A strain of antagonistic bacteria MK94-B2 against Pinus massoniana red blight and its application

Abstract

The present application discloses a strain of antagonistic bacteria MK94‑B2 against red blight of Masson pine and its application, and relates to the field of microbial technology. The present application isolates and obtains antagonistic bacteria MK94‑B2 against red blight of Masson pine from branches of Masson pine, and the deposit number is CCTCC NO: M 20241586. Experiments have confirmed that the strain has a significant inhibitory effect on the conidia germination and germ tube elongation of the red blight pathogen of Masson pine. When treated with MK94‑B2 fermentation filtrate diluted 2.5 times, the spore germination rate of the pathogen of red blight of Masson pine is 0%, and the inhibitory effect is significant, and it can be used to prevent and treat red blight of Masson pine. In addition, the strain has an inhibitory effect on 8 important forest pathogenic fungi, especially on teak brown spot pathogen Alternaria alternata, Masson pine red blight pathogen Lushan pseudodisc multi-hairy spore and poplar leaf multi-septate sclerotinia, and can be used as a broad-spectrum antibacterial agent.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a biocontrol bacterium and a biological preparation for preventing and treating red blight of Masson pine. Background Art

[0002] Masson pine (Pinus massoniana Lamb.) belongs to the Pinus genus of the Pinaceae family. It not only plays a pioneering role in afforestation, but also has a wide range of uses and high economic value. The distribution area of ​​Masson pine is extremely wide, mainly in the south of the Qinling Mountains and the Huaihe River, and the east of the Yunnan-Guizhou Plateau. This tree species is not only used in construction projects, but also has medicinal value. It is also the main afforestation tree species in southern my country. The serious occurrence of Masson pine red blight not only damages the economic value of Masson pine, but also destroys the balance of the forestry ecosystem. Therefore, the prevention and control of Masson pine red blight is particularly important.

[0003] The pathogen of Masson pine red blight is the fungus of the genus Pestatiopsis, which, as a pathogen, lives in plant tissues. This disease mainly harms the new leaves of young forests, causing dead branches, resulting in a red forest stand, and affecting the stability of the ecosystem. In the early stage of pine needle damage, water-soaked yellow spots appear on the affected area, and then the spots gradually turn brown, and finally form gray or dark gray spots. A dark red ring is often used as a mark of the boundary between disease and health, and black conidia protrude from the diseased part. The shedding rate increases with the severity of the disease, seriously affecting the growth of tree height and forest quality. July is the peak period of Masson pine red blight, when the temperature is about 20 degrees Celsius and the humidity is 85% to 86%. After July, the incidence rate will show a downward trend. For a long time, Masson pine forests have been frequently threatened by red blight, and a large number of Masson pine needles have been damaged.

[0004] In recent years, due to a series of problems caused by improper application of chemical pesticides, biological control has become increasingly important in the prevention and control of Pinus massoniana red blight. Endophytic bacteria are a type of microbial group that is widely distributed in healthy plant tissues or intercellular spaces and is rich in diversity. As the main biocontrol microbial resource, endophytic bacteria, like other biocontrol microorganisms, mainly play a biocontrol role through competition, antagonism, antibiotics and induction of plant systemic disease resistance. Endophytic bacteria and host plants have influenced each other in the long-term co-evolution process, and gradually formed a harmonious coexistence and mutually beneficial relationship. The host plant provides the necessary nutrients and living places for the growth and reproduction of endophytes, and endophytes promote the growth of the host by secreting related substances and improving the host's ability to resist pests and diseases and adversity. Plant endophytes are not only rich in species, but also produce a variety of secondary metabolites, which almost include all natural compounds, and have great research value. A large number of studies have shown that endophytes can produce the same or similar biologically active substances as the host. At the same time, endophytes can also produce novel natural substances with disease prevention and antibacterial activities, providing a very rich microbial resource for the discovery of pesticide active substances and new medicines. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a biological control bacteria for preventing and controlling the red blight of Masson pine. The technical problem to be solved by the present invention is to provide a biological agent for preventing and controlling the red blight of Masson pine.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A strain of antagonistic bacteria MK94-B2 against red blight of Masson pine, named Bacillus velezensis MK94-B2, has been deposited in the China Center for Type Culture Collection, with the deposit number: CCTCC NO: M20241586, the deposit date: July 17, 2024, and the deposit address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] Application of antagonistic bacteria MK94-B2 against Masson pine red blight in the prevention and control of Masson pine red blight.

[0009] Application of antagonistic bacteria MK94-B2 against Pinus massoniana red blight in inhibiting the growth of pathogens.

[0010] The pathogens include teak brown spot pathogen Alternaria alternata, willow leaf spot pathogen Botryosphaeria dothidea, magnolia leaf spot pathogen Colletotrichum gloeosporioides, soapberry leaf spot pathogen Diaporthe biconispora, pine dieback pathogen Diplodia sapinea, cryptomeria fortunei stem canker pathogen Nerfusicoccumcryptomeriae, Masson pine red spot pathogen Pestalotiopsis lushanensis, and poplar leaf spot pathogen Septotinia populiperda.

[0011] A biological preparation for preventing and treating Masson pine red blight, comprising fermentation filtrate of Masson pine red blight antagonistic bacteria MK94-B2.

[0012] The application of the biological preparation in preventing and treating Pinus massoniana red blight.

[0013] The method for preventing and controlling the Masson pine red blight is to reduce the incidence of the Masson pine red blight.

[0014] Application of fermentation filtrate of antagonistic bacteria MK94-B2 against Masson pine red blight in the prevention and treatment of Masson pine red blight.

[0015] The preparation method of the fermentation filtrate of the Masson pine red blight antagonistic bacteria MK94-B2 is as follows: a single colony of the strain MK94-B2 grown on an LB culture medium for 3 days is selected and inoculated into an LB liquid culture medium, and the culture is placed on a shaker at 28°C and 200 r / min for 24 hours to obtain the fermentation broth of the strain MK94-B2; the fermentation broth of the strain MK94-B2 is placed in a high-speed centrifuge at 10,000 r / min, centrifuged for 10 minutes, and the supernatant is filtered through a 0.22 μm microporous filter membrane to obtain a sterile fermentation filtrate.

[0016] The method for preventing and controlling the Masson pine red blight is to reduce the incidence of the Masson pine red blight.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1) In the present application, the antagonistic bacteria MK94-B2 against red blight of Masson pine was isolated from branches of Masson pine, and its classification name is Bacillus velezensis MK94-B2 MK94-B2, and its preservation number is: CCTCC NO: M20241586. The results of the examples of the present application confirmed that the strain MK94-B2 inhibited the growth of hyphae of 8 important forest pathogenic fungi: Alternaria alternata, the leaf spot pathogen of teak, Botryosphaeria dothidea, Colletotrichum gloeosporioides, Diaporthe biconispora, Diplodia sapinea, Nerfusicoccum cryptomeriae, Pestalotiopsis lushanensis, and Septotinia populiperda. Among them, it has obvious inhibitory effect on teak brown spot pathogen Alternaria alternata, Masson pine red spot pathogen Pestalotiopsis lushanensis and poplar leaf spot pathogen Septotinia populiperda, with inhibition rates of 64%, 79% and 88% respectively, indicating that strain MK94-B2 has a broad-spectrum antibacterial ability.

[0019] 2) The examples of the present application confirm that the fermentation filtrate of strain MK94-B2 has a significant inhibitory effect on the conidia germination and germ tube elongation of Masson pine red blight pathogen, which can cause the germ tubes to swell into a sphere or no germ tubes to extend, proving that the inhibitory effect is significant; when the fermentation filtrate of MK94-B2 diluted 2.5 times is used for treatment, the spore germination rate of Masson pine red blight pathogen is 0%. Therefore, strain MK94-B2 and its fermentation filtrate have a good effect on preventing and controlling Masson pine red blight.

[0020] 3) The antagonistic bacteria against red blight of Masson pine isolated and screened in the present application can effectively prevent and control the red blight of Masson pine, while reducing the incidence of Masson pine, without causing damage to the environment and the host; the results of the potted experiment of the present application confirmed that after spraying the fermentation filtrate of strain MK94-B2 for 48 hours and inoculating the pathogen, there were no obvious lesions after 5 days, and the lesions were smaller after 15 days. The incidence rate was 40.8%, and the lesions were 3.61±0.7 mm. The degree of disease was significantly reduced compared with that without spraying the fermentation filtrate of strain MK94-B2, and it has a good antagonistic function against red blight of Masson pine. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the colony morphology of five antagonistic bacteria after rescreening;

[0022] Figure 2 This is a graph showing the inhibition of mycelial growth of the pathogen Pestalotiopsis lushanensis by five antagonistic bacteria;

[0023] Figure 3 This is the colony morphology of strain MK94-B2 after being cultured on LB medium for 2 days;

[0024] Figure 4 Phylogenetic tree of strain MK94-B2 constructed based on 16S rRNA sequence BLAST results ( Figure 4 A) and the phylogenetic tree of strain MK 94-B2 constructed based on the BLAST results of the gyrA gene sequence ( Figure 4 B);

[0025] Figure 5 The antibacterial spectrum of strain MK94-B2 is measured (A is Pestalotiopsis lushanensis, B is Botryosphaeria dothidea, C is Alternaria alternat, D is Colletotrichum gloeosporioides, E is Septotinia populiperda, F is Diplodia sapinea, G is Diaporthebiconispora, and H is Nerfusicoccum cryptomeriae);

[0026] Figure 6 The graph is a graph showing the inhibitory effect of the fermentation filtrate of strain MK94-B2 on the conidia germination of Pestalotiopsis lushanensis LZ1-1, the pathogen of Pinus massoniana red blight (A is CK, B is the spore germination after the fermentation filtrate of strain MK94-B2 was diluted 5 times, and C is the spore germination after the fermentation filtrate of strain MK94-B2 was diluted 2.5 times);

[0027] Figure 7 This is a graph showing the control effect of the fermentation filtrate of strain MK94-B2 on Pinus massoniana red blight (the first group is CK, which was not inoculated with pathogens and sprayed with sterile LB culture medium; the second group was sprayed with sterile LB culture medium for 48 hours and then inoculated with pathogens; the third group was sprayed with the fermentation filtrate of strain MK94-B2 for 48 hours and then inoculated with pathogens). DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0029] Example 1

[0030] 1. Strain Isolation

[0031] Using the tissue separation method, the Masson pine branches collected from the state-owned Washan Forest Farm in Quanjiao County, Chuzhou City, Anhui Province were rinsed with tap water for 20 minutes, the Masson pine stem phloem was cut on a sterile operating table, cut into 0.5cm×0.5cm tissue blocks, disinfected with 75% ethanol solution for 30S, transferred to 1% sodium hypochlorite solution for disinfection for 90S, then rinsed with sterile water 3 times, and finally dried with sterile filter paper. The surface-sterilized tissue blocks were clamped with sterile tweezers and placed on potato dextrose agar (PDA) plates and incubated in a 25°C incubator for 4-5 days in the dark. The growth and morphology of the bacterial colonies were observed, and pure cultures were obtained by transferring the hyphae tips to new PDA culture media. Finally, 251 strains of bacteria were isolated from 50 Masson pine clone branches and stored at 4°C for future use.

[0032] 2. Strain screening

[0033] The plate confrontation method was used to screen the isolated endophytic bacterial strains of Masson pine. The specific steps are as follows:

[0034] A 5 mm diameter Pestalotiopsis lushanensis LZ1-1 bacterial block (preserved in the Pathology Laboratory of Nanjing Forestry University) was inoculated in the center of the PDA plate, and the purified bacterial strain was inoculated at equal distances (2.5 cm) around it. The plate was cultured in a 25°C incubator. When the control colony grew to 3 / 4 of the size of the plate, the diameter of the test colony was measured using the cross method. Each treatment was repeated 3 times.

[0035] Based on the size of colony diameter, 5 strains with antagonistic effects were finally obtained from 251 endophytic bacteria, such as Figure 1 shown.

[0036] The isolated highly effective antagonistic strains were rescreened using the plate confrontation method. The specific steps are as follows:

[0037] After the purified bacterial strains were inoculated at equal distances (2.5 cm) around the PDA for 24 h, 48 h, and 72 h, a 5 mm diameter Pestalotiopsis lushanensis LZ 1-1 bacterial block was inoculated in the center of the PDA plate and cultured in a 25 ° C incubator. When the control colony grew to 3 / 4 of the size of the plate, the diameter of the test colony was measured using the cross method, and the inhibition rate was calculated. Each treatment was repeated 3 times.

[0038]

[0039] The results of rescreening of 5 highly antagonistic strains showed that strain MK94-B2 had the best antibacterial effect 72 hours after inoculation, with an inhibition rate of up to 90%. The inhibition rates of each strain are shown in Table 1 and Figure 2 shown.

[0040] Table 1 Results of rescreening antibacterial rate

[0041] Strain number MK 17-B1 MK 51-B2 MK 63-B1 MK 94-B2 MK 120-B1 24h antibacterial rate (%) 58 62 66 73 70 48h antibacterial rate (%) 79 84 78 89 80 72h antibacterial rate (%) 82 89 85 90 70

[0042] Example 2

[0043] 1. Strain identification and preservation

[0044] 1) Morphological identification

[0045] After culturing strain MK94-B2 on NA medium for 2 days, the colonies were milky white with wrinkles and irregular edges ( Figure 3 ). Gram staining results in purple, indicating Gram-positive bacteria.

[0046] 2) Molecular biological identification

[0047] The 16s rDNA gene sequence of strain MK 94-B2 is shown in SEQ ID NO.1; the gyrA gene sequence of strain MK 94-B2 is shown in SEQ ID NO.2.

[0048] In order to further clarify the taxonomic status of strain MK94-B2, genomic DNA of strain MK94-B2 was extracted and 16S rDNA and gyrA gene sequences were amplified by PCR. Homologous alignment was performed using Blast on the NCBI website, closely related species were downloaded, and the ML phylogenetic tree was constructed using the GTR+G4+F model. The phylogenetic results showed that strain MK 94-B2 and Bacillus velezensis FZB24 were clustered into a branch with a support rate of 97( Figure 4). Combined with morphological characteristics and phylogenetic analysis, strain MK94-B2 was identified as Bacillus velezensis, and its classification name was Bacillus velezensis MK94-B2, which has been deposited in the China Center for Type Culture Collection with the deposit number: CCTCCNO: M20241586, deposit date: July 17, 2024, and deposit address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection.

[0049] Example 3

[0050] The pathogenic bacteria tested in this embodiment (all of which were preserved in the Pathology Laboratory of Nanjing Forestry University) were Alternaria alternata, which causes brown spot of teak, Botryosphaeria dothidea, which causes leaf spot of weeping willow, Colletotrichum gloeosporioides, which causes leaf spot of Magnolia grandiflora, Diaporthe biconispora, which causes leaf spot of Sapindus mukorossi, Diplodia sapinea, which causes pine dieback, Nerfusicoccum cryptomeriae, which causes cedar stem canker, Pestalotiopsis lushanensis, which causes red blight of Masson pine, and Septotinia populiperda, which causes large spot of poplar.

[0051] The plate confrontation method was used to detect the inhibitory effect of MK94-B2 on 8 pathogenic fungi and determine the antibacterial spectrum of MK94-B2 strain. The specific steps are as follows:

[0052] 1. Determination of the antibacterial spectrum of MK94-B2

[0053] The plate confrontation method was used to detect the inhibitory effect of MK94-B2 on 8 pathogenic fungi. A 5 mm pathogenic fungus block was inoculated in the center of the PDA plate, and the purified strain MK94-B2 was inoculated at an equal distance (2.5 cm) around it. The plate was placed in a 25°C incubator for cultivation. When the control colony grew to 3 / 4 of the size of the plate, the colony to be tested was measured using the cross method. Each treatment was repeated 3 times.

[0054] The results are as follows Figure 5As shown in the results, strain MK94-B2 inhibited the growth of mycelium of 8 important forest pathogenic fungi. Among them, the inhibition of teak brown spot pathogen Alternaria alternata, masson pine red spot pathogen Pestalotiopsis lushanensis and poplar leaf spot pathogen Septotinia populiperda was particularly obvious, with inhibition rates of 64%, 79% and 88% respectively. This shows that strain MK94-B2 has a broad-spectrum antibacterial ability.

[0055] 2. Inhibitory effect of MK94-B2 fermentation filtrate on spore germination

[0056] The concave slide method was used to detect the inhibitory effect of strain MK94-B2 on the spore germination of Pestalotiopsis lushanensis LZ 1-1, the pathogen of Masson pine red blight.

[0057] 1) Preparation of pathogen spore solution

[0058] After the spores of Pestalotiopsis lushanensis LZ 1-1, the pathogen of Pinus massoniana red blight, were produced, the spore suspension was prepared to a concentration of 1×10 6 Pieces / mL.

[0059] 2) Preparation of fermentation filtrate of strain MK94-B2

[0060] A single colony of strain MK94-B2 grown on LB medium for 3 days was picked and inoculated into LB liquid medium, and placed on a shaker at 28°C and 200 r / min for 24 h to obtain the fermentation broth of strain MK94-B2; the fermentation broth of strain MK94-B2 was placed in a high-speed centrifuge at 10000 r / min, centrifuged for 10 min, and the supernatant was filtered through a 0.22 μm microporous filter membrane to obtain a sterile fermentation filtrate.

[0061] 3) Spore germination inhibition by fermentation filtrate of strain MK94-B2

[0062] 20 μL and 40 μL of fermentation filtrate were added to 80 μL and 60 μL of spore suspension of Pestalotiopsis lushanensis LZ 1-1, the pathogen of red blight of Masson pine, respectively, to prepare 100 μL of mixed solution. 20 μL of the mixed solution was pipetted and dropped on the center of the concave glass slide, and LB medium was used instead of fermentation filtrate as a control. After 24 hours, the spore germination was observed, and each treatment was repeated 3 times.

[0063] The results are as follows Figure 6As shown in the figure, the fermentation filtrate of strain MK94-B2 has a significant inhibitory effect on the spore germination and germ tube elongation of the pathogen of Pinus massoniana. The spore germination rate of the pathogen of Pinus massoniana in the control group was 100%, and the germ tube was slender ( Figure 6 A); After treatment with MK94-B2 fermentation filtrate diluted 5 times, the spore germination rate of Masson pine red blight pathogen was 85.09±0.12%. Although the germination rate was high, the bud tubes swelled into a spherical shape, proving that the inhibitory effect was significant ( Figure 6 B); after treatment with MK94-B2 fermentation filtrate diluted 2.5 times, the spore germination rate of the Masson pine red blight pathogen was 0% ( Figure 6 C).

[0064] Example 4

[0065] The control function of MK94-B2 fermentation filtrate on Pinus massoniana red blight:

[0066] A wound was made on the top needles of healthy potted Masson pine seedlings with a sterile needle. The Masson pine seedlings were divided into three groups. The first group (CK) was the control group and was sprayed with sterile LB medium. The second group (pathogen group) was sprayed with sterile LB medium for 48 hours and then the conidia suspension of Pestalotiopsis lushanensis LZ 1-1 (concentration of 10 6 The third group of pathogens + antagonists was sprayed with the fermentation filtrate of strain MK94-B2 for 48 hours, and the conidia suspension of Pestalotiopsis lushanensis LZ 1-1 (concentration of 10 6 All inoculated seedlings were bagged for moisture retention and placed in a greenhouse at 25±2℃ for continuous observation for 10 days. All experiments were repeated 3 times.

[0067] The results are as follows Figure 7 As shown, the control group sprayed with sterile LB culture medium potted plants did not get sick on the 5th and 15th days; the second group of pathogen group was sprayed with sterile LB culture medium for 48 hours and then inoculated with pathogens. There were no obvious lesions after 5 days, but the lesion area was significantly enlarged after 15 days, the incidence rate was 100%, and the lesions were 35.88±0.33mm; the third group of pathogen + antagonistic bacteria group was sprayed with the fermentation filtrate of strain MK94-B2 for 48 hours and then inoculated with pathogens. There were no obvious lesions after 5 days, and the lesions were smaller after 15 days, the incidence rate was 40.8%, and the lesions were 3.61±0.7mm. The degree of disease was significantly reduced compared with the second group inoculated with pathogens alone.

[0068] From the above test results, it can be seen that the strain MK94-B2 has a good inhibitory effect on the pathogen of Masson pine red blight, effectively reducing the occurrence and development of the disease, and the prevention and control effect is significant.

[0069] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A strain of antagonistic bacteria against Pinus massoniana red blight, MK94-B2, is named Bacillus velezensis MK94-B2 has been deposited in the China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 20241586, the deposit date: July 17, 2024, and the deposit address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. Use of the Masson pine red blight antagonistic bacterium MK94-B2 according to claim 1 in preventing and treating Masson pine red blight.

3. Use of the Masson pine red blight antagonistic bacteria MK94-B2 according to claim 1 in inhibiting the growth of pathogenic bacteria, wherein the pathogenic bacteria include Alternaria alternata , Botryosphaeria dothidea , Colletotrichum gloeosporioides , Diaporthe biconispora , Diplodia sapinea , Nerfusicoccum Cryptomeriae , Pestalotiopsis lushanensis , Septotinia populiperda .

4. A biological agent for preventing and treating Pinus massoniana red blight, characterized in that: A fermentation filtrate containing the Masson pine red blight antagonistic bacteria MK94-B2 according to claim 1.

5. Use of the biological agent according to claim 4 in preventing and treating Pinus massoniana red blight.

6. Use of the fermentation filtrate of the Masson pine red blight antagonistic bacteria MK94-B2 according to claim 1 in preventing and treating Masson pine red blight.

7. The biological agent according to claim 4, characterized in that The fermentation filtrate of the Masson pine red blight antagonistic bacteria MK94-B2 is prepared by the following method: picking a single colony of the strain MK94-B2 and inoculating it into an LB liquid culture medium, and placing it on a shaker at 28°C and 200 r / min for shaking culture for 24 hours to obtain the fermentation liquid of the strain MK94-B2; placing the fermentation liquid of the strain MK94-B2 in a high-speed centrifuge at 10000 r / min, centrifuging for 10 minutes, and filtering the supernatant through a 0.22 μm microporous filter membrane to obtain a sterile fermentation filtrate.

8. The use according to claim 6, characterized in that: The fermentation filtrate of the Masson pine red blight antagonistic bacteria MK94-B2 is prepared by the following method: picking a single colony of the strain MK94-B2 and inoculating it into an LB liquid culture medium, and placing it on a shaker at 28°C and 200 r / min for shaking culture for 24 hours to obtain the fermentation liquid of the strain MK94-B2; placing the fermentation liquid of the strain MK94-B2 in a high-speed centrifuge at 10000 r / min, centrifuging for 10 minutes, and filtering the supernatant through a 0.22 μm microporous filter membrane to obtain a sterile fermentation filtrate.

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