A strain of antagonistic bacteria AH2-38 against Pinus massoniana red blight and its application

Abstract

The present application discloses a strain of Masson pine red blight antagonistic bacteria AH2‑38 and its application, and relates to the field of microbial technology. The present application isolates and obtains Masson pine red blight antagonistic bacteria AH2‑38 from the rhizosphere soil of Masson pine, and the deposit number is CCTCC NO: M 20241587. Experiments have confirmed that the strain has a significant inhibitory effect on the conidia germination and germ tube elongation of Masson pine red blight pathogens, and can make the germ tubes swell into a sphere or no germ tubes extend. When treated with AH2‑38 fermentation filtrate diluted 2.5 times, the spore germination rate of Masson pine red blight pathogens is 0%, and the inhibitory effect is significant, which can be used to prevent and treat Masson pine red blight. In addition, the strain can inhibit 8 important forest pathogenic fungi, especially the Masson pine red blight pathogen Pseudomonas aeruginosa, the coral leaf spot pathogen Alternaria alternata and the poplar leaf spot pathogen Populus multispore sclerotinia, which has a particularly significant inhibitory effect and can be used as a broad-spectrum antibacterial agent.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and more specifically relates to a Masson pine red blight antagonistic bacterium AH2-38 and an application thereof. Background Art

[0002] Masson pine (Pinus massoniana Lamb.) is a tree plant of the genus Pinus in the Pinaceae family. It is drought-resistant and barren-resistant, and is a pioneer tree species for restoring forests on barren hills. Masson pine has a wide range of uses, high economic value, and is widely distributed. It is distributed in the south of the Qinling Mountains and the Huaihe River, and in the east of the Yunnan-Guizhou Plateau. It is the main afforestation tree species in southern my country.

[0003] Pine blight caused by the fungus Pestatiopsis is one of the common diseases of pine trees, usually occurring on new leaves in young forests. Pine blight is widespread in my country, affecting a large area and harming many tree species. It has been reported on Pinus massoniana, Pinus yunnanensis, Pinus thunbergii, Pinus taeda, Pinus tabulaeformis, Pinus sylvestris var. mongolica and Pinus elliottii, making prevention and control very difficult.

[0004] At present, my country's prevention and control of Pinus massoniana red blight caused by Pseudomonas fungi mainly rely on afforestation measures and chemical control. Afforestation measures are still theoretical. Although chemical control is effective, it not only pollutes soil and water bodies, but also makes Pseudomonas fungi resistant to drugs, which in severe cases will harm human health. With the continuous advancement of science and technology, biological control has gradually revealed its unique advantages. It has the characteristics of safety, pollution-free, low cost, and difficulty in pathogens to develop resistance. Its role in forest disease prevention and control is becoming increasingly prominent, showing a broad and far-reaching development prospect. 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 strain of antagonistic bacteria AH2-38 against red blight of Masson pine. The technical problem to be solved by the present invention is to provide an application of antagonistic bacteria AH2-38 against red blight of Masson pine for preventing and controlling 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 pine red blight antagonistic bacteria AH2-38, whose classification name is Bacillus velezensis AH2-38, has been deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M20241587, a deposit date of July 17, 2024, and a deposit address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0008] The application of the Masson pine red blight antagonistic bacteria AH2-38 in preventing and controlling the Masson pine red blight.

[0009] The fermentation filtrate of the Masson pine red blight antagonistic bacteria AH2-38 is used in preventing and treating Masson pine red blight.

[0010] The application is that the fermentation filtrate of the antagonistic bacteria AH2-38 against red blight of Masson pine is prepared by the following method: a single colony of the strain AH2-38 is picked and inoculated into an LB liquid culture medium to obtain a fermentation broth of the strain AH2-38; the fermentation broth is centrifuged to separate the solid and liquid, and the obtained liquid is filtered through a 0.22 μm microporous filter membrane to obtain the supernatant, which is the fermentation filtrate.

[0011] The application of the Masson pine red blight antagonistic bacteria AH2-38 in inhibiting pathogenic bacteria.

[0012] The pathogens include teak brown spot pathogen Alternaria alternata, willow leaf spot pathogen Botryosphaeria dothidea, coral phoenix leaf spot pathogen Colletotrichumsiamense, soapberry leaf spot pathogen Diaporthe eres, pine dieback pathogen Diplodia sapinea, cedar stem canker pathogen Neofusicoccum cryptomeriae, Masson pine red spot pathogen Pestalotiopsis jiangsuensis and poplar leaf spot pathogen Septotinia populiperda.

[0013] The method for preparing the fermentation filtrate of the Masson pine red blight antagonistic bacteria AH2-38 is specifically as follows: a single colony of the strain AH2-38 grown on a NA medium for 3 days is picked and inoculated into an LB liquid medium, and the culture is placed on a shaker at 28° C. and 200 r / min for 24 hours to obtain a fermentation broth of the strain AH2-38; the fermentation broth of the strain AH2-38 is placed in a high-speed centrifuge at 10,000 r / min, centrifuged for 8 minutes, and the supernatant is filtered through a 0.22 μm microporous filter membrane to obtain a sterile fermentation filtrate.

[0014] The application of the Masson pine red blight antagonistic bacteria AH2-38 in the preparation of a broad-spectrum antibacterial agent.

[0015] The fermentation filtrate of the Masson pine red blight antagonistic bacteria AH2-38 is used in the preparation of a broad-spectrum antibacterial agent.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1) In the present application, the antagonistic bacteria AH2-38 against red blight of Masson pine was isolated from the rhizosphere soil of Masson pine, which was classified and named Bacillus velezensis AH2-38 and has been deposited in the China Center for Type Culture Collection with the deposit number: CCTCC NO: M 20241587. The results of the embodiment show that the strain AH 2-38 can inhibit the growth of mycelium of 8 important forest pathogenic fungi: teak brown spot pathogen Alternaria alternata, willow leaf spot pathogen Botryosphaeria dothidea, coral leaf spot pathogen Colletotrichum siamense, soapberry leaf spot pathogen Diaporthe eres, pine blight pathogen Diplodiasapinea, cedar stem canker pathogen Neofusicoccum cryptomeriae, pine red blight pathogen Pestalotiopsis jiangsuensis and poplar leaf spot pathogen Septotinia populiperda. It shows that the strain AH 2-38 has a broad-spectrum antibacterial ability and can be used to prepare a broad-spectrum antibacterial agent. Among them, strain AH2-38 has a particularly significant inhibitory effect on the hyphae growth of Pestalotiopsis jiangsuensis, the coral leaf spot pathogen Alternaria alternata, and the poplar leaf spot pathogen Septotinia populiperda, with outstanding results.

[0018] 2) The examples of the present application confirm that the fermentation filtrate of strain AH2-38 has a significant inhibitory effect on the germination of conidia and the elongation of germ tubes 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 AH2-38 diluted 2.5 times is used for treatment, the spore germination rate of Masson pine red blight pathogen is 0%. Therefore, strain AH2-38 and its fermentation filtrate have a good effect on preventing and controlling Masson pine red blight.

[0019] 3) The antagonistic bacteria against red blight of Masson pine separated and screened by the present invention can effectively prevent and treat red blight of Masson pine, and while reducing the incidence of Masson pine, does not cause damage to the environment and the host; it plays an important role in reducing the use of fertilizers and pesticides and alleviating agricultural non-point source pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1This is a screen diagram for antagonistic bacteria against Masson pine red blight (A is CK, B is strain AH 2-6, C is strain NL 2-52, D is strain AH 2-19, E is strain AH 2-8, and F is strain AH2-38);

[0021] Figure 2 This is the colony morphology of strain AH 2-38 after culturing on NA medium for 2 days;

[0022] Figure 3 is the phylogenetic tree of strain AH 2-38 constructed based on 16S rRNA sequence;

[0023] Figure 4 The antibacterial spectrum of strain AH 2-38 is measured (A is Alternaria alternata, B is Pestalotiopsis jiangsuensis, C is Diplodia sapinea, D is Neofusicoccumcryptomeriae, E is Colletotrichum siamense, F is Botryosphaeria dothidea, G is Diaporthe eres, and H is Septotinia populiperda);

[0024] Figure 5 The figure is the inhibitory effect of the fermentation filtrate of strain AH 2-38 on the conidia germination of Pestalotiopsisjiangsuensis BM1-1, the pathogen of red blight of Masson pine (A is CK, B is the spore germination after the fermentation filtrate of strain AH 2-38 was diluted 5 times, and C is the spore germination after the fermentation filtrate of strain AH 2-38 was diluted 2.5 times);

[0025] Figure 6 The control effect of the fermentation filtrate of strain AH 2-38 on Pinus massoniana red blight (the first group is CK, which is not inoculated with pathogens and sprayed with sterile LB culture medium; the second group is inoculated with pathogens 48 hours later and then sprayed with sterile LB culture medium; the third group is inoculated with pathogens 48 hours later and then sprayed with the fermentation filtrate of strain AH 2-38). DETAILED DESCRIPTION

[0026] 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.

[0027] The Masson pine rhizosphere soil samples used in the present invention were taken from three locations: Bengbu, Anhui, Quanjiao, Anhui, and Nanjing, Jiangsu.

[0028] Example 1

[0029] 1. Strain Isolation

[0030] Six healthy Masson pine rhizosphere soil samples were collected from three locations: Bengbu, Anhui, Quanjiao, Anhui, and Nanjing, Jiangsu. 10 g of soil sample was placed in a triangular flask containing 90 mL of sterile water, and an appropriate amount of sterile glass beads was added. The sample was placed on a shaker at 28°C and 180 r / min for 30 min to fully mix the soil sample with the sterile water. The soil sample was diluted to 10 using the gradient dilution method. -4 , 10 -5 , 10 -6 , take 100 μL of 10 -4 , 10 -5 , 10 -6 The soil suspension was spread on NA medium using a spreader rod and repeated three times. The plate was placed in a 28°C incubator for 2 days. Bacterial colonies of different shapes, colors and sizes were selected, purified and cultured using the plate streak method, and numbered. Finally, 477 bacterial strains were isolated and stored in a 4°C refrigerator.

[0031] 2. Strain screening

[0032] The isolated bacterial strains were screened using the plate confrontation method. The specific steps are as follows:

[0033] A 5-mm-diameter Pestalotiopsis jiangsuensisBM1-1 bacterial block (preserved in the Forest 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.

[0034] Based on the size of colony diameter, 5 bacterial strains were initially screened out from 477 bacterial strains, which had a high antagonistic effect on Pseudomonas jiangsuensis. Figure 1 shown.

[0035] The isolated highly effective antagonistic strains were rescreened using the plate confrontation method. The specific steps are as follows:

[0036] A 5-mm-diameter Pestalotiopsis jiangsuensisBM1-1 bacterial block was inoculated in the center of the PDA plate, and the purified bacterial strain was inoculated at an equal distance (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, and the inhibition rate was calculated. Each treatment was repeated 3 times.

[0037]

[0038] Through rescreening of 5 highly antagonistic strains, the results showed that strain AH2-38 had the best antibacterial effect, with an inhibition rate of up to 79% (Table 1).

[0039] Table 1 Results of rescreening antibacterial rate

[0040] Strain number CK AH 2-6 NL 2-52 AH 2-19 AH 2-8 AH2-38 Antibacterial rate (%) 0 59 69 63 74 79

[0041] Example 2

[0042] 1. Strain identification and preservation

[0043] 1) Morphological identification

[0044] After strain AH2-38 was cultured on NA medium for 2 days, the colonies were milky white, wrinkled, and had irregular edges ( Figure 2 ). Gram staining results in purple, indicating Gram-positive bacteria.

[0045] 2) Molecular biological identification

[0046] In order to further clarify the taxonomic status of strain AH2-38, genomic DNA of strain AH2-38 was extracted and 16S rDNA gene sequence was amplified by PCR (as shown in SEQ ID NO.1). 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 AH2-38 and Bacillus velezensis RA-9 were clustered into one branch with a support rate of 100 ( Figure 3 ). Combined with morphological characteristics and phylogenetic analysis, strain AH2-38 was identified as Bacillus velezensis, and its classification name was Bacillus velezensis AH2-38, which has been deposited in China Center for Type Culture Collection with the deposit number: CCTCCNO: M 20241587, deposit date: July 17, 2024, and deposit address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China Center for Type Culture Collection.

[0047] Example 3

[0048] The pathogenic bacteria tested in this example (preserved in the Forest Pathology Laboratory of Nanjing Forestry University) are Alternaria alternata, which causes brown spot disease of teak, Botryosphaeria dothidea, which causes leaf spot disease of weeping willow, Colletotrichum siamense, which causes leaf spot disease of coral paulownia, Diaporthe eres, which causes leaf spot disease of soapberry, Diplodia sapinea, which causes dieback disease of white bark pine, Neofusicoccum cryptomeriae, which causes stem base canker disease of Cryptomeria fortunei, and Pseudomonas sylvestris, which causes red blight disease of Masson pine.

[0049] (Pestalotiopsis jiangsuensis), and Sclerotinia sclerotiorum, which causes poplar leaf spot disease

[0050] (Septotinia populiperda).

[0051] The plate confrontation method was used to detect the inhibitory effect of AH 2-38 on 8 pathogenic fungi. The specific steps are as follows:

[0052] 1. Determination of the antibacterial spectrum of AH 2-38

[0053] The plate confrontation method was used to detect the inhibitory effect of AH 2-38 on 8 pathogenic fungi. A 5mm diameter pathogenic fungal block was inoculated in the center of the PDA plate, and the purified strain AH 2-38 was inoculated at an equal distance (2.5cm) 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 4 As shown in the figure, strain AH 2-38 inhibited the mycelial growth of 8 pathogenic fungi. Among them, the inhibitory effect on Pestalotiopsis jiangsuensis, Alternaria alternata and Septotinia populiperda was particularly obvious, indicating that strain AH 2-38 has a broad-spectrum antibacterial ability ( Figure 4 ).

[0055] 2. Inhibitory effect of AH2-38 fermentation filtrate on spore germination

[0056] The concave slide method was used to detect the inhibitory effect of strain AH2-38 on the spore germination of Pestalotiopsis jiangsuensis BM1-1, the pathogen of Pinus massoniana red blight.

[0057] 1) Preparation of pathogen spore solution

[0058] After the spores of Pestalotiopsis jiangsuensis BM1-1, the pathogen of Pinus massoniana red blight, are produced, the spore suspension is prepared to a concentration of 10 6 Pieces / mL.

[0059] 2) Preparation of fermentation filtrate of strain AH2-38

[0060] A single colony of strain AH2-38 grown on NA medium for 3 days was picked up and inoculated into LB liquid medium, and placed on a shaker at 28°C and 200 r / min for 24 hours to obtain the fermentation broth of strain AH2-38; the fermentation broth of strain AH2-38 was placed in a high-speed centrifuge at 10000 r / min, centrifuged for 8 minutes, and the supernatant was filtered through a 0.22 μm microporous filter membrane to obtain a sterile fermentation filtrate.

[0061] 3) Strain AH 2-38 fermentation filtrate inhibits spore germination

[0062] 20 μL and 40 μL of fermentation filtrate were added to 80 μL and 60 μL of spore suspension of Pestalotiopsis jiangsuensis BM1-1, 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 5 As shown in the figure, the fermentation filtrate of strain AH2-38 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 5 A); After treatment with AH2-38 fermentation filtrate diluted 5 times, the spore germination rate of Masson pine red blight pathogen was 89.8±0.04%. Although the germination rate was high, the bud tubes swelled into a spherical shape, proving that the inhibitory effect was significant ( Figure 5 B); After being treated with the 2.5-fold diluted AH2-38 fermentation filtrate, the spore germination rate of the Masson pine red blight pathogen was 0%. It can be seen that the last cell of the three middle cells was slightly swollen, but no germ tube extended ( Figure 5 C).

[0064] Example 4

[0065] Effect of AH2-38 fermentation filtrate on control of Pinus massoniana red blight

[0066] A wound was made on the top needle of healthy potted Masson pine seedlings with a sterile needle. The Masson pine seedlings were divided into three groups. The first group was the control group, which was sprayed with sterile LB medium; the second group was treated with conidia suspension of Pestalotiopsis jiangsuensis BM1-1 (10 6 The third group was sprayed with conidia suspension (10 6 The inoculated seedlings were bagged for moisture retention and placed in a greenhouse at 25±2℃ for 10 days. All the experiments were repeated 3 times.

[0067] The results are as follows Figure 6 As shown, the control group sprayed with sterile LB medium had no disease on the 5th and 10th days; the second group was inoculated with the pathogen for 48 hours and then sprayed with sterile LB medium. After 5 days, the lesions were obvious, and after 10 days, the lesion area expanded, the incidence rate was 100%, and the lesion size was 34.88±0.34mm; the third group was inoculated with the pathogen for 48 hours and then sprayed with the fermentation filtrate of strain AH 2-38. After 5 days, there were no obvious lesions, and after 10 days, the lesions were smaller, the incidence rate was 48.4%, and the lesion size was 3.51±0.72mm. The disease severity was significantly lower than that of the second group.

[0068] From the above test results, it can be seen that strain AH 2-38 has a good inhibitory effect on the pathogen of Masson pine red blight, significantly reducing the occurrence and development of the disease, and the prevention and control effect is remarkable.

[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 bacterium AH2-38 antagonistic to Pinus massoniana red blight, which is classified and named Bacillus velezensis AH2-38 has been deposited in the China Center for Type Culture Collection, with the deposit number: CCTCC NO: M 20241587, the deposit date: July 17, 2024, and the deposit address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. Use of the antagonistic bacteria AH2-38 against red blight of Masson pine according to claim 1 in preventing and treating red blight of Masson pine.

3. Use of the fermentation filtrate of the Masson pine red blight antagonistic bacteria AH2-38 according to claim 1 in preventing and treating Masson pine red blight.

4. The use according to claim 3, characterized in that: The fermentation filtrate of the Masson pine red blight antagonistic bacteria AH2-38 is prepared by the following method: a single colony of the strain AH2-38 is picked and inoculated into an LB liquid culture medium to obtain a fermentation broth of the strain AH2-38; the fermentation broth is centrifuged to separate the solid and liquid, and the obtained liquid is filtered through a 0.22 μm microporous filter membrane to obtain the supernatant, which is the fermentation filtrate.

5. Use of the antagonistic bacteria AH2-38 against red blight of Masson pine according to claim 1 in inhibiting pathogenic bacteria, wherein the pathogenic bacteria are Alternaria alternata, Botrytis cinerea, Colletotrichum siamese, Prunella vulgaris, Diplodia pine, Neothracena fortunei, Polytrichomonas jiangsuensis or Sclerotinia sclerotiorum of poplar leaves.

6. Use of the antagonistic bacteria AH2-38 against red blight of Masson pine according to claim 1 in the preparation of a broad-spectrum antibacterial agent, wherein the antibacterial agent is used to inhibit Alternaria alternata, Botrytis cinerea, Colletotrichum siamese, Psoralea corylifolia, Diplodia pine, Neothracena fortunei, Polytrichomonas jiangsuensis or Sclerotinia sclerotiorum of poplar leaves.

Images