Prevention and treatment method of bacillus siamensis in pine shoot disease

Abstract

The invention discloses a prevention and treatment method of bacillus siamensis in pine shoot disease, QX3-4 has relatively strong bacteriostatic activity on the pine shoot disease of masson pine, the bacteriostatic rates can reach 74.38%, 80.1% and 85.17% respectively when bacteria are inoculated for 0 hour, 24 hours and 48 hours later, and the bacteriostatic rate can reach 100% when the bacteria are inoculated for 72 hours later, so that the bacillus siamensis can be used for preventing and treating the pine shoot disease of masson pine, and the bacillus siamensis can be used for preventing and treating the pine shoot disease of masson pine. The invention relates to the technical field of biocontrol bacteria. According to the prevention and treatment method of the bacillus siamensis in the pine wilt disease, the sterile fermentation filtrate of the QX3-4 still has an inhibition effect on the aschersonia championii when being diluted by 100 times, the bacteriostasis rate is kept at 90% or above when being diluted by 50 times or below, the bacteriostasis rate can reach 87.49% when the sterile fermentation filtrate of the QX3-4 is diluted by 100 times after all components of a culture medium are optimized, and in a potting prevention effect test, the bacteriostasis rate of the sterile fermentation filtrate of the QX3-4 can reach 87.49%. The QX3-4 can relieve the disease symptom of the pinus massoniana seedling pine wilt in the early stage and basically stop the disease development in the later stage, and the bacterial suspension, the fermentation liquor and the fermentation filtrate of the QX3-4 have prevention and treatment effects on the aschersonia confusa.

Description

Technical Field

[0001] The invention relates to the technical field of biocontrol bacteria, in particular to a method for preventing and controlling pine dieback disease with siam bacillus. Background Art

[0002] Pine dieback is caused by the pathogen Sphaeropsis sapinea. It is one of the most common pine diseases in the world and was listed as an important forest disease in China in 1999. The pathogen of Sphaeropsis sapinea is one of the most common fungal pathogens in more than 65 countries including Asia, Africa, Europe, Oceania, North America and South America. Red pine, Scots pine, Masson pine, Chinese pine, larch and other tree species are the main hosts of pine dieback. Once these trees are infected, their branches will show a series of symptoms such as dieback, ulcer spots and dead needles. Sphaeropsis sapinea can directly invade the tender shoots of pine trees, causing the tender shoots to wilt. As the symptoms of the disease continue to deepen, branches that are not born in the current year will also be affected, eventually leading to the death of the entire tree. At the same time, Sphaeropsis sapinea will also reproduce through conidiophores during the infection process, further expanding the scope of pine dieback.

[0003] Pine dieback is a host-dominant disease that usually infects trees with poor growth and weak vigor. It is hidden and sudden. Infected pine branches wither, stem ulcers, dead tops, blue discoloration and even death. Its pathogen Aspergillus pinaster is a common fungal pathogen that can cause dieback and ulcers in naturally occurring and planted pine and other coniferous trees around the world. In severe cases, branches and branches die back, causing considerable losses. According to research, the loss of timber caused by felling immature diseased trees due to the disease is as high as 28%, and the potential loss of timber is as high as 55%. Over the years, the host range of pine dieback has continued to expand in my country, and the damage has continued to increase. In mild cases, it affects the growth of individual seedlings, and in severe cases, it causes large-scale disease and even death of trees, causing major ecological damage and economic losses. It has become a major forest disease in my country, so the prevention and control of the disease is becoming increasingly important.

[0004] At present, based on the self-control ability of the ecosystem and the epidemic laws of pine dieback disease, the main prevention and control technology adopted is a comprehensive prevention and control technology that focuses on forestry prevention and control and combines biological control and chemical control.

[0005] In the review of pine dieback, it is pointed out that the main measures for afforestation and prevention of pine dieback include: clearing dead trees, dying trees and depressed trees in the forest, pruning the lower branches of seriously diseased plants, removing diseased leaves, cleaning up fallen diseased leaves and cones, etc.; creating mixed coniferous and broad-leaved forests and isolation belts to avoid planting large areas of pure pine forests; at the same time, Bordeaux liquid can be sprayed during the spring growth period. In the study of pine dieback prevention and control strategies, it is clear that the prevention and control of this disease should pay attention to the selection of planting resistant varieties, suitable trees for suitable places, and strengthening quarantine. However, the current application of afforestation measures for the prevention and control of pine dieback is still in theory, and further promotion and practice are needed.

[0006] Chemical control is a practical and common method of pest control in forestry, which can quickly and timely prevent the spread of diseases. Zhao Meiling et al. found that fungicides such as Senbao No. 1 + Miebingwei (1:2) 1000 times liquid and sodium pentachlorophenol smoke agent have good control effects on larch dieback. Chang Jiayu et al. found that most quaternary ammonium salt compounds containing dihydrogenated nopside have an inhibition rate of more than 90% on pinocytic Aspergillus at a mass concentration of 50 mg / L, and the inhibition effect is much higher than the commonly used fungicides thiophanate-methyl and carbendazim. Chemical control can play an emergency control role in forest stands with serious disease, and control the continued spread of diseases in a short period of time. Although this has brought high benefits to forestry production, many disadvantages such as environmental pollution and destruction of the natural balance of the ecosystem have also followed.

[0007] Biological control is an important new disease control technology that neither affects the ecological environment nor produces drug resistance in host plants. It is also an important part of comprehensive management. People are gradually paying attention to its role in forestry pest control. The research on biological control has a good development prospect. At present, the biological control of pine dieback is mainly carried out from two aspects, namely the screening of antagonistic bacteria and the separation and extraction of antibacterial substances produced by biological metabolism. However, there are currently no commercial biocontrol agents used in the control of pine dieback.

[0008] Chemical control plays an important role in the integrated prevention and control of pests and diseases. However, the long-term use of stable chemical pesticides will not only enhance the resistance of pathogens and reduce the effectiveness of prevention and control, but also pollute agricultural products, air, soil and water, endangering the health and safety of humans and animals and the ecological environment.

[0009] As most pine forests are large and the trees are tall, manual clearing is difficult, labor-intensive, inefficient and costly, and the effects are not as good as other prevention and control measures. Therefore, it is difficult to implement and remains largely at the theoretical level. Summary of the invention

[0010] 1. Technical issues to be resolved

[0011] In view of the deficiencies of the prior art, the present invention provides a method for controlling siam bacillus in pine dieback, which solves the problem that the existing chemical control will not only enhance the drug resistance of pathogens and reduce the control effect when using chemical pesticides with stable properties for a long time, but also pollute agricultural products, air, soil and water, endangering the health and safety of people and livestock and the ecological environment. The forestry control measures for pine dieback are difficult to carry out manually because most pine forests are large in area and the trees are tall, which consumes manpower, is inefficient and costly, and is not as effective as other control measures. It is difficult to carry out and basically remains at the theoretical level.

[0012] (II) Technical solution

[0013] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for preventing and treating siam bacillus in pine dieback, specifically comprising the following steps:

[0014] S1. Take healthy Masson pine needles, rinse with water, disinfect with 75% alcohol for 1 min, rinse with sterile water 3 times, and use the sterile water from the last rinse as a blank control to apply on the surface of the culture medium;

[0015] S2. Take the sample tissue blocks that have been thoroughly sterilized on the surface and grind them thoroughly in a mortar. Use sterile water to prepare the grinding solution into dilution solutions of different concentrations.

[0016] S3, take 30 μL of sample dilution and spread it on LB medium plates respectively, evenly spread it and put it upside down in a constant temperature incubator at 28℃ in the dark for 2-3 days, pick colonies with different morphological characteristics and streak them on the medium, purify them 3 times and store them for later use;

[0017] S4, using Aspergillus pinecone A1-2 as the target, a total of 10 strains of endophytic bacteria with antagonistic effects were obtained by plate confrontation method, and the inhibition rate was between 62.34-74.38%;

[0018] S5. Since the growth rate of pathogens is faster than that of antagonistic bacteria, the subsequent re-screening is to inoculate pathogens 24h, 48h, and 72h after inoculation of antagonistic bacteria, and calculate the inhibition rate;

[0019] S6. 188 bacterial strains were isolated and purified from healthy pine needle samples of Masson pine by the dilution spreading plate method. QX3-4 had a strong antibacterial activity against Masson pine dieback disease. The inhibition rates were 74.38%, 80.1% and 85.17% respectively when inoculated with Conchocera pinaster 0h, 24h and 48h after inoculation of bacteria. The inhibition rate reached 100% when Conchocera pinaster was inoculated 72h after inoculation of bacteria. The sterile fermentation filtrate of QX3-4 still had an inhibitory effect on Conchocera pinaster when diluted 100 times, and the inhibition rate remained above 90% when diluted 50 times or less.

[0020] Preferably, the initial screening of pine needle endophytic bacteria specifically comprises the following steps:

[0021] a1. Pathogens cultured for 3 days were selected, and strains with antibacterial activity were screened by the five-point confrontation method. A 2.5 mm bacterial cake was prepared with a hole puncher and inoculated in the center of the PDA medium. 2 μL of purified endophytic bacterial solution was dripped in the four directions of the cake, top, bottom, left, and right. Each treatment was repeated 3 times, and the control group was not dripped with bacterial solution.

[0022] a2. After culturing at 25°C for 3 days, the colony diameter of the pathogen was measured by the cross method and the inhibition rate was calculated. The strains with an inhibition rate greater than 50% were antagonistic bacteria and were then rescreened.

[0023] Preferably, the calculation formula for calculating the antibacterial rate in step a2 is as follows:

[0024] Inhibition rate = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%.

[0025] Preferably, the rescreening of endophytic bacteria in pine needles is as follows: the endophytic bacteria obtained in the initial screening are again subjected to the five-point confrontation method. First, 2 μL of bacterial solution is inoculated on the PDA culture medium plate. After constant temperature culture at 28°C for 24h, 48h, and 72h, the pathogen is inoculated in the center of the PDA culture medium plate. Each treatment is repeated 3 times, and the control group without bacterial solution is used as the control group. The inhibition rate is calculated after constant temperature culture at 25°C for 3d.

[0026] Preferably, the candidate antagonistic bacteria protection evaluation specifically comprises the following steps:

[0027] b1. Inoculate the strain on LB solid medium by streaking, incubate at 28°C for 24 hours, pick a single colony and inoculate it into LB liquid medium, incubate at 28°C, 180r / min for 12 hours to obtain seed solution, and incubate the seed solution at 0.2% inoculation volume and 50% filling volume in a shake flask at 28°C, 180r / min for 72 hours;

[0028] b2. Select Masson pine seedlings with similar growth potential and spray 2 mL of bacterial suspension of ten effective strains on each plant using the foliar spray method. The bacterial concentration is 0.8*10 8 cfu / mL, spraying only sterile water was used as a negative control, and moisturizing treatment was done well, and each treatment was repeated 3 times;

[0029] b3. After 7 days, use a sterile needle to pierce holes in the needles of the upper and middle parts of the Masson pine seedlings, and then spray the pathogen solution on the pine needles. Observe and record the disease situation 7 days and 15 days after inoculation.

[0030] Preferably, the morphological identification is as follows: the screened antagonistic bacteria are streaked and inoculated on the corresponding culture medium plate, and cultured at a constant temperature of 28°C for 3 days, and a single colony is grown, and the colony size, color, whether the edge is flat, whether the surface is smooth, the degree of transparency and the state of protrusion are observed, and Gram staining is performed at the same time.

[0031] Preferably, molecular biology and physiological and biochemical identification, using freeze-thaw method to extract antagonistic bacterial DNA, the specific operation is as follows:

[0032] c1. Inoculate the screened single colony into liquid LB medium and culture at 28°C with shaking overnight;

[0033] c2. Take 1 mL of bacterial solution and centrifuge at 12000 r / min for 1 min, discard the supernatant, and resuspend with 100 μL of ultrapure water;

[0034] c3. Place in a 100℃ water bath for 5 minutes, freeze at -20℃ for 10 minutes, repeat the above steps, centrifuge at 12000r / min for 10 minutes, take the supernatant, which is the bacterial DNA, and perform PCR amplification after measuring the concentration;

[0035] c4. Compare the 16S rRNA sequencing results in NCBI, conduct phylogenetic analysis with closely related strains, select an outgroup strain, and construct a molecular phylogenetic tree using MEGA11.0 software;

[0036] c5. The 16S rDNA fragment of strain QX3-4 was amplified and sequenced. The sequence was then compared with the NCBI database using BLAST. The similarity between strain QX3-4 and Bacillus siamensis was 90%. Based on the morphological characteristics, physiological and biochemical properties, and molecular biological identification results, strain QX3-4 was identified as Bacillus siamensis.

[0037] Preferably, the method for applying the inhibitory effect of the antagonistic bacteria aseptic fermentation filtrate on Aspergillus pinocybe is as follows:

[0038] d1. The strain was streaked on LB solid medium, cultured and activated at 28°C for 24 hours, a single colony was picked and inoculated into LB liquid medium, cultured at 28°C, 180 r / min for 12 hours to obtain seed solution, the seed solution was cultured in a shake flask at 28°C, 180 r / min for 72 hours at a rate of 0.2% inoculation volume and 50% liquid volume, and then centrifuged at 10,000 r / min for 15 minutes;

[0039] d2, using a 0.22 pm bacterial filter to filter and sterilize the supernatant to obtain a sterile fermentation filtrate, mixing the fermentation filtrate with PDA medium, and preparing plates diluted 5-100 times;

[0040] d3. After the culture medium solidifies, inoculate the pathogen cake in the center of the plate, use the mixed LB liquid culture medium as the control, and calculate the inhibition rate of the fermentation filtrate of the strain on Aspergillus niger.

[0041] (III) Beneficial effects

[0042] The present invention provides a method for preventing and treating pine dieback disease with Siamese Bacillus. Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) The control method of pine dieback disease by using the Siamese Bacillus sp. QX3-4 has strong antibacterial activity against Masson pine dieback disease, and the antibacterial rates can reach 74.38%, 80.1% and 85.17% respectively when inoculated with Conchocera pine cones 0h, 24h and 48h after inoculation of bacteria, and the antibacterial rate can reach 100% when inoculated with Conchocera pine cones 72h after inoculation of bacteria, and the sterile fermentation filtrate of QX3-4 still has an inhibitory effect on Conchocera pine cones when diluted 100 times, and the antibacterial rate is maintained above 90% when diluted 50 times or less.

[0044] (2) The control method of pine dieback disease by using Siamese Bacillus is as follows: after optimizing the components of the culture medium, the antibacterial rate of the sterile fermentation filtrate of QX3-4 can reach 87.49% when diluted 100 times; in the potted plant control test, QX3-4 can alleviate the symptoms of pine dieback disease in Masson pine seedlings in the early stage and basically stop the development of the disease in the later stage; the bacterial suspension, fermentation liquid and fermentation filtrate thereof all have control effects on Aspergillus niger, among which the control effect of the bacterial suspension of QX3-4 on Aspergillus niger reaches 90.90%, which is the best effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 For the present invention Figure 1 Schematic diagram of the initial screening of antagonistic bacteria of Aspergillus pinecone, in which A: NC1; B: QX1-9; C: QX2-23; D: QX3-4; E: QX5-2; F: QZ10-1; G: QZ10-4; H: QX10-12; I: QX11-3; J: QX12-17;

[0046] Figure 2 It is a schematic diagram of the multiple screening of antagonistic bacteria of Aspergillus pinocene of the present invention;

[0047] Figure 3 It is a schematic diagram of the effect of the effective bacterial strain suspension of the present invention on Aspergillus pinaster on Pinus massoniana;

[0048] Figure 4 Schematic diagram of the morphological observation results of the strain QX3-4 of the present invention, wherein A is the colony morphology after 3 days of cultivation, and B is the Gram staining result of the strain QX3-4;

[0049] Figure 5This is a schematic diagram of the phylogenetic tree of QX3-4 constructed based on the 16S rRNA sequence of the present invention;

[0050] Figure 6 Schematic diagram of the inhibitory effect of the fermentation filtrate of the strain QX3-4 of the present invention on Conchocera pinecone;

[0051] Figure 7 Schematic diagram of the effect of six different fermentation media on the antibacterial rate of the present invention;

[0052] Figure 8 Schematic diagram of the effect of different carbon sources on the antibacterial rate of fermentation filtrate in the present invention;

[0053] Fig. 9 Schematic diagram of the effect of different nitrogen sources on the antibacterial rate of fermentation filtrate in the present invention;

[0054] Fig.10 Schematic diagram of the effect of different inorganic salts on the antibacterial rate of fermentation filtrate in the present invention;

[0055] Fig.11 This is a schematic diagram of a comparative test of the antibacterial rate of the strains before and after the optimization of the culture medium of the present invention;

[0056] Fig.12 This is a schematic diagram showing the effect of adding sterile water to the control group, and performing alfalfa test and tobacco test on QX3-4 in the present invention;

[0057] Fig.13 This is a schematic diagram of the antibacterial effect of the strain QX3-4 of the present invention on 6 tested plant pathogenic fungi;

[0058] Fig.14 This is a schematic diagram of the CK of the present invention, inoculating pathogens 3 days after applying QX3-4 bacterial suspension, inoculating pathogens 3 days after applying QX3-4 fermentation liquid, and inoculating pathogens 3 days after applying QX3-4 fermentation filtrate. DETAILED DESCRIPTION

[0059] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0060] See also Figure 1-14 The embodiment of the present invention provides a technical solution: a method for preventing and treating siam bacillus in pine dieback, which specifically comprises the following steps:

[0061] S1. Take healthy Masson pine needles, rinse with water, disinfect with 75% alcohol for 1 min, rinse with sterile water 3 times, and use the sterile water from the last rinse as a blank control to apply on the surface of the culture medium;

[0062] S2. Take the sample tissue blocks that have been thoroughly sterilized on the surface and grind them thoroughly in a mortar. Use sterile water to prepare the grinding solution into dilution solutions of different concentrations.

[0063] S3, take 30 μL of sample dilution and spread it on LB medium plates respectively, evenly spread it and put it upside down in a constant temperature incubator at 28℃ in the dark for 2-3 days, pick colonies with different morphological characteristics and streak them on the medium, purify them 3 times and store them for later use;

[0064] S4. Using Aspergillus pinecone A1-2 as the target, a total of 10 strains of endophytic bacteria with antagonistic effects were obtained by plate confrontation method, with an inhibition rate between 62.34% and 74.38% ( Figure 1 );

[0065] S5. Since the growth rate of pathogens is faster than that of antagonistic bacteria, the subsequent re-screening is to inoculate pathogens 24h, 48h, and 72h after inoculation of antagonistic bacteria, and calculate the inhibition rate;

[0066] S6. 188 bacterial strains were isolated and purified from healthy pine needle samples of Masson pine by the dilution spread plate method.

[0067] In the embodiment of the present invention, the initial screening of pine needle endophytic bacteria specifically includes the following steps:

[0068] a1. Pathogens cultured for 3 days were selected, and strains with antibacterial activity were screened by the five-point confrontation method. A 2.5 mm bacterial cake was prepared with a hole puncher and inoculated in the center of the PDA medium. 2 μL of purified endophytic bacterial solution was dripped in the four directions of the cake, top, bottom, left, and right. Each treatment was repeated 3 times, and the control group was not dripped with bacterial solution.

[0069] a2. After culturing at 25°C for 3 days, the diameter of the pathogen colony was measured by the cross method and the inhibition rate was calculated. The strains with an inhibition rate greater than 50% were antagonistic bacteria, and then rescreened. The calculation formula for the inhibition rate was as follows:

[0070] Inhibition rate = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%.

[0071] In the embodiment of the present invention, the rescreening of endophytic bacteria in pine needles is as follows: the endophytic bacteria obtained from the initial screening are again subjected to the five-point confrontation method. First, 2 μL of bacterial solution is inoculated on the PDA culture medium plate. After constant temperature culture at 28°C for 24h, 48h, and 72h, the pathogenic bacteria are inoculated in the center of the PDA culture medium plate. Each treatment is repeated 3 times, and the control group without bacterial solution is used as the control group. After constant temperature culture at 25°C for 3 days, the antibacterial rate is calculated. After rescreening, the present invention screened out 5 strains with strong antagonistic effects on pathogens. Figure 2 It can be seen that the inhibition rates of NC1, QX3-4, QX5-2, QZ10-1 and QZ10-4 against Aspergillus pinecone were significantly higher than those of other strains, and the inhibition rates reached 93.84-96.34% respectively 72 hours after inoculation of antagonistic bacteria.

[0072] In the embodiment of the present invention, the evaluation of the protective effect of candidate antagonistic bacteria specifically includes the following steps:

[0073] b1. Inoculate the strain on LB solid medium by streaking, incubate at 28°C for 24 hours, pick a single colony and inoculate it into LB liquid medium, incubate at 28°C, 180r / min for 12 hours to obtain seed solution, and incubate the seed solution at 0.2% inoculation volume and 50% filling volume in a shake flask at 28°C, 180r / min for 72 hours;

[0074] b2. Select Masson pine seedlings with similar growth potential and spray 2 mL of bacterial suspension of ten effective strains on each plant using the foliar spray method. The bacterial concentration is 0.8*10 8 cfu / mL, spraying only sterile water was used as a negative control, and moisturizing treatment was done well, and each treatment was repeated 3 times;

[0075] b3. After 7 days, use a sterile needle to pierce holes in the needles of the upper and middle parts of the Masson pine seedlings, and then spray the pathogen solution on the pine needles. Observe and record the disease situation 7 days and 15 days after inoculation.

[0076] The results are as follows Figure 3 As shown in the figure, 7 days after inoculation with Aspergillus niger, CK showed different degrees of pine dieback symptoms, while the symptoms of Masson pine seedlings of strains NC1, QX3-4, QX5-2, QZ10-1, QZ10-4, QX10-12, and QX11-3 in the treatment group were milder. 15 days after inoculation with the antagonistic strain, the disease development of strains QX3-4 and QZ10-4 in the treatment group basically stopped. The test results show that strains QX3-4 and QZ10-4 have better prevention effects.

[0077] In the embodiment of the present invention, the morphological identification is specifically as follows: the screened antagonistic bacteria are streaked and inoculated on the corresponding culture medium plate, and cultured at a constant temperature of 28° C. for 3 days, and a single colony is grown. The colony size, color, whether the edge is flat, whether the surface is smooth, the degree of transparency and the state of protrusion are observed, and Gram staining is performed at the same time.

[0078] The results are as follows Figure 4 The colonies of strain QX3-4 are milky white, round, matte, opaque and easy to pick up. There are irregular small protrusions on the surface of the colonies, the edges are jagged, and there is a pungent smell. The results of Gram staining showed that strain QX3-4 appeared blue-purple under a microscope and was a Gram-positive bacterium.

[0079] In the embodiment of the present invention, molecular biology and physiological and biochemical identification were performed, and the freeze-thaw method was used to extract the antagonistic bacterial DNA, and the specific operation was as follows:

[0080] c1. Inoculate the screened single colony into liquid LB medium and culture at 28°C with shaking overnight;

[0081] c2. Take 1 mL of bacterial solution and centrifuge at 12000 r / min for 1 min, discard the supernatant, and resuspend with 100 μL of ultrapure water;

[0082] c3. Place in a 100℃ water bath for 5 minutes, freeze at -20℃ for 10 minutes, repeat the above steps, centrifuge at 12000r / min for 10 minutes, take the supernatant, which is the bacterial DNA, and perform PCR amplification after measuring the concentration;

[0083] c4. Compare the 16S rRNA sequencing results in NCBI, conduct phylogenetic analysis with closely related strains, select an outgroup strain, and construct a molecular phylogenetic tree using MEGA11.0 software;

[0084] c5. The 16S rDNA fragment of strain QX3-4 was amplified and sequenced. The sequence was then compared with the NCBI database by BLAST. The similarity between strain QX3-4 and Bacillus siamensis was 90%. Based on the morphological characteristics, physiological and biochemical properties and molecular biological identification results, strain QX3-4 was identified as Bacillus siamensis.

[0085] In the embodiment of the present invention, the application method of the inhibitory effect of the antagonistic bacteria aseptic fermentation filtrate on Aspergillus pinocybe is as follows:

[0086] d1. The strain was streaked on LB solid medium, cultured and activated at 28°C for 24 hours, a single colony was picked and inoculated into LB liquid medium, cultured at 28°C, 180 r / min for 12 hours to obtain seed solution, the seed solution was cultured in a shake flask at 28°C, 180 r / min for 72 hours at a rate of 0.2% inoculation volume and 50% liquid volume, and then centrifuged at 10,000 r / min for 15 minutes;

[0087] d2, using a 0.22 pm bacterial filter to filter and sterilize the supernatant to obtain a sterile fermentation filtrate, mixing the fermentation filtrate with PDA medium, and preparing plates diluted 5-100 times;

[0088] d3. After the culture medium solidifies, inoculate the pathogen cake in the center of the plate, use the mixed LB liquid culture medium as the control, and calculate the inhibition rate of the fermentation filtrate of the strain on Aspergillus niger.

[0089] Depend on Figure 6 It can be seen that the aseptic fermentation filtrate of strain QX3-4 has a high inhibition rate on Conchocera pinecone when diluted 5-30 times, all reaching 100%. The aseptic fermentation filtrate of strain QX3-4 still has an inhibitory effect on Conchocera pinecone when diluted 100 times, and the inhibition rate remains above 90% when diluted 50 times.

[0090] Physiological and biochemical identification: The results of physiological and biochemical tests of strain QX3-4 are shown in Table 1. According to the Bergey's Manual of Bacterial Identification, QX3-4 was preliminarily identified as a species of Bacillus based on its morphological characteristics and physiological and biochemical reaction characteristics.

[0091] Table 1 Physiological and biochemical test results of strain QX3-4

[0092] Tab.2-1Physiological and biochemical test results of strain

[0093] QZ10-1

[0094]

[0095] Note: (“+” represents positive, “-” represents negative)

[0096] Screening of basic fermentation culture medium: By consulting the literature, the following 6 culture media were selected for screening of basic culture medium, with the antibacterial rate as the screening index.

[0097] Improved medium 1: glucose 20.0 g / L, soluble starch 20.0 g / L, ammonium chloride 10.0 g / L, peptone 10.0 g / L, magnesium sulfate 1.0 g / L;

[0098] Improved medium 2: sucrose 30.0 g / L, ammonium sulfate 10.0 g / L, yeast extract 3.0 g / L, potassium dihydrogen phosphate 0.3 g / L, magnesium sulfate 5.0 g / L;

[0099] TSB: tryptone 15.0 g / L, soy peptone 5.0 g / L, sodium chloride 5.0 g / L;

[0100] NA: beef extract 3.0 g / L, peptone 10.0 g / L, sodium chloride 5.0 g / L;

[0101] Improved medium 5: glucose 10.0 g / L, peptone 5.0 g / L, yeast extract 8.0 g / L, potassium chloride 5.0 g / L;

[0102] Improved culture medium 6: peptone 20.0 g / L, dipotassium hydrogen phosphate 1.5 g / L, magnesium sulfate heptahydrate 1.5 g / L.

[0103] Bacillus QX3-4 was shaken in a 50 mL Erlenmeyer flask in the above basic fermentation medium at a liquid volume of 50%, an inoculation amount of 0.2%, a rotation speed of 200 r / min, and 28°C for 72 h to obtain the fermentation filtrate, which was diluted 100 times with PDA medium and poured onto plates. Each treatment was repeated 3 times, the colony diameter of the pathogen was determined, the inhibition rate was calculated, and the optimal basic fermentation medium was determined.

[0104] The screening results of basic fermentation medium are as follows Figure 7 Among the six selected basic culture media, the antibacterial rate of strain QX3-4 selected by modified medium No. 5 was the highest, so modified medium No. 5 was selected as the basic culture medium for culture medium optimization.

[0105] Optimization of different components of the culture medium: Select several carbon sources (glucose, sucrose, soluble starch, mannitol and corn flour), nitrogen sources (protein pulse, yeast powder, beef extract, soybean protein pulse, ammonium sulfate, ammonia water and yeast extract) and inorganic salts (potassium dihydrogen phosphate, calcium carbonate, calcium chloride, ferrous sulfate and zinc sulfate) for testing, and replace the corresponding components in the basic culture medium in equal amounts. Bacillus QX3-4 was shaken in a 50mL Erlenmeyer flask at 50% liquid volume, 0.2% inoculation volume, 200r / min, and 28°C for 72h to obtain the fermentation filtrate, which was diluted 80 times with PDA culture medium and poured into plates. Each treatment was repeated 3 times, the colony diameter of the pathogen was measured, the inhibition rate was calculated, and the optimal carbon source, nitrogen source and inorganic salt type were determined.

[0106] Depend on Figure 8It can be seen that when the improved medium No. 5 was used as the basic fermentation medium, when glucose was used as the carbon source, the fermentation filtrate of strain QX3-4 had the highest inhibition rate against Aspergillus niger, reaching 94.18%. Therefore, glucose should be selected as the optimal carbon source for the culture medium of strain QX3-4 to antagonize Aspergillus niger.

[0107] Depend on Fig. 9 It can be seen that when the improved culture medium No. 5 after carbon source optimization was used as the basic fermentation medium, the fermentation filtrate of strain QX3-4 had the highest inhibition rate against Aspergillus pinecone, reaching 93.44%. Therefore, tryptone should be selected as the optimal nitrogen source for the culture medium of strain QX3-4 to antagonize Aspergillus pinecone.

[0108] Depend on Fig.10 It can be seen that when the improved culture medium No. 5 after carbon source optimization was used as the basic fermentation medium, when potassium chloride was used as the inorganic salt, the fermentation filtrate of strain QX3-4 had the highest inhibition rate against Aspergillus niger, reaching 86.42%. Therefore, potassium chloride should be selected as the optimal inorganic salt component of the culture medium for strain QX3-4 to antagonize Aspergillus niger.

[0109] Fig.11 This is the result of optimization of each component of the culture medium. The antibacterial rate of QX3-4 sterile fermentation filtrate increased from 27.98% to 87.49% when diluted 100 times.

[0110] Bacterial safety testing: Fig.12 As shown, QX3-4 was tested on alfalfa and tobacco respectively, and sterile water was added to the control group. The results showed that the QX3-4 bacterial solution did not have any adverse effects on the growth of tobacco and alfalfa.

[0111] The sensitivity of the strain to antibiotics is an important indicator for evaluating the safety of the strain. The sensitivity of QX3-4 to 10 antibiotics was within the quality control range, and it was highly sensitive to 9 antibiotics (the diameter of the inhibition zone was above 15 mm). The drug sensitivity results are shown in Table 2.

[0112] Table 2 Results of drug sensitivity test of strain QX3-4

[0113]

[0114]

[0115] S. Highly sensitive I. Intermediately sensitive R. Resistant.

[0116] Broad spectrum of bacterial inhibition: Under the above optimized fermentation culture conditions, the fermentation broth was obtained, and the six plant pathogens (Fusarium oxysporum, Alternaria alternata, Botrytis cinerea, Alternaria ovata, Polytrichomoniasis, Fusarium graminearum) were used as the inhibition objects, and the inhibition rate was determined and calculated using the method in 4. The antibacterial effect of strain QX3-4 on the six plant pathogens was statistically analyzed, and the results showed good inhibitory activity (see Fig.13 ), QX3-4 has a broad-spectrum antibacterial activity.

[0117] QX3-4 potted plant control effect: prepare QX3-4 fermentation liquid, fermentation filtrate and bacterial suspension respectively, and adjust the concentration of fermentation liquid and bacterial suspension to OD600 = 0.8. Take two-year-old healthy Masson pine potted seedlings with similar growth, take 2ml of each of the above treatments and spray them evenly on the surface of Masson pine needles. After 3 days, spray 2ml of Conchosporon mycelium liquid evenly for each treatment, and spray the same amount of sterile water and liquid LB medium as a control, do a good job of moisturizing, and repeat each treatment 4 times. Record the degree of disease every 7 days after inoculation.

[0118] The severity of the disease is divided into four levels: Level 0 is when the whole plant is disease-free, Level 1 is when the withered and yellowed part of the whole plant is ≤25%, Level 2 is when the withered and yellowed part of the whole plant is ≥25.1%-50%, Level 3 is when the withered and yellowed part of the whole plant is ≥59.1%-75%, and Level 4 is when the withered and yellowed part of the whole plant is ≥75.1%.

[0119] Disease index = 100 × ∑ (number of diseased plants at each level × representative value of each level) / (total number of plants surveyed × highest representative value)

[0120] Relative control effect = 100 × (disease index when only pathogens are applied - disease index when both biocontrol bacteria and pathogens are applied) / disease index when only pathogens are applied.

[0121] The potted plant efficacy test results of strain QX3-4 are shown in Fig.14 As shown in Table 3, the incidence of CK reached 100%. The bacterial suspension, fermentation liquid and fermentation filtrate of QX3-4 all had a certain control effect on Aspergillus niger. Among them, the control effect of QX3-4 bacterial suspension on Aspergillus niger reached 90.90%, which was the best effect.

[0122] Table 3 Relative control effect of strain QX3-4

[0123]

[0124] The biological control of the present invention is an important new disease control technology that will neither affect the ecological environment nor produce drug resistance in the host plant. It is also an important part of comprehensive management. Endophytic bacteria in plants have a high living space in the plant body, are not easily affected by external environmental conditions, reproduce quickly, and are numerous; and most bacteria can produce a variety of secondary metabolites, which have the effect of fighting pathogens and promoting plant growth. Bacillus has the advantages of being able to secrete a variety of secondary metabolites, good root colonization ability, and large and stable spore production. The bacteria screened out in this experiment are endophytic bacteria of healthy Masson pine needles, and they can be determined to be Siamese Bacillus after identification.

[0125] Bacillus siameensis is widely distributed in nature, easy to separate and culture, non-toxic and harmless to humans and animals, does not pollute the environment, has broad-spectrum antibacterial activity and strong stress resistance, and is an important biocontrol bacteria resource. It has a very broad application prospect in the development of biofertilizers and biopesticides. The strain QX3-4 used in this study is an endophytic bacterium from Pinus massoniana in Quanjiao, Anhui Province. It is non-pathogenic to plants, has simple culture conditions, strong antibacterial activity, and good control effect on pine dieback. After culturing in LB solid medium for 24h, 48h, and 72h, the inhibition rate of mycelial growth of Aspergillus pine cones can reach 80.1%, 85.17%, and 100%, respectively; the 72h fermentation filtrate of strain QX3-4 has a very high inhibition rate on Aspergillus pine cones. After the optimization of the culture medium components, the inhibition rate of its sterile fermentation filtrate on Aspergillus pine cones can still reach 87.49% when diluted 100 times, which has the potential for development and utilization as a biopesticide. However, the experiment was only conducted under in vitro conditions, and further verification of its effectiveness in preventing and controlling pine dieback in different field environmental conditions is needed.

[0126] Strain QX3-4 not only has an inhibitory effect on pine dieback pathogens, but also has high antibacterial activity against a variety of plant pathogens. It has a broad-spectrum antibacterial property and the potential to prevent and control a variety of plant diseases. It can be used in the development and application of biological control of a variety of garden plant diseases.

[0127] Tobacco tests, alfalfa tests and antibiotic sensitivity tests are all important indicators for evaluating the safety of potential biocontrol bacteria. It can be determined that QX3-4 has a certain degree of safety.

[0128] In the pot experiment, the bacterial suspension, fermentation liquid and fermentation filtrate of strain QX3-4 all had certain control effects on Aspergillus niger. Among them, the control effect of QX3-4 bacterial suspension on Aspergillus niger reached 90.90%, which was the best effect.

[0129] Meanwhile, the contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0130] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0131] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for controlling siam bacillus in pine dieback, characterized in that: The isolation of endophytic bacteria from pine needles specifically includes the following steps: S1. Take healthy Masson pine needles, rinse with water, disinfect with 75% alcohol for 1 min, rinse with sterile water 3 times, and use the sterile water from the last rinse as a blank control to apply on the surface of the culture medium; S2. Take the sample tissue blocks that have been thoroughly sterilized on the surface and grind them thoroughly in a mortar. Use sterile water to prepare the grinding solution into dilution solutions of different concentrations. S3, take 30 μL of sample dilution and spread it on LB medium plates respectively, evenly spread it and put it upside down in a constant temperature incubator at 28℃ in the dark for 2-3 days, pick colonies with different morphological characteristics and streak them on the medium, purify them 3 times and store them for later use; S4, using Aspergillus pinecone A1-2 as the target, a total of 10 strains of endophytic bacteria with antagonistic effects were obtained by plate confrontation method, and the inhibition rate was between 62.34-74.38%; S5. Since the growth rate of pathogens is faster than that of antagonistic bacteria, the subsequent re-screening is to inoculate pathogens 24h, 48h, and 72h after inoculation of antagonistic bacteria, and calculate the inhibition rate; S6. 188 bacterial strains were isolated and purified from healthy pine needle samples of Masson pine by the dilution spread plate method.

2. A method for controlling pine dieback of Siamese Bacillus according to claim 1, characterized in that: The initial screening of pine needle endophytic bacteria specifically includes the following steps: a1. Pathogens cultured for 3 days were selected, and strains with antibacterial activity were screened by the five-point confrontation method. A 2.5 mm bacterial cake was prepared with a hole puncher and inoculated in the center of the PDA medium. 2 μL of purified endophytic bacterial solution was dripped in the four directions of the cake, top, bottom, left, and right. Each treatment was repeated 3 times, and the control group was not dripped with bacterial solution. a2. After culturing at 25°C for 3 days, the colony diameter of the pathogen was measured by the cross method and the inhibition rate was calculated. The strains with an inhibition rate greater than 50% were antagonistic bacteria and were then rescreened.

3. A method for controlling pine dieback of Siamese Bacillus according to claim 2, characterized in that: The calculation formula for calculating the antibacterial rate in step a2 is as follows: Inhibition rate = (control colony diameter - treated colony diameter) / (control colony diameter - bacterial cake diameter) × 100%.

4. The method for controlling siam bacillus in pine dieback according to claim 3, characterized in that: The rescreening of endophytic bacteria in pine needles was as follows: the endophytic bacteria obtained in the initial screening were again subjected to the five-point confrontation method. First, 2 μL of bacterial solution was inoculated on the PDA culture medium plate. After constant temperature culture at 28°C for 24h, 48h, and 72h, the pathogen was inoculated in the center of the PDA culture medium plate. Each treatment was repeated 3 times, and the control group without bacterial solution was used. The inhibition rate was calculated after constant temperature culture at 25°C for 3 days.

5. The method for controlling pine dieback of Siamese Bacillus according to claim 1, characterized in that: The evaluation of candidate antagonistic bacteria efficacy specifically includes the following steps: b1. Inoculate the strain on LB solid medium by streaking, incubate at 28°C for 24 hours, pick a single colony and inoculate it into LB liquid medium, incubate at 28°C, 180r / min for 12 hours to obtain seed solution, and incubate the seed solution at 0.2% inoculation volume and 50% filling volume in a shake flask at 28°C, 180r / min for 72 hours; b2. Select Masson pine seedlings with similar growth potential and spray 2 mL of bacterial suspension of ten effective strains on each plant using the foliar spray method. The bacterial concentration is 0.8*10 8 cfu / mL, spraying only sterile water was used as a negative control, and moisturizing treatment was done well, and each treatment was repeated 3 times; b3. After 7 days, use a sterile needle to pierce holes in the needles of the upper and middle parts of the Masson pine seedlings, and then spray the pathogen solution on the pine needles. Observe and record the disease situation 7 days and 15 days after inoculation.

6. The method for controlling pine dieback of Siamese Bacillus according to claim 1, characterized in that: The morphological identification is as follows: the selected antagonistic bacteria are streaked and inoculated on the corresponding culture medium plate, and cultured at a constant temperature of 28°C for 3 days. When a single colony grows, the colony size, color, whether the edge is flat, whether the surface is smooth, the degree of transparency and the condition of the protrusions are observed, and Gram staining is performed at the same time.

7. The method for controlling pine dieback of Siamese Bacillus according to claim 1, characterized in that: Molecular biology and physiological and biochemical identification, the freeze-thaw method was used to extract the antagonistic bacterial DNA, the specific operation is as follows: c1. Inoculate the screened single colony into liquid LB medium and culture at 28°C with shaking overnight; c2. Take 1 mL of bacterial solution and centrifuge at 12000 r / min for 1 min, discard the supernatant, and resuspend with 100 μL of ultrapure water; c3. Place in a 100℃ water bath for 5 minutes, freeze at -20℃ for 10 minutes, repeat the above steps, centrifuge at 12000r / min for 10 minutes, take the supernatant, which is the bacterial DNA, and perform PCR amplification after measuring the concentration; c4. Compare the 16S rRNA sequencing results in NCBI, conduct phylogenetic analysis with closely related strains, select an outgroup strain, and construct a molecular phylogenetic tree using MEGA11.0 software; c5. The 16S rDNA fragment of strain QX3-4 was amplified and sequenced. The sequence was then compared with the NCBI database by BLAST. The similarity between strain QX3-4 and Bacillus siamensis was 90%. Based on the morphological characteristics, physiological and biochemical properties and molecular biological identification results, strain QX3-4 was identified as Bacillus siamensis.

8. The method for controlling pine dieback of Siamese Bacillus according to claim 1, characterized in that: The application method of the inhibitory effect of the antagonistic bacteria aseptic fermentation filtrate on Aspergillus niger is as follows: d1. The strain was streaked on LB solid medium, cultured and activated at 28°C for 24 hours, a single colony was picked and inoculated into LB liquid medium, cultured at 28°C, 180 r / min for 12 hours to obtain seed solution, the seed solution was cultured in a shake flask at 28°C, 180 r / min for 72 hours at a rate of 0.2% inoculation volume and 50% liquid volume, and then centrifuged at 10,000 r / min for 15 minutes; d2, using a 0.22 pm bacterial filter to filter and sterilize the supernatant to obtain a sterile fermentation filtrate, mixing the fermentation filtrate with PDA medium, and preparing plates diluted 5-100 times; d3. After the culture medium solidifies, inoculate the pathogen cake in the center of the plate, use the mixed LB liquid culture medium as the control, and calculate the antibacterial rate of the strain fermentation filtrate against Aspergillus niger.

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