Application of Bacillus amyloliquefaciens JL54 in controlling larch shoot blight

Abstract

The present invention discloses the application of Bacillus amyloliquefaciens JL54 in controlling larch shoot blight, belonging to the field of microbial technology. The Bacillus amyloliquefaciens JL54 of the present invention is classified and named as Bacillus amyloliquefaciens JL54, and has been deposited in the China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 2023793, the deposit date: May 22, 2023, and the deposit address: Wuhan University, Wuhan, China. The antibacterial rate of the Bacillus amyloliquefaciens JL54 of the present invention against the pathogen of larch shoot blight is 63.16%; the inoculation pot experiment shows that the control effect of the treatment group inoculated with the endophyte JL54 on larch shoot blight is 50.0%. It can be seen that the Bacillus amyloliquefaciens JL54 of the present invention as a biocontrol bacterium will have wide applications in controlling larch shoot blight.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and more specifically, relates to the application of Bacillus amyloliquefaciens JL54 in controlling larch shoot blight. Background Art

[0002] Larch Larix gmelinii (Rupr.) Kuzen.] belongs to the genus Larix of the family Pinaceae Larix Mill.), and has excellent characteristics such as strong adaptability, fast growth, wide distribution, and resistance to pests and diseases. It is one of the three major coniferous timber tree species in Northeast China. The wood of larch is heavy and solid, with high compressive and bending strength, and is resistant to decay. It has high wood processing value and is excellent timber. At the same time, due to the tall and straight tree shape, beautiful crown shape, and well-developed root system of larch, it is also an excellent landscaping tree species.

[0003] Larch shoot blight Neofusicoccum laricinum (Sawada) Y. Hattori&C. Nakash] is one of the serious forest diseases in the world and a dangerous disease of larch plantations in China. It was first discovered in Hokkaido, Japan in 1938. By 1995, the disease had spread to Inner Mongolia, Hebei, Shanxi, Shandong, Shaanxi and other regions in China, with an occurrence area of more than 667,000 hm 2 , and the severely affected areas are mainly in the three northeastern provinces, with extremely serious harm. It is distributed in Japan, Korea, Russia and other places abroad. This disease rages in larch plantations in northern China. It is a very important fungal disease with great harm and extremely fast spread, and has been officially listed as a quarantine object in China. Different from the main inducement of larch shoot blight in Japan being strong wind, in China, the main forest ecosystem factors affecting this disease are topography, soil and forest age.

[0004] Plant endophytes refer to a class of microorganisms that can colonize inside plant cells or in the cell gaps and can establish a harmonious symbiotic environment with the host. Endophytes have a large variety and strong functions, and are mainly divided into three categories: endophytic fungi, endophytic bacteria and endophytic actinomycetes. During the coexistence time with the host plant, endophytes and their metabolites can promote the growth of the host plant and enhance the stress resistance and disease resistance of the host. Jiang Shilin et al. tested and studied the antibacterial properties of endophytic actinomycetes in the intestine of Periplaneta americana and found that endophytic actinomycetes have innate antibacterial advantages against intestinal bacteria; in field experiments, scientists showed through greenhouse root irrigation inoculation experiments B.subtilis against the wheat take-all pathogen Gaeumannomyces graminis var.tritici (Ggt)] with a control effect of 55.3%.

[0005] Chinese Patent CN 113897319 A discloses the application of Bacillus amyloliquefaciens G-7 in antagonizing Fusarium moniliforme and degrading fumonisin; Chinese Patent CN 115322928 A discloses the application of Bacillus amyloliquefaciens ZLP-0 in controlling plant pathogens, aquatic pathogens and human pathogens, as well as in controlling Lepidoptera pests, Blattodea pests, Hymenoptera pests, Scolopendromorpha pests, Hemiptera pests, Coleoptera pests or Orthoptera pests. However, there is no document studying the effect of Bacillus amyloliquefaciens in controlling larch shoot blight. Summary of the Invention

[0006] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide the application of Bacillus amyloliquefaciens JL54 in controlling larch shoot blight, for controlling larch shoot blight.

[0007] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] Bacillus amyloliquefaciens JL54 is classified and named as Bacillus amyloliquefaciens JL54, which has been deposited in the China Center for Type Culture Collection, with the deposit number: CCTCC: M 2023793, the deposit date: 20230522, and the deposit address: Wuhan University, China. The control of larch shoot blight is to reduce the disease index of larch shoot blight.

[0009] The control of larch shoot blight is to reduce the incidence of larch shoot blight.

[0010] The application of the bacterial fermentation broth of Bacillus amyloliquefaciens JL54 in controlling larch shoot blight.

[0011] The preparation method of the bacterial fermentation broth is as follows: The JL 54 endophyte stored at 4°C is streaked and inoculated on an NA plate, and activated and cultured at a constant temperature of 28°C for 36 h; One loop of single colonies is picked and inoculated into LB culture medium, and the seed liquid of the JL 54 endophyte is obtained after 1 d at 28°C and 200 r / min; The seed liquid of the JL 54 endophyte is inoculated into the sterilized and cooled LB culture medium at an inoculation amount of 1%, and cultured by shaking at 200 r / min and 28°C in the dark for 3 d to obtain the bacterial fermentation broth of Bacillus amyloliquefaciens JL54.

[0012] The application of Bacillus amyloliquefaciens JL54 in inhibiting the pathogen of larch shoot blight.

[0013] The pathogen of larch shoot blight is Neofusicoccum laricinum (Sawada) Y.Hattori&C. Nakash, with the strain number DHKS 6-3.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] For Bacillus amyloliquefaciens JL54 of the present invention, its antibacterial rate against the pathogen of larch shoot blight is 63.16%; when inoculating the bacterial fermentation broth of larch shoot blight for 14 days, the incidence rate of the pathogen inoculation control group reaches 100%, and the disease index reaches 72.22; in the treatment group inoculated with endophyte JL54, some pine seedlings have dry and falling needles. The number of diseased pine needles is less than that of the pathogen inoculation control group. The incidence rate is 80%, the control effect is 50.0%, and the disease index is 36.11. The pine seedlings in the blank control group grow normally and there are no obvious changes in the pine needles. It can be seen that Bacillus amyloliquefaciens JL54 of the present invention as a biocontrol bacterium will have a wide range of uses in the prevention and control of larch shoot blight. Description of the Drawings

[0016] Figure 1 It is a diagram showing the disease symptoms of larch after 14 days of inoculation with different treatments. Detailed Embodiments

[0017] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the technical means used are all conventional means well-known to those skilled in the art.

[0018] The test pathogens are as follows:

[0019] The pathogen of larch shoot blight is Neofusicoccum laricinum (Sawada) Y. Hattori & C. Nakash, with the strain number DHKS 6-3, provided by the Jilin Academy of Forestry Sciences, and isolated and identified from Larix olgensis plants with larch shoot blight in the larch forest area of Dunhua City, Yanbian Korean Autonomous Prefecture, Jilin Province.

[0020] The test larch is as follows:

[0021] Healthy larch branches and leaves for isolating endophytic bacteria were collected from 13 sampling points in China (Yunnan, Hunan, Hebei, Jilin, Liaoning, Heilongjiang, Inner Mongolia, Shandong). The test larch seedlings are 3-year-old Japanese larch, purchased from Gaoqiao Town, Huixian County, Longnan City, Gansu Province in October 2022, and transplanted into greenhouse flower pots on October 15, 2022.

[0022] The culture media used are as follows:

[0023] PDA medium: 200 g of potato, 20 g of glucose, 20 g of agar, 1 L of distilled water.

[0024] NA medium: 10 g of peptone, 5 g of sodium chloride, 3 g of beef extract, 20 g of agar, pH 7.3, 1 L of distilled water.

[0025] LB medium: 10 g of tryptone, 5 g of yeast extract, 4 g of sodium chloride, 1 L of distilled water.

[0026] After the above medium is prepared according to the formula, it is aliquoted and sterilized at 121 °C for 20 min.

[0027] The culture and antagonism test of Botryosphaeria laricina adopt PDA medium, and the isolation and culture of endophytic bacteria adopt NA medium and LB medium.

[0028] The test agents are as follows:

[0029] All chemical reagents are of analytical grade and are purchased from chemical reagent companies. The Ezup column bacterial genomic DNA extraction kit is purchased from Shanghai Sangon Biotech Co., Ltd., Taq enzyme is purchased from Nanjing Novoprotein Biological Co., Ltd., and PCR primers are synthesized by Shanghai Sangon Biotech Co., Ltd.

[0030] The experimental instruments are as follows:

[0031] Laminar flow cabinet (AC2-4S1, Artel High-Tech Co., Ltd., Singapore), autoclave (SX-500, Tomy Digital Biology Co., Ltd., Japan), oven (MOV-212F-PC, Panasonic Healthcare Co., Ltd., Japan), shaker (Multitron Standard, Evison Biotechnology Co., Ltd., China), incubator (MIR-553, Sanyo Electric Co., Ltd., Japan), refrigerator (HYCD-290, Haier Group, China), PCR instrument (nexus GSX1, Eppendorf AG, Germany), optical microscope (DM500, Leica Microsystems GmbH, Germany), electrophoresis apparatus (DYY-12C, Beijing Liuyi Instrument Factory), centrifuge (Centrifuge 5424R, Eppendorf AG, Germany). Example

[0032] 1. Culture of endophytic bacteria

[0033] Healthy larch branches and leaves were collected from 13 sampling points in 8 provinces respectively. The larch plant tissues were rinsed with running water to remove surface dirt, and then cleaned and air-dried. The leaves and branches were disinfected separately. For the branches, the outer bark needed to be peeled off before disinfection. They were successively washed with sterile water, disinfected with 75% ethanol for 60 s, then disinfected with 3% sodium hypochlorite for 30 s, and finally rinsed with sterile water 3 times. The sterile water from the last wash was used as a control and spread on NA solid medium. The disinfected branches were cut into lengths of about 2 cm and placed in NA solid medium. A total of 5 segments of branches were placed in the center and around each petri dish. For the leaves, there were two treatment methods: direct placement and homogenate spreading. The direct placement treatment method was the same as that of the branches. The disinfected leaves were cut into lengths of about 2 cm and placed in NA solid medium. A total of 5 pieces of leaves were placed in the center and around each petri dish. For homogenate spreading, the leaves were first ground into leaf pulp with a sterilized mortar, 200 μL of leaf pulp was aspirated with a pipette and placed in NA solid medium, and then the leaf pulp was evenly spread with a spreader. After sealing the treated NA medium, it was placed in an incubator at 28 °C for cultivation. After 2 - 3 d, the bacterial colonies grown on the medium were picked and streaked for cultivation. After 1 - 2 d, the growth of the colonies was observed, and single colonies were picked and inoculated on NA solid medium for purification. The purification was repeated twice, and a total of 391 endophytic bacteria were isolated.

[0034] The number of strains and strain numbers collected from each sampling point are shown in Table 1.

[0035] Table 1 Isolation of endophytic bacteria from larch

[0036]

[0037] 2. Screening of endophytic bacteria

[0038] The pathogen of larch shoot blight was inoculated on PDA medium and cultured in an environment of 28 °C for 5 d. The 391 obtained endophytic bacteria were preliminarily screened and re-screened by the plate confrontation method.

[0039] 1) Preliminary screening: The two-side streaking confrontation culture method was adopted. A vigorously growing mycelial cake (diameter 6 mm) of the pathogen of larch shoot blight was inoculated in the center of the PDA plate. Single colonies of endophytic bacteria were picked and streaked on both sides of the PDA plate. At the same time, plates with no endophytic bacteria streaking and only inoculated with pathogenic fungi were set as controls. Each of the above treatments was repeated 3 times. It was cultured in an incubator at 28 °C. When the control group filled the 90-mm petri dish, it was observed whether an inhibition zone was formed, and the colony diameters of the pathogens in the treatment group and the control group were measured.

[0040] 2) Re-screening: Using the four-point confrontation culture method, place a vigorously growing mycelial disc of Botryosphaeria laricina (inoculation diameter: 6 mm) in the center of a PDA plate (90 mm in diameter) containing 25 mL. Pick a single colony of endophytic bacteria and inoculate it at four diagonal points 3 cm away from the mycelial disc of the pathogen. Repeat each treatment 3 times. At the same time, set up a control plate with only the mycelial disc of the pathogen placed in the center of the plate without inoculation of endophytic bacteria. Incubate in a constant temperature incubator at 28 °C. When the control group fills the 90-mm petri dish, measure the colony diameter of the pathogen in the treatment group and the control group, and calculate the inhibition rate.

[0041] Measure the colony diameter of the control group and the treatment group using the cross method and calculate the inhibition rate. The formula for calculating the inhibition rate is as follows:

[0042] 。

[0043] Record the inhibition rate of the endophyte. The re-screening results show that the bacterium with the best antagonistic effect is JL 54, with a colony diameter of the pathogen of 33.16 mm and an inhibition rate of 63.16%. Example

[0044] 1. Preparation of endophyte fermentation broth

[0045] Streak inoculate the JL 54 endophyte stored at 4 °C on an NA plate and incubate it in a constant temperature incubator at 28 °C for 36 h for activation; pick a loop of single colony and inoculate it into a conical flask containing LB culture medium, and incubate it on a shaker at 28 °C and 200 r / min for 1 d to obtain the seed liquid of the JL 54 endophyte; use a pipette to take the seed liquid of the JL 54 endophyte and inoculate it into the sterilized and cooled LB culture medium at an inoculation amount of 1%, and incubate it on a shaker at 200 r / min and 28 °C in the dark for 3 d to obtain the fermentation broth of the JL 54 endophyte.

[0046] 2. Extraction of endophyte genomic DNA

[0047] Extract the genomic DNA of the JL 54 endophyte using a commercial DNA extraction kit (Sangon Biotech, Shanghai) according to the method for extracting endophyte genomic DNA. The specific steps are as follows:

[0048] 1) Take 0.5 - 1 mL of the overnight culture of bacterial liquid, add it to a 1.5-mL centrifuge tube, centrifuge at 8,000 rpm at room temperature for 1 min, discard the supernatant, and collect the bacterial cells. Add 180 μL of Buffer Digestion, then add 20 μL of Proteinase K solution, and shake well. Incubate in a water bath at 56 °C for 1 h until the cells are completely lysed.

[0049] Volume of bacterial solution: when OD600 ≥ 3, 500 µL of bacterial solution is sufficient; when OD600 < 3, 0.5 - 1 mL of bacterial solution is used. During the water bath process, invert and mix well every 10 minutes to promote sample lysis.

[0050] 2) Add 200 µL of Buffer BD, invert and mix well thoroughly, and incubate in a 70°C water bath for 10 minutes.

[0051] 3) Add 200 µL of absolute ethanol, invert and mix well thoroughly.

[0052] 4) Place the adsorption column into the collection tube, and use a pipette to add all the solution and the translucent fibrous suspension into the adsorption column. Let it stand for 2 minutes, then centrifuge at 12,000 rpm at room temperature for 1 minute, and pour out the waste liquid in the collection tube.

[0053] 5) Put the adsorption column back into the collection tube, add 500 µL of PW Solution, centrifuge at 10,000 rpm for 30 seconds, and pour out the filtrate.

[0054] 6) Put the adsorption column back into the collection tube, add 500 µL of Wash Solution, centrifuge at 10,000 rpm for 30 seconds, and pour out the filtrate.

[0055] 7) Put the adsorption column back into the collection tube, centrifuge at 12,000 rpm at room temperature for 2 minutes to remove the residual Wash Solution. Open the lid of the adsorption column and place it at room temperature for several minutes to thoroughly dry the residual Wash Solution in the adsorption material. The residual Wash Solution will affect the yield of genomic DNA and subsequent experiments.

[0056] 8) Take out the adsorption column, put it into a new 1.5 mL centrifuge tube, add 50 - 100 µL of CE Buffer, let it stand for 3 minutes, centrifuge at 12,000 rpm at room temperature for 2 minutes, and collect the DNA solution. The extracted DNA can be immediately used for the next experiment or stored at -20°C.

[0057] 3. PCR Amplification

[0058] Select the universal bacterial primers 27 - F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492 - R (5’-GGTTACCTTGTTACGACTT-3’). For the PCR amplification of the gyrB gene sequence, use the gyrB gene primers UP - 1 (5’-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3’) and UP - 2r (5’-AGCAGGATACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3’).

[0059] PCR reaction system (20 µL): 10 µL of Green Taq Mix, 1 µL each of forward and reverse primers, 2 µL of template DNA, 6 µL of ddH2O.

[0060] 16S rDNA PCR reaction procedure: Pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 48 °C for 30 s, extension at 72 °C for 90 s, 30 cycles, final extension at 72 °C for 10 min.

[0061] gyrB gene sequence PCR reaction procedure: Pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 90 s, 30 cycles, final extension at 72 °C for 5 min.

[0062] 4. Sequencing

[0063] Using strain JL 54 as a template, after PCR amplification with 16S rDNA specific primers, the amplification results were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The two sequencing results of the forward and reverse primers were spliced using DNA MAN software, and the obtained target sequence is shown in SEQ ID NO.1, resulting in a 1445 bp sequence. Alignment with known sequences on NCBI showed that the similarity between strain JL 54 and Bacillus amyloliquefaciens Bacillus amyloliquefaciens (accession number: MG548650.1) was 99.72%. It has been deposited in the China Center for Type Culture Collection, and its taxonomic name is Bacillus amyloliquefaciens JL54, deposit number: CCTCC: M 2023793, deposit date: 20230522, deposit address: Wuhan University, China. Example

[0064] For root irrigation inoculation, use a pipette to inject 25 mL of the endophytic bacteria fermentation broth (1×10 7 CFU / mL) into the root soil of larch potted seedlings in 5 aliquots of 25 mL each. After 2 days, inoculate the larch shoot blight pathogen cake; for each seedling, select 1 main branch and 2 - 3 well-growing lateral branches, make a tangential cut at the lower part of the shoot tip with a sterile scalpel, then attach the pathogen cake to the cut surface so that the pathogen contacts the tangential cut surface of the shoot tip. Use a sealing film to make an inverted conical shape to wrap the cake, and then add distilled water to it for moisturizing treatment to maintain the activity of the pathogen. The negative control group was first inoculated with 25 mL of sterile water and then the larch shoot blight pathogen cake. The blank control group was only inoculated with 25 mL of sterile water, and 1 main branch and 2 - 3 well-growing lateral branches were selected to make wounds with a sterile scalpel.

[0065] The processed larch seedlings were placed in an incubator at 28 °C. A humidifier was used in the greenhouse to create a hot and rainy environment. The disease incidence was observed daily, and distilled water was added to the sealing film. After 14 days, the disease incidence, disease index, and control effect were calculated.

[0066] Grading criteria for larch shoot blight: Grade 0: The whole plant is disease-free; Grade 1: Less than 1 / 4 of the needles are diseased; Grade 2: 1 / 4 - 1 / 2 of the needles are diseased; Grade 3: 1 / 2 - 3 / 4 of the needles are diseased; Grade 4: More than 3 / 4 of the needles are diseased; Grade 5: The diseased plant is basically dead.

[0067] The calculation formulas for disease incidence, disease index, and control effect are as follows:

[0068] 。

[0069] 。

[0070] 。

[0071] The results are as Figure 1 shown. Seven days after inoculation with the larch shoot blight pathogen, the control group inoculated with the pathogen began to show symptoms, with yellowing needles, while the seedlings in the treatment group did not show disease. Nine days after inoculation, the symptoms in the control group inoculated with the pathogen were obvious, and the needles of the diseased seedlings began to wither and fall off. Yellowing signs appeared in the seedlings of the treatment group. Fourteen days after inoculation, the disease incidence in the control group inoculated with the pathogen reached 100%, and the disease index reached 72.22. In the treatment group inoculated with the endophyte JL54, some needles of the seedlings withered and fell off. The number of diseased needles was less than that in the control group inoculated with the pathogen. The disease incidence was 80%, the disease index was 36.11, and the control effect was 50.0%. The disease index was 36.11 (Table 2). The seedlings in the blank control group grew normally, and there were no obvious changes in the needles.

[0072] Table 2 Control effect of antagonistic strains on larch shoot blight

[0073]

Claims

1. Application of Bacillus amyloliquefaciens JL54 in controlling larch shoot blight, wherein the Bacillus amyloliquefaciens JL54 is taxonomically named Bacillus amyloliquefaciens JL54, which has been deposited in the China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 2023793, the deposit date: May 22, 2023, and the deposit address: Wuhan University, Wuhan, China; The pathogen of the larch shoot blight is Neofusicoccum laricinum (Sawada) Y. Hattori & C. Nakash, and the strain number is DHKS 6-3.

2. Use of Bacillus amyloliquefaciens JL54 according to claim 1 in controlling larch shoot blight, characterized in that, The prevention and control of larch shoot blight mentioned above is to reduce the incidence index of larch shoot blight.

3. Use of Bacillus amyloliquefaciens JL54 according to claim 1 in the prevention and control of larch shoot blight, characterized in that, The prevention and control of larch shoot blight mentioned above is to reduce the incidence rate of larch shoot blight.

4. Application of the bacterial fermentation broth of Bacillus amyloliquefaciens JL54 in controlling larch shoot blight, wherein the Bacillus amyloliquefaciens JL54 is taxonomically named Bacillus amyloliquefaciens JL54, which has been deposited with the China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 2023793, the deposit date: May 22, 2023, and the deposit address: Wuhan University, Wuhan, China; The pathogen of the larch shoot blight is Neofusicoccum laricinum (Sawada) Y. Hattori &C. Nakash, and the strain number is DHKS 6-3.

5. Use of the bacterial fermentation broth of Bacillus amyloliquefaciens JL54 according to claim 4 in the prevention and control of larch shoot blight, characterized in that, The preparation method of the bacterial fermentation broth is as follows: Streak inoculate the JL 54 endophyte preserved at 4°C on an NA plate and incubate it at a constant temperature of 28°C for 36 h for activation; Pick a loop of single colonies and inoculate them into an LB culture medium, and obtain the seed liquid of the JL 54 endophyte after 1 day at 28°C and 200 r / min; Take the seed liquid of the JL 54 endophyte and inoculate it into the sterilized and cooled LB culture medium at an inoculation amount of 1%, and shake and culture it at 200 r / min and 28°C in the dark for 3 days to obtain the bacterial fermentation broth of Bacillus amyloliquefaciens JL54.

6. Application of Bacillus amyloliquefaciens JL54 in inhibiting the pathogen of larch shoot blight, and the Bacillus amyloliquefaciens JL54 is classified and named as Bacillus amyloliquefaciens JL54, which has been deposited in the China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 2023793, the deposit date: May 22, 2023, and the deposit address: Wuhan University, Wuhan, China; The pathogen of larch shoot blight is Neofusicoccum laricinum (Sawada) Y. Hattori & C. Nakash, and the strain number is DHKS 6-3.

Images