Application of bacillus velezensis yn2 in preventing and treating larch canker

Abstract

The application discloses an application of bacillus velezensis YN2 in preventing and treating larch canker disease, and belongs to the technical field of microorganisms.The classification and naming of the bacillus velezensis YN2 of the application is Bacillus velezensis YN2, which has been preserved in the China Center for Type Culture Collection, with a preservation number of CCTCC NO: M 2023792, a preservation date of May 22, 2023, and a preservation address of Wuhan University in Wuhan, China.The bacillus velezensis YN2 of the application has an inhibition rate of 65.08% on a larch canker disease pathogen.Pot experiment shows that the bacillus velezensis YN2 has a control effect of 57.7% on the larch canker disease.It can be seen that the bacillus velezensis YN2 of the application will have a wide application as a biocontrol agent in preventing and treating the larch canker disease.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microorganisms, and more particularly relates to application of bacillus velezensis YN2 in prevention and treatment of larch canker. BACKGROUND

[0002] Larch canker [Neofusicoccum laricinum (Sawada) Y. Hattori & C. Nakash] is one of the most serious forest diseases in the world, and is a dangerous disease of larch plantations in China. It was first discovered in Hokkaido, Japan in 1938. As of 1995, the disease has spread to more than 130 cities in nine provinces and regions of China, including Inner Mongolia, Hebei, Shanxi, Shandong and Shaanxi, with an occurrence area of more than 66.7 million hm 2 , and the main heavy-occurrence areas are in the three northeastern provinces, with extremely serious harm. It is distributed in Japan, North Korea, South Korea and Russia abroad. The disease is rampant in larch plantations in northern China and is a very important fungal disease with great harm and rapid spread, and has been officially listed as a quarantine object. Unlike the main cause of larch canker in Japan, which is strong wind, the main ecological factors affecting the disease in China are topography, soil and stand age.

[0003] Larch canker is caused by Neofusicoccum laricinum (Sawada) Y. Hattori & C. Nakash, and is a dangerous fungal disease. The ascocarps of the pathogen begin to be buried under the epidermis of the diseased new shoots and the top residual needles, and are single or clustered, mature and break through the epidermis in the spring of the second year after overwintering, and are black and nearly spherical; the ascospores are club-shaped and double-walled, arranged in rows at the base of the ascus cavity, and have a large number of pseudoparenchyma; the ascospores are colorless, single-celled and oval. The conidiophores are black and spherical, and are generated under the epidermis of the top branch leaves and diseased shoots; the conidiospores are colorless and single-celled; the anamorphs are colorless and long.

[0004] The pathogen overwinters in the epidermis of the diseased new shoots and the top residual leaves with immature ascocarps and mycelium, and the ascospores and conidiospores are the initial infection sources. In early June, the ascocarps will break through the host, and the ascospores will appear between the pseudoparenchyma in the ascus cavity, and gradually produce ascospores, which mature in the middle and late of the month and show obvious pathogenicity. The conidiophores appear on the top branch leaves 10-20 days after the larch is infected. The pathogen can be transmitted by wind and rain, and enters the pine through the wound. Frost damage and frost damage can also make the pathogen invade the wound, and at the same time, weaken the growth of larch, which is more conducive to the infection of larch canker.

[0005] Chinese patent CN 115651857 A discloses the application of Bacillus velezensis HS1 in the preparation of tea tree amino acid foliar fertilizer; Chinese patent CN 116042436 A discloses the application of Bacillus velezensis SF334 in the preparation of a preparation for inhibiting Colletotrichum siamense and / or Colletotrichum australisinense, and the application of its fermentation liquor in the preparation of a plant growth promoting preparation; Chinese patent CN 115181693 A discloses the application of Bacillus velezensis 021TIBBST23 in inhibiting Fusarium oxysporum, Botryosphaeria dothidea, Rhizoctonia solani, Ralstonia solanacearum and Diapthosphae citri in solanaceous plants. However, there is no document researching the effect of Bacillus velezensis in preventing and treating larch dieback. SUMMARY

[0006] In view of the above problems existing in the prior art, the technical problem to be solved by the present application is to provide the application of Bacillus velezensis YN2 in preventing and treating larch dieback, for preventing and treating larch dieback.

[0007] To solve the above technical problems, the technical scheme adopted by the present application is as follows:

[0008] The application of Bacillus velezensis YN2 in preventing and treating larch dieback, wherein the Bacillus velezensis YN2 is classified and named as Bacillus velezensis YN2, has been preserved in the China Center for Type Culture Collection, with the preservation number CCTCC: M 2023792, the preservation date 20230522, and the preservation address Wuhan University, China.

[0009] The larch dieback to be prevented and treated is to reduce the disease index of larch dieback.

[0010] The larch dieback to be prevented and treated is to reduce the incidence of larch dieback.

[0011] The application of the bacterial fermentation liquor of Bacillus velezensis YN2 in preventing and treating larch dieback.

[0012] The preparation method of the bacterial fermentation liquor is as follows: the YN 2 endophyte preserved at 4℃ is streak inoculated on a NA plate, and activated and cultured at 28℃ for 36h; a single colony is picked and inoculated in LB culture solution, and seed solution of the YN 2 endophyte is obtained under the condition of 28℃ and 200r / min for 1d; the seed solution of the YN 2 endophyte is inoculated into sterilized and cooled LB culture solution at an inoculation amount of 1%, and the bacterial fermentation liquor of Bacillus velezensis YN2 is obtained by oscillation culture under the condition of 200r / min and 28℃ in the dark for 3d.

[0013] Application of Bacillus velezensis YN2 in inhibiting the pathogen of larch canker.

[0014] The pathogen of larch canker is Neofusicoccum laricinum (Sawada) Y. Hattori & C. Nakash, and the strain number is DHKS 6-3.

[0015] Compared with the prior art, the beneficial effects of the present application are:

[0016] The bacteriostatic rate of the Bacillus velezensis YN2 of the present application on the pathogen of larch canker is 65.08%; 14 days after inoculation of the larch canker bacterial fermentation liquor, the morbidity of the inoculated pathogen control group reaches 100%, and the disease index reaches 72.22; the needles of part of the pine seedlings in the endophyte YN2 treatment group are dry and shed, the diseased pine needles are less than those in the inoculated pathogen control group, the morbidity is 60%, the control effect is 57.7%, and the disease index is 30.56. The pine seedlings in the blank control group grow normally, and the pine needles have no obvious change. It can be seen that the Bacillus velezensis YN2 of the present application as a biocontrol agent will have a wide application in the prevention and control of larch canker. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Figure 2 is a picture of the disease symptoms of larch after inoculation for 14 days under different treatments. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described below in combination with specific examples. In the following examples, the technical means used are all conventional means known to those skilled in the art unless otherwise specified.

[0019] The test pathogen is as follows:

[0020] The pathogen of larch canker is Neofusicoccum laricinum (Sawada) Y. Hattori & C. Nakash, and the strain number is DHKS 6-3, which is provided by Jilin Provincial Institute of Forestry Science, and is isolated and identified from the larch canker plant Larix olgensis in larch forest area of Dunhua City, Yanbian Korean Autonomous Prefecture, Jilin Province.

[0021] The test larch is as follows:

[0022] Healthy larch branches and leaves for isolating endophytic bacteria were collected from 13 sampling sites in 8 provinces (Yunnan, Hunan, Hebei, Jilin, Liaoning, Heilongjiang, Inner Mongolia, Shandong) of China. The test larch seedlings were 3-year-old Japanese larches purchased from Gaotun County, Longnan City, Gansu Province in October 2022, and transplanted into greenhouse pots on October 15, 2022.

[0023] The culture medium used is as follows:

[0024] PDA culture medium: potato 200 g, glucose 20 g, 20 g agar, distilled water 1 L.

[0025] NA culture medium: peptone 10 g, sodium chloride 5 g, beef extract 3 g, agar 20 g, pH 7.3, distilled water 1 L.

[0026] LB culture medium: tryptone 10 g, yeast extract 5 g, sodium chloride 4 g, distilled water 1 L.

[0027] After the above-mentioned culture medium is prepared according to the formula, it is divided and sterilized at 121 DEG C for 20 min.

[0028] The culture of the needle blight pathogen of Larix kaempferi and the antagonistic test use PDA culture medium, and the isolation and culture of the endophytic bacteria use NA culture medium and LB culture medium.

[0029] The test agents are as follows:

[0030] The chemical agents are all analytical pure and purchased from a chemical reagent company. The Ezup column bacterial genome DNA extraction kit is purchased from Shanghai Shengong Biological Company, the Taq enzyme is purchased from Nanjing Nuowezan Biological Company, and the PCR primer is synthesized by Shanghai Shengong Biological Company.

[0031] The experimental instruments are as follows:

[0032] Ultra-clean workbench (AC2-4S1, Singapore Yisigao Technology Co., Ltd.), high-pressure steam sterilization pot (SX-500, Japan Tomy Digital Biology Co., Ltd.), oven (MOV-212F-PC, Japan Panasonic Health Care Appliances Co., Ltd.), shaking bed (Multitron Standard, China Yisensheng Biological Technology Co., Ltd.), constant-temperature incubator (MIR-553, Japan Sanyo Electric Co., Ltd.), refrigerator (HYCD-290, China Haier Group), PCR instrument (nexus GSX1, Germany Eppendorf Co.), optical microscope (DM500, Germany Leica Co.), electrophoresis instrument (DYY-12C, Beijing Liuyi Instrument Factory), centrifuge (Centrifuge 5424R, Germany Eppendorf Co.).

[0033] Example 1

[0034] 1. Endophytic bacteria culture

[0035] The healthy larch branches and leaves collected from 13 collection points in 8 provinces were washed with running water to remove surface dirt, cleaned and dried. The leaves and branches were sterilized separately, and the outer skin of the branches was peeled off before sterilization. The branches were washed with sterile water, sterilized with 75% ethanol for 60 seconds, then sterilized with 3% sodium hypochlorite for 30 seconds, and finally rinsed with sterile water three times. The last time the sterile water was used as a control and was coated on the NA solid medium. The sterilized branches were cut into lengths of about 2 cm and placed on the NA solid medium, with 5 branches placed in the center and around each dish. The leaves were placed directly and homogenized for coating. The direct placement treatment was the same as the branch treatment. The sterilized leaves were cut into lengths of about 2 cm and placed on the NA solid medium, with 5 leaves placed in the center and around each dish. For homogenate coating, the leaves were ground into leaf homogenate using a sterilized mortar, 200 μL of leaf homogenate was taken with a pipette gun and coated evenly on the NA solid medium using a coater. The treated NA medium was sealed and placed in a 28°C constant temperature incubator for culture. After 2-3 days, the bacterial colonies growing in the medium were picked and streaked for culture. After 1-2 days, 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 strains of endophytic bacteria were isolated. The number of strains collected from each sample point and the strain number are shown in Table 1.

[0036] Table 1 Isolation of larch endophytic bacteria

[0037]

[0038]

[0039] 2. Screening of endophytic bacteria

[0040] The larch branch blight pathogen was inoculated on PDA medium and cultured at 28°C for 5 days. The 391 strains of endophytic bacteria obtained were subjected to primary screening and secondary screening by plate confrontation method.

[0041] 1) Primary screening: Two-side streaking confrontation culture method was used. A 6 mm diameter of larch branch blight pathogen cake was inoculated in the center of the PDA plate, and single colonies of endophytic bacteria were streaked on both sides of the plate. Plates with no endophytic bacteria and only pathogenic fungi were used as controls, and each treatment was repeated three times. The plates were cultured in a 28°C constant temperature incubator. When the control group was full, the diameter of the pathogenic fungal colony was measured. Through two-side streaking confrontation culture method, 78 strains of larch endophytic bacteria with good antagonistic effect were obtained.

[0042] 2) Rescreening, four-point confrontation culture method was used, the central of PDA plate (diameter of 90 mm) containing 25 mL was placed with the growing vigorously Larch canker disease pathogen fungus cake (inoculation diameter of 6 mm), the endophytic bacteria single colony was picked and inoculated at four diagonal lines 3 cm away from the pathogen fungus cake, each treatment was repeated 3 times, and the plate with only pathogen fungus cake in the center and no endophytic bacteria inoculation was set as a control. Incubation in a 28°C constant temperature incubator, when the control group was full of 90 mm culture dish, the pathogen colony diameter of the treatment group and the control group was measured, and the inhibition rate was calculated.

[0043] The colony diameters of the control group and the treatment group were measured by cross method, and the inhibition rate was calculated. The inhibition rate calculation formula is as follows:

[0044]

[0045] Rescreening was carried out by four-point confrontation culture method, and 10 strains of endophytic bacteria with better antagonistic effect were further obtained, and the inhibition rate was more than 57%. On the basis of rescreening, the endophytic bacteria YN 2 isolated from larch in Yunnan Province with better antagonistic effect was selected for further experiment, and the inhibition rate of endophytic bacteria was recorded, the pathogen colony diameter was 31.43 mm, and the inhibition rate was 65.08%.

[0046] Example 2

[0047] 1. Preparation of endophyte fermentation broth

[0048] The YN 2 endophyte stored at 4°C was streaked on NA plate and incubated in a 28°C constant temperature incubator for 36 h. A single colony was inoculated in a conical flask containing LB culture solution and incubated in a 28°C, 200 r / min shaking incubator for 1 d to obtain the seed solution of YN 2 endophyte. The seed solution of YN 2 endophyte was taken by a pipette and inoculated into sterilized and cooled LB culture solution at an inoculation amount of 1%, and then incubated in a shaking incubator at 200 r / min, 28°C in the dark for 3 d to obtain the fermentation broth of YN 2 endophyte.

[0049] 2. Extraction of endophyte genomic DNA

[0050] The genomic DNA of YN 2 endophyte was extracted according to the commercial DNA extraction kit (Shanghai Genechem Co., Ltd.) for endophyte genomic DNA extraction, and the specific steps were as follows:

[0051] 1) Take 0.5-1 mL bacterial solution of overnight culture, add to 1.5 mL centrifuge tube, centrifuge at 8,000 rpm for 1 min at room temperature, discard supernatant, collect bacterial cells. Add 180 μL Buffer Digestion, add 20 μL Proteinase K solution, shake well to mix. Incubate at 56°C for 1 h to completely lyse the cells.

[0052] The amount of bacterial solution used is 500 μL when OD600 value is ≥3, and 0.5-1 mL when OD600 value is <3. During the water bath process, invert the solution every 10 min to promote lysis of the sample.

[0053] 2) Add 200 μL Buffer BD, invert well to mix, and incubate at 70°C for 10 min.

[0054] 3) Add 200 μL anhydrous ethanol, invert well to mix.

[0055] 4) Place the adsorption column in a collection tube, and use a pipette to add the solution and translucent fibrous suspension into the adsorption column. Let stand for 2 min, then centrifuge at 12,000 rpm for 1 min at room temperature, and discard the waste solution in the collection tube.

[0056] 5) Place the adsorption column back into the collection tube, add 500 μL PW Solution, and centrifuge at 10,000 rpm for 30 s to discard the filtrate.

[0057] 6) Place the adsorption column back into the collection tube, add 500 μL Wash Solution, and centrifuge at 10,000 rpm for 30 s to discard the filtrate.

[0058] 7) Place the adsorption column back into the collection tube, centrifuge at 12,000 rpm for 2 min at room temperature to remove residual Wash Solution. Open the lid of the adsorption column and let stand at room temperature for several minutes to completely dry the residual Wash Solution in the adsorption material. Residual Wash Solution will affect the yield of genomic DNA and subsequent experiments.

[0059] 8) Take out the adsorption column and place it into a new 1.5 mL centrifuge tube, add 50-100 μL CE Buffer, let stand for 3 min, centrifuge at 12,000 rpm for 2 min at room temperature, and collect the DNA solution. The extracted DNA can be immediately used for the next step or stored at -20°C.

[0060] 3. PCR amplification

[0061] The bacterial universal primers 27-F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492-R (5'-GGTTACCTTGTTACGACTT-3') were selected. The PCR amplification of the gyrB gene sequence was performed using the gyrB gene primers UP-1 (5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3') and UP-2r

[0062] (5'-AGCAGGATACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3').

[0063] The PCR reaction system (20 μL) was as follows: Green Taq Mix 10 μL, each 1 μL of the upstream and downstream primers, 2 μL of template DNA, and 6 μL of ddH2O.

[0064] The 16S rDNA PCR reaction program was as follows: 95 °C pre-denaturation for 5 min, 95 °C denaturation for 30 s, 48 °C annealing for 30 s, 72 °C extension for 90 s, 30 cycles, and 72 °C final extension for 10 min.

[0065] The gyrB gene sequence PCR reaction program was as follows: 94 °C pre-denaturation for 5 min, 94 °C denaturation for 30 s, 60 °C annealing for 30 s, 72 °C extension for 90 s, 30 cycles, and 72 °C final extension for 5 min.

[0066] 4. Sequencing

[0067] After the 16S rDNA specific primer PCR amplification with the YN 2 strain as the template, the amplification results were sent to Shanghai Shengong Biological Company for sequencing. The two sequencing result sequences of the two primers were spliced using the DNA MAN software, the obtained target sequence was shown as SEQ ID NO. 1, and a 1452 bp sequence was obtained. The sequence alignment on the NCBI showed that the sequence similarity of the strain YN 2 and Bacillus velezensis (accession number: OP223018.1) was 100%. It has been preserved in the China Center for Type Culture Collection, and its classification and naming is Bacillus velezensis YN2, the preservation number is CCTCC: M 2023792, the preservation date is 20230522, and the preservation address is Wuhan University, China.

[0068] Example 3

[0069] The root irrigation inoculation was performed, 25 mL of endophytic bacterial fermentation liquor (1 × 10 7Inject 25 mL of sterile water (CFU / mL) into the soil around the roots of potted larch seedlings in five divided doses. Two days later, inoculate with larch twig blight fungal cakes. For each seedling, select one main branch and 2-3 healthy lateral branches. Make a tangential cut at the lower part of the branch tip with a sterile blade. Then, place the fungal cake against the cut surface, ensuring contact between the pathogen and the tangential cut. Wrap the cake with an inverted cone shape using sealing film, and then add distilled water to maintain the activity of the pathogen. The negative control group was inoculated with 25 mL of sterile water before inoculation with larch twig blight fungal cakes. The blank control group was inoculated with only 25 mL of sterile water, and wounds were made on one main branch and 2-3 healthy lateral branches using a sterile blade.

[0070] The treated larch seedlings were placed in a 28°C room temperature environment and a humidifier was used in the greenhouse to create a hot and rainy environment. The disease situation was observed every day, and distilled water was added to the sealing film. After 14 days, the incidence rate, disease index and control effect were calculated.

[0071] Grading standards for larch twig blight: Grade 0: No disease in the whole plant; Grade 1: Less than 1 / 4 of needles are diseased; Grade 2: 1 / 4-1 / 2 of needles are diseased; Grade 3: 1 / 2-3 / 4 of needles are diseased; Grade 4: More than 3 / 4 of needles are diseased; Grade 5: Diseased plant is basically dead.

[0072] The formulas for calculating incidence rate, disease index, and prevention efficacy are as follows:

[0073]

[0074]

[0075]

[0076] The results are as follows Figure 1 As shown in Table 2, 7 days after inoculation with *Larix chinensis*, the control group inoculated with the pathogen began to show symptoms, with needles turning yellow, while the treated pine seedlings showed no symptoms. 9 days after inoculation, the control group showed obvious symptoms, with needles beginning to dry and fall off, while the treated seedlings showed signs of yellowing. At 14 days, the incidence rate in the control group reached 100%, with a disease index of 72.22. In the endophytic fungus YN2 treatment group, some pine seedlings experienced needle drying and falling off, and the number of diseased needles was less than in the control group, with an incidence rate of 60%, a control effect of 57.7%, and a disease index of 30.56. The blank control group showed normal pine seedling growth and no significant changes in needles.

[0077] Table 2. Control effects of antagonistic strains on larch twig blight.

[0078]

[0079]

Claims

1. Use of Bacillus velezensis YN2 in inhibiting the pathogen of larch canker disease, characterized in that, The pathogen of the larch canker disease is Neofusicoccum laricinum (Sawada) Y. Hattoii & C. Nakash, and the strain number is DHKS 6-3; the taxonomic name of the Bacillus velezensis YN2 is Bacillus velezensis YN2, and the preservation number is CCTCC NO: M2023792.

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