed in recent years, and is a new point for increasing income of farmers. In the production of the lotus seeds, the lotus root pond creates conditions for the occurrence of the rot disease induced by the infection of the lotus seeds by pathogenic bacteria in successive years. Pathogenic bacteria are accumulated in the lotus root pond year by year due to residue of lotus rhizome nodes and other residues, so that the number and variety of the pathogenic bacteria in the lotus root pond are large, and the occurrence degree of the rot disease of the lotus seeds is increased year by year. Due to dilution and diffusion of a water body, pathogenic bacteria are transferred along with water flow, so that the whole pond becomes a disease attack infection point, the disease attack of the whole pond is often presented, and the major loss of the whole pond is caused.

The causative agent of the lotus seed rot disease is Fusarium spp, of which Fusarium oxysporum is the most common, and Fusarium equiseti (Fusarium equiseti) isolated from diseased plants by the applicant can also cause typical diseases. After the pathogens infect plant tissues, they often cause rot of the infected tissue parts, which then spread throughout the plant, greatly reducing the quality and yield of the crop. The pathogeny comes from soil or lotus root-carried bacteria, and can not be eradicated, so that the breeding of disease-free lotus root and the elimination of the pathogeny in the lotus root field are the keys for preventing and treating the rot disease of the seed lotus. At present, the prevention and treatment of the disease mainly uses chemical agents, and the prevention and treatment methods such as agricultural auxiliary measures, ecological prevention and treatment and the like have less investment and little effect.

For a long time, the pest control of crops in China mainly depends on chemical pesticides, and in the production of the lotus seeds, the rot disease is mainly controlled chemically. As the rot disease of the lotus seed belongs to vascular bundle disease, the prevention and treatment period is generally at the early stage of the disease. The main application method of the chemical agent comprises the measures of disinfecting soil and lotus roots by using the chemical agent, spraying leaf surfaces, injecting stems and the like. The chemical agent is efficient and easy to use, but the pesticide pollution is serious, and the pesticide detection rate in food is up to more than 90%, which seriously harms the physical health of consumers. Therefore, pesticide residue is an urgent problem to be solved in food safety production. Compared with chemical pesticides, biological pesticides have the advantages of safety, environmental protection, no residue and the like. The development of biopesticides focusing on low toxicity, high efficiency and strong selectivity has become one of the current research hotspots.

Currently, many researchers advocate the development of biocontrol technologies to control blight of plants. The biological control measures are environment-friendly, safe to people and livestock and can effectively control the occurrence and spread of diseases, so the biological control measures are favored. The biocontrol bacteria reported at present are Bacillus (Bacillus spp.), Trichoderma (Trichoderma spp.), and the like. The bacillus is a biological control bacterium with the advantages of wide distribution, strong bacteriostatic broad-spectrum performance and wide application, and has good prevention and control effect.

Due to the water habit of the lotus seed, the biological control process of the putrefaction disease is difficult to operate, and related research is lacked. Research on the use of bacillus amyloliquefaciens for preventing and treating the rot disease of the lotus seedpod has not been reported.

Disclosure of Invention

The first purpose of the invention is to provide a bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B-36 (hereinafter referred to as B-36 strain), which has stronger control effect on the lotus rot disease.

The second purpose of the invention is to provide the application of the bacillus amyloliquefaciens B-36 or the fermentation product thereof in preparing a biological preparation medicament for preventing and treating the lotus seed rot disease.

The third purpose of the invention is to provide the application of the bacillus amyloliquefaciens B-36 in promoting the growth of the lotus seed.

In order to achieve the purpose, the invention adopts the following technical measures:

bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B-36 obtained by the following steps:

the applicant obtains a strain with strong control effect on the lotus rot disease by separating and screening from the lotus pond base of China agriculture university in Wuhan City, Hubei province, China, and names the strain B-36. Identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) through morphological and molecular biological identification. The strain is preserved in China Center for Type Culture Collection (CCTCC) in 2017 at 11 and 3 months, wherein the preservation address is as follows: wuhan university in Wuhan, China, the preservation number: CCTCC NO: m2017652.

Bacterial colony characteristics of the bacillus amyloliquefaciens B-36 strain: the bacterial colony is milky white, flat, smooth and nearly round, the edge is jagged, the bacterial colony is opaque and dull, the texture is dry, and no pigment is generated; gram staining was positive.

The application of the bacillus amyloliquefaciens B-36 and the fermentation product thereof in preparing a biological preparation medicament for preventing and treating the lotus seed rot disease comprises the step of preparing the medicament for preventing and treating the lotus seed rot disease by using the strain or the fermentation product thereof as the only effective component or one of the effective components.

The application of the bacillus amyloliquefaciens B-36 in promoting the growth of the lotus seedpod comprises the step of using the bacillus amyloliquefaciens B-36 fermentation liquor to prepare a lotus seedpod growth promoting preparation.

Compared with the prior art, the invention has the following advantages:

1. the invention screens out a new strain B-36 for preventing and treating the lotus rot disease, the strain is a bacillus amyloliquefaciens, and no relevant report about the strain for preventing and treating the lotus rot disease exists at present.

2. In a flat plate confronting test of the B-36 strain screened by the invention and the Fusarium equiseti indoors, the growth inhibition rate of the strain on the Fusarium equiseti reaches 74.07%. The inhibition rates of the fermentation supernatant of B-36 with the concentrations of 1%, 5%, 10% and 20% on the growth of fusarium equiseti are 42.74%, 74.03%, 89.78% and 90.6% respectively. The results of tests on the potted lotus rot disease by the B-36 bacterial suspension show that the incidence rates of the rot disease of the lotus are respectively 37.35%, 31.19% and 37.92% after the lotus is treated by the B-36 bacterial suspension in the early stage, the middle stage and the later stage of the growth of the lotus, and the relative prevention effects of the B-36 bacterial suspension on the rot disease of the lotus are respectively as follows: 47.19%, 53.52%, 46.20%.

3. The strain used by the invention has higher prevention effect on the rot disease of the lotus seeds and is nontoxic to people and livestock; the cost is low.

4. The invention utilizes the zymocyte liquid of the bacillus amyloliquefaciens B-36 strain to directly prevent and treat the rot disease of the lotus seed, has very simple production and use, does not use organic solvent in the production, reduces the pollution in the production process and products, does not produce solvent poisoning, and has the advantage that the biopesticide is safe to people and livestock.

Drawings

FIG. 1 is a micrograph of colony morphology and gram staining of strain B-36 on NA plates.

FIG. 2 shows the colony morphology of strain B-36 against a Fusarium equiseti indoor plate for 3 days.

FIG. 3 is a colony morphology of the effect of 20% concentration fermentation broth of strain B-36 on the growth of Fusarium equiseti.

Detailed Description

The technical scheme of the invention is the conventional technology in the field if not particularly stated. The reagents or materials used in the present invention, if not specifically mentioned, are commercially available.

Example 1: isolation and identification of Bacillus amyloliquefaciens B-36 Strain

1. Isolation of B-36 Strain

The applicant obtains a strain with strong control effect on the lotus root rot disease by separating and screening from lotus root pond-based blueberries of agriculture university in Huazhong, Wuhan City, Hubei province in China, and the strain is named as B-36. The B-36 strain of the present invention is isolated and purified by a dilution coating plate method, plate streaking, bioassay, or the like. The specific method comprises the following steps:

(1) collecting 30 pieces of lotus seed tubers as materials, cleaning the materials by using running water, adding a detergent during cleaning or treating the materials by using ultrasonic waves during the washing by using the running water in order to ensure the cleaning effect, and finally washing the materials by using sterile water;

(2) soaking the tissue cut blocks washed by sterile water in 75% alcohol for 5min, soaking in 2% sodium hypochlorite for 5min, rinsing with sterile water for 3 times, placing in 80 deg.C sterile water bath for 10min, and removing excess water on sterile filter paper; cutting the sterilized plant material into small pieces by using a sterile tool, grinding or adding a certain amount of quartz sand and grinding again to obtain homogenate, and then coating a PDA flat plate;

(3) screening the separated bacillus by adopting a plate confronting method, and selecting antagonistic bacteria with the largest radius of a bacteriostatic zone (from the center of a bacterial colony to the edge of a pathogenic bacterium hypha) to obtain the bacillus amyloliquefaciens B-36 with the highest growth inhibition rate on the fusarium equiseti.

2. Identification of strains

Morphological identification of the strains: the color, morphology, edges, transparency, gloss, color of the medium, etc. of the colonies were observed, and the morphological characteristics of the cells were observed by gram staining.

The B-36 strain was streaked on the NA medium plate, and it can be seen from FIG. 1 that: b-36 bacterial colony is milky white, flat, smooth and nearly round, the edge is jagged, the bacterial colony is opaque, dull, dry in texture and free of pigment; gram staining was positive, and the above morphological characteristics indicated that strain B-36 belongs to the genus Bacillus.

Molecular characterization of Strain B-36: b-36 single colonies are picked by an inoculating loop in a subpackaged NA liquid culture medium in a sterile operating platform, shake culture is carried out for 12h under the conditions of 35 ℃ and 180rpm, and bacterial liquid PCR amplification is carried out by using gyrA gene design primers gyrA-f (5'-CAGTCA GGA AAT GCG TAC GTC CTT-3') and gyrA-r (5'-CAA GGT AAT GCT CCA GGC ATT GCT-3'). The PCR amplification procedure was: pre-denaturation at 95 deg.C for 5min, 30s at 95 deg.C, 30s at 55 deg.C, 45s at 72 deg.C, 30 cycles, 10min at 72 deg.C, and storage at 4 deg.C. And detecting the PCR reaction product by 1.5% agarose gel electrophoresis, and sending the PCR reaction product to Wuhan Tianyihuiyuan company for sequencing after the PCR reaction product is qualified.

The gyrA gene fragment of the strain B-36 obtained by sequencing has the size of 1004bp, the sequence of the gyrA gene fragment is shown as SEQ ID NO.1, the strain with higher homology with the gyrA gene fragment is selected by Blast analysis, and phylogenetic analysis and an NJ method are utilized to construct a phylogenetic tree by using software MEGA 6.0. The result shows that B-36 and Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) are polymerized on one branch, the statistical support rate is 100%, and B-36 is identified as the Bacillus amyloliquefaciens by combining the prior morphological characteristics and physiological and biochemical experiments.

The Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) B-36 is preserved in China Center for Type Culture Collection (CCTCC) at the preservation address: wuhan university in Wuhan, China, the preservation date: 11/3/2017, accession number: CCTCC NO: m2017652.

The NA culture solution used for culturing the B-36 strain comprises the following components and formula: 5g of peptone, 2.5g of glucose and 3g of beef extract, supplementing distilled water to 1000mL, adjusting the pH value to 7.0, and sterilizing for 30min at 121 ℃ by high-pressure steam.

3. Shake flask fermentation culture

A single bacterial colony is picked up by an inoculating loop, inoculated in a 10mL centrifuge tube containing 4mL NA culture solution, shaken at 28 ℃ and 280r/min for 24h, transferred into a conical flask containing 100mL PDB culture medium by 1% of volume ratio, and shaken at 28 ℃ and 280r/min for 72 h.

The PDB culture medium comprises the following components: 200g of potato, 20g of glucose and 1000mL of distilled water, and sterilizing at 121 ℃ for 30 min.

After the fermentation broth after 72h of shake culture was centrifuged at 12000r/min for 10min, the supernatant was collected, filtered by a cloth funnel, filtered through a filter membrane with a pore size of 0.45 μm, and the fermentation broth was finally passed through a disposable 0.22 μm sterile filter under sterile conditions to obtain a sterile filtrate, which was used in example 3.

Example 2: effect of B-36 Strain on the growth of Fusarium equiseti in plates

Selecting B-36 single colony, drawing two straight lines at positions 2cm away from the center of the plate, simultaneously transferring agar blocks of Fusarium equiseti with diameter of 0.3cm into the center of the plate, culturing in 25 deg.C incubator, and repeating each strain for 4 times. Controls were not streaked and only Fusarium equiseti hypha blocks were inoculated. After 3 days, the radius of the inhibition zone (from the center of the B-36 colony to the edge of the hypha of the pathogenic bacteria) is measured, and the inhibition rate is calculated.

Inhibition rate ═ (growth rate of control fusarium-growth rate of treated fusarium)/growth rate of control fusarium × 100%.

TABLE 1B-36 Effect on Fusarium equiseti growth in plates (3d, 25 ℃ C.)

The results of the effect of B-36 strain on the growth of Fusarium equiseti in plates show that: the B-36 strain can obviously inhibit the growth of the fusarium equiseti in a flat plate (see figure 2), and the inhibition rate of the B-36 strain on the fusarium equiseti reaches 74.07%.

Example 3: effect of B-36 Strain fermentation supernatant on the growth of Fusarium equiseti

Mixing the sterile filtrate B-36 with a proper-temperature PDA culture medium according to the volume ratio (V/V) of 1%, 5%, 10% and 20% respectively to prepare a PDA flat plate, inoculating activated fusarium equiseti mycelium blocks, culturing and observing in the dark at 25 ℃, measuring the diameter of fusarium colonies by using a cross method after 7 days, and calculating the bacteriostasis rate. The PDA medium was inoculated with Fusarium equiseti as a control.

TABLE 2B-36 Effect of fermentation supernatant on Fusarium equiseti growth (7d, 25 deg.C)

The effect of the B-36 strain fermentation supernatant on the growth of Fusarium equiseti shows that: the B-36 strain fermentation supernatant with different concentrations has certain inhibition effect on the growth of fusarium equiseti, the inhibition rate is increased along with the increase of the concentration of the fermentation liquid, and the inhibition rate is the highest and reaches 90.46% when the concentration is 20% (Table 2). The test shows that the B-36 strain fermentation supernatant has strong inhibition effect on the growth of fusarium equiseti.

Example 4: plastic greenhouse potting test for preventing and treating rot disease of lotus seedpod by B-36 strain

Planting the seed lotus: the soil is prepared from non-diseased lotus pond sludge in a test field and field soil according to the volume ratio of 1: 1 and mixing. In the middle and late ten days of 4 months, disease-free lotus tubers (space lotus No. 6) with consistent sizes are screened as test materials, planted in flowerpots with the caliber of 30cm and grown under natural conditions.

B-36 bacterial suspension preparation: and (3) selecting a single B-36 colony, inoculating the single B-36 colony into a triangular flask filled with 200mL of NA liquid culture medium, and shaking and culturing at 28 ℃ and 180rpm for 72 hours to obtain a B-36 bacterial suspension.

Inoculating pathogenic bacteria, inoculating Fusarium oxysporum F-8 (isolated from Amur Lotus rhizome field of agriculture university in Huazhong of Wuhan, Hubei province) which is a putrefactive pathogenic bacterium after the first vertical leaf of the seed lotus grows out, and sucking the Fusarium oxysporum 1 × 10 with a syringe⁴CFU/mL spore suspension was injected into the tubers of the lotus seeds at 100. mu.L/strain.

Inoculating antagonistic strain B-36 by injection: inoculating the seed lotus with fusarium oxysporum spore liquid for 15d and then inoculating the antagonistic strain. A total of 3 treatments were set, each treatment was repeated 3 times, and a positive control of 500mL of carbendazim spray and a control of pathogen only were set. And counting the morbidity after 1, 2 and 3 months after the injection and inoculation of the antagonistic strain. During the third statistics after inoculation, the disease occurrence condition is only counted on the vertical leaves due to the physiological decay of the floating leaves of the lotus seeds, and the disease incidence and the disease prevention effect are calculated.

The process settings were as follows:

the incidence (%) is diseased leaf number/total leaf number × 100%

Control effect (%) - (control incidence-treatment incidence)/control incidence × 100%

TABLE 3 determination of disease incidence and disease prevention effect for different treatments at different growth stages

Note: spray carbendazim treatment as a positive control; the same letters after the same column of data indicate that the differences are not significant (LSD, P ≦ 0.05).

The investigation result of the growth condition of the lotus seeds shows that: comparing the incidence and disease prevention effect of rot disease in the early, middle and later stages of the growth of the lotus after inoculation of antagonistic strains. As shown in table 3: in 3 investigation periods, the incidence of the B-36 strain injection treatment group is reduced compared with that of a control group, and the difference is obvious; along with the growth, the incidence of the injection treatment is reduced to a greater extent compared with the control group, and the injection treatment of the B-36 strain shows a significant difference compared with the control group to the later growth stage. The incidence of the positive control group increases along with the increase of the growth time, the incidence of the positive control group in each group treated by inoculation shows a decreasing trend, and the incidence of the positive control group in the middle and later growth period is higher than that of the group treated by the B-36 strain injection.

TABLE 4 biological Properties of the treated cotyledons at the late growth stage

The biological characters of the lotus seeds in each group at the later growth stage are observed, and the results are shown in table 4: compared with a control only inoculated with pathogenic bacteria, the ratio of the stand leaves of the treated group is increased by 13.7-26.1%, wherein the B-36 strain injection treated group achieves a significant difference compared with the control; the treatment groups are higher than the control group in the height of petioles, the diameter of leaves and the ratio of vertical leaves, and have obvious difference on the level of 0.05, thereby further indicating that the B-36 strain has a certain promotion effect on the growth of the agapanthus.

Sequence listing

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Claims

1. Bacillus amyloliquefaciens (A)Bacillus amyloliquefaciens) The method is characterized in that: the preservation number of the bacillus amyloliquefaciens is CCTCC NO: m2017652.

2. Use of a bacillus amyloliquefaciens strain according to claim 1 for the preparation of a medicament for the treatment or prevention of a disease caused by fusarium oxysporum (f) f (f) or (f) spFusarium oxysporum) Or Fusarium equiseti (F.), (Fusarium equiseti) Application of the preparation for treating the caused lotus seed rot disease.

3. The use of a fermentation broth of bacillus amyloliquefaciens according to claim 1 for inhibiting the growth of a microorganism selected from the group consisting of fusarium oxysporum (f) fFusarium oxysporum) Or Fusarium equiseti (F.), (Fusarium equiseti) Application in treating rhizoma et radix Veratri Thalictrifoliae putrefaction.

4. The use of bacillus amyloliquefaciens according to claim 1 for promoting the growth of lotus seedpod.