Antagonistic strain Bjerkandera adusta F2 capable of efficiently preventing and controlling pepper phytophthora blight and application of antagonistic strain Bjerkandera adusta F2

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to an antagonistic strain Bjerkandera adusta F2 capable of efficiently preventing and controlling pepper phytophthora blight and application of the antagonistic strain Bjerkandera adusta F2. The invention relates to an antagonistic strain F2, the taxonomic name of the antagonistic strain F2 is Bjerkandera adusta, the antagonistic strain F2 is preserved in Guangdong Microbial Culture Collection Center on August 23, 2024, and the preservation number is GDMCC No: 65051. The screened antagonistic strain F2 has the effect of efficiently preventing and controlling the pepper phytophthora blight, the colonization ability of the strain in soil is high, the lasting period is long, the effect is stable, support can be provided for biological prevention and control of the pepper phytophthora blight and development of related biopesticides, bacterial fertilizers and bio-organic fertilizers, and the antagonistic strain F2 has important practical value for guaranteeing sustainable development of the pepper industry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and particularly relates to a high-efficiency antagonistic strain Bjerkandera adusta F2 for preventing and controlling pepper blight and application thereof. BACKGROUND

[0002] Pepper is one of the pillar industries in Guizhou, Hunan and Jiangxi, etc. However, in recent years, with the development of intensive agriculture, unreasonable use of chemical fertilizers and pesticides, and long-term continuous cropping, various soil-borne diseases have broken out, which has caused a serious threat to the development of pepper industry. Among them, pepper blight caused by Phytophthora capsici Leonian is one of the most serious soil-borne diseases in pepper production, which has fast transmission, heavy incidence and strong destructiveness, and even causes the absolute loss of pepper. Meanwhile, the pathogenic fungus can not only infect Solanaceae crops such as pepper, but also pose a threat to the production of legume crops and Cucurbitaceae crops. Therefore, how to efficiently prevent and control pepper blight is a major problem in the actual production process of pepper. Phytophthora capsic

[0003] At present, China mainly uses traditional methods such as chemical pesticide spraying and disease-resistant variety breeding to control pepper blight. However, due to the strong drug resistance of the pathogenic fungus, the long breeding cycle of varieties, and the irreversible damage of chemical pesticides to the agricultural environment, food safety and human health, it is difficult to achieve the goals of efficient disease control, environmental friendliness and safety and health, and new solutions are urgently needed.

[0004] Microbial control is an environmentally friendly disease control technology that uses the complex interactions between plants and microorganisms and between microorganisms to inhibit the infection of pathogenic fungi on plants by applying one or a type of soil beneficial microorganism. It is currently an internationally recognized safe and effective measure for the prevention and control of major agricultural and forestry diseases, and can play a role of "four ounces to move a thousand catties" in protecting crop health. Suppressive soil is a treasure house of naturally enriched beneficial microbial resources, and antagonistic strains play a key role in inhibiting the growth of pathogenic fungi, regulating the composition of rhizosphere communities and protecting plant health, which is far beyond their low abundance. At present, the screening of antagonistic microorganisms in suppressive soil and the construction of artificial synthetic microbial flora are international new frontiers and hotspots for the prevention and control of crop diseases and the maintenance of soil health. Studies have shown that microbial agents such as Pseudomonas, Bacillus subtilis, Trichoderma hamatum, Chaetomium aureum and Bacillus amyloliquefaciens have a significant effect on the prevention and control of pepper blight, with a disease control rate of more than 70%. However, due to the difficulty of resisting biological and non-biological stress conditions in complex soil environments and the competition with indigenous microorganisms, the widely used antagonistic microbial agents have weak rhizosphere colonization ability, low survival rate and unstable function, which limits the large-area application of the products and technologies. Therefore, it is crucial to creatively mine special microbial agents for preventing and controlling pepper blight with high microbial activity to ensure that biological control plays a high-efficiency and stable disease control role in the field. ​

[0005] Currently, there is no related report on the prevention and control of pepper blight by using P. nigroporus. Bjerkandera adusta

[0006] The information disclosed in this BACKGROUND section is only for the purpose of increasing the understanding of the background of the application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art that is publicly known. SUMMARY

[0007] The present application aims to provide an antagonistic strain P. nigroporus F2 for efficiently preventing and controlling pepper blight. Bjerkandera adusta F2 and its application.

[0008] To achieve the above-mentioned purpose, the present application provides the following technical solutions. The first object of the present application is to provide an antagonistic strain F2, which is taxonomically named P. nigroporus (P. nigroporus) and was deposited in the Guangdong Microbial Culture Collection Center on August 23, 2024, with the accession number GDMCC No: 65051. Bjerkandera adusta

[0009] The second object of the present application is to provide the application of the antagonistic strain F2 in efficiently preventing and controlling plant diseases.

[0010] Further, the plant disease includes pepper blight.

[0011] Further, the pathogenic bacteria of pepper blight is pepper Phytophthora.

[0012] The third object of the present application is to provide a biological pesticide, microbial fertilizer or biological organic fertilizer, which comprises the antagonistic strain F2 of claim 1.

[0013] The fourth object of the present application is to provide a method for efficiently preventing and controlling pepper blight, comprising the following steps: applying the antagonistic strain F2 to the rhizosphere of pepper seedlings.

[0014] Further, the concentration of the antagonistic strain F2 is 1.0×10 8 cfu / mL, and the inoculation amount is 1.0×10 8 cfu / g of soil.

[0015] Compared with the prior art, the present application has the following beneficial effects: (1) High antagonistic efficiency: the P. nigroporus F2 of the present application has a high antagonistic effect on pepper Phytophthora, with a plate antagonistic rate of 86.1% and a fermentation liquid inhibition rate of 93.7%, and the effect is remarkable.

[0016] ​​(2) stable and lasting effect: pot experiment for 5 consecutive seasons showed that after inoculation of the present invention F2, the incidence of pepper blight was reduced by 61.9% compared with the control group, and the number of pathogenic bacteria in the soil was reduced from 1.23x10 7 cfu / g to 6.17x10 3 cfu / g, about 10000 times, solving the problem of weak colonization ability and unstable effect of existing antagonistic bacteria.

[0017] In summary, the antagonistic strain F2 screened by the present invention has high efficiency in preventing and controlling pepper blight, and has strong colonization ability in soil, long lasting period and stable effect, which can provide support for biological control of pepper blight and development of related biological pesticides, bacterial manure and biological organic fertilizer, and has important practical value for ensuring the sustainable development of pepper industry.

[0018] Preservation information The taxonomic name of strain F2 is Physisporinus pachyphorus, which was preserved in Guangdong Microbial Culture Collection Center on August 23, 2024, the preservation address is No. 59, Building 5, Guangzhou Xianlie Middle Road 100, Institute of Microbiology, Guangdong Academy of Sciences, and the preservation number is GDMCC No: 65051. Bjerkandera adusta BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The occurrence of pepper blight in the high incidence soil and disease suppression soil of the test base; Figure 2 The antagonistic strain obtained after 2 rounds of screening; Figure 3 The results of re-screening and plate confrontation; Figure 4 The inhibition effect of F2 fermentation on pathogenic bacteria Figure 5 The phylogenetic tree of molecular identification; Figure 6 The occurrence and growth health of pepper in different treatment groups in pot experiment; Figure 7 The incidence of pepper blight and the number of pathogenic bacteria in soil in 5 consecutive seasons of planting; DETAILED DESCRIPTION

[0020] The technical solutions of the present invention will be described below in a clear and complete manner. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present invention.

[0021] ​In this example, the high-incidence soil samples used for isolating and identifying the pathogen and the disease-suppressing soil samples used for isolating antagonistic strains were both obtained from a long-term organic fertilization experimental base in Feicheng County, Tai'an City, Shandong Province. This experimental base is one of the most important organic agricultural production areas in Shandong Province, China. From 1994 to 2020, it conducted field trials of organic fertilizer (cow manure organic fertilizer) and chemical fertilizer for 26 consecutive years. The experimental field has passed EU and Japanese organic certifications. The occurrence of pepper blight in the high-incidence soil and disease-suppressing soil of the experimental base is as follows: Figure 1 As shown, in 2022, the incidence rates of disease in chili peppers grown in high-disease soils and disease-suppressing soils with long-term application of organic fertilizers were 87.5% and 8.5%, respectively, with pathogen counts of 4.56 × 10⁻⁶. 6 cfu / g and 6.45×10 2 cfu / g soil.

[0022] 1. Isolation, identification, and culture of Phytophthora capsici, the pathogen of pepper. 1.1 Separation 1.0g of rhizosphere soil was collected from pepper plants in Changsha County, Hunan Province, where long-term application of chemical fertilizers and continuous cropping led to a high incidence of Phytophthora blight. A suspension was prepared by placing the soil in an Erlenmeyer flask containing 45mL of sterile water and incubating it for 1 hour at 28℃ and 180rpm in a constant-temperature shaker. 1mL of the soil suspension was then added to 9mL of sterile water, mixed thoroughly, and serially diluted to 10⁻⁶. -3 10 -4 10 -5 10 -6 10 -7 10 -8 The solutions were prepared by taking 50 μL of suspensions of different gradients, spreading them on PDA plates, and incubating them in a constant temperature incubator at 28°C for about 48 hours. Single colonies from each plate were then picked up with a syringe needle and transferred to a new PDA plate for further incubation.

[0023] 1.2 Identification After the bacteria had completely covered the petri dish, microbial morphology was observed. Strains with similar morphology to the pathogen were further cultured until conidia were produced. 10 mL of sterile water was added, and germinating spores were scraped from the plate using a sterile spreader. The resulting spore suspension was passed through four layers of sterile gauze for single-cell isolation and sequencing. The obtained sequences were compared using BLAST in the NCBI database, and the pathogen was identified as *Phytophthora capsici*. Phytophthora capsic ).

[0024] 1.3 Cultivation The pathogen was inoculated onto a PDA plate and cultured in a constant temperature incubator at 28°C for 7 days. 10 mL of sterile water was added to obtain a spore suspension. The spore concentration in the spore suspension was determined using a hemocytometer. The spore suspension was then mixed with an equal volume of sterile 30% glycerol and stored at -80°C for later use.

[0025] 2. Isolation of antagonistic strains 2.1 Separation A 5.0g sample of rhizosphere soil from chili pepper plants grown in healthy soil (long-term application of organic fertilizer) sourced from Hunan Xiangyan Seed Industry in Changsha County, Hunan Province, was placed in a 250mL sterile Erlenmeyer flask containing 45mL of sterile water and incubated at 30℃ and 180rpm for 1 hour in a constant temperature shaker. 1mL of the soil suspension was then added to 9mL of sterile water, mixed thoroughly, and serially diluted to 10⁻⁶. -3 10 -4 10 -5 10 -6 10 -7 10 -8 The solutions were prepared by adding 50 μL of each solution to beef extract peptone agar (NA) plates and PDA plates, spreading them evenly with a spreader. Each treatment group was replicated in triplicate.

[0026] The coated NA and PDA plates were incubated in constant temperature incubators at 37℃ and 28℃, respectively. Single colonies with different colony characteristics were picked and cultured and purified until single colonies appeared on the plates.

[0027] 2.2 Flat-panel standoff 1 μL of pathogenic bacteria and fungal spores, preserved in glycerol at -80℃, were inoculated onto PDA plates and incubated at 28℃. Once the colonies were fully grown, 2 mm bacterial discs were punched out. The pathogen was inoculated at the center of the plate, and purified single bacterial strains were inoculated at opposite ends, 30 mm apart, for plate confrontation. Additionally, isolated bacterial colonies were inoculated with an inoculation loop at 30 mm intervals between the centers of the plates to confront the pathogen. Six replicates were set up for each treatment, along with a control group, and the plates were incubated for another 7 days.

[0028] The degree of antagonism was determined by the distance from the bacterial or fungal inoculation point to the outer edge of the *Phytophthora capsici* colony. Colonies showing significant inhibition of pathogen growth were selected, numbered, and recorded. Strains exhibiting significant antagonistic activity against *Phytophthora capsici* were screened, and the screening results are shown in […]. Figure 2 .

[0029] Depend on Figure 2 It is known that after two rounds of screening, 28 fungi and 17 bacteria have been selected from 182 isolated strains (102 fungi and 80 bacteria).

[0030] 3. Antagonistic rescreening of antagonistic fungi and Phytophthora capsici. 3.1 Flat-panel standoff The pathogenic bacteria and the antagonistic bacteria were inoculated in PDA plates at a distance of 1 cm from the edge of the plate, 1 strain per plate, 6 repeats per colony, and a control group was set up, and the culture was continued for 7 days. The fungi with strong antagonistic ability were recorded and preserved. The results of the confrontation of the F2 plate are shown in Figure 3 .

[0031] It can be seen from Figure 3 that the antagonistic strain F2 has a significant antagonistic effect on the pepper Phytophthora, and as the confrontation culture time increases, the antagonistic rate of the pathogenic bacteria reaches 86.1% on the 10th day.

[0032] 3.2 Inhibition rate of fermentation liquor After the antagonistic strain was cultured with PDA medium, the fungus cake was punched with a puncher and inoculated in the center of the PDA medium, and after 7 days of culture in a 28°C constant temperature incubator, 10 μL of sterile water was added to obtain a spore suspension, and the spore concentration in the strain spore solution was determined with a hemocytometer. 2 μL of the antagonistic strain spore solution (concentration 1.0×0 7 cfu / mL) was inoculated into a triangular flask containing 100 mL of PDB medium, and the culture was carried out in a 28°C constant temperature shaker at 180 rpm for 7 days. The fermentation product was centrifuged, the supernatant was filtered through a 0.22 μm sterile water system filter head, and the product was stored in a 4°C refrigerator.

[0033] 10 mL of the filtered fermentation liquor was taken and poured into 90 mL of pre-melted PDA medium to make a flat plate. After solidification, the pathogenic fungus cake was punched with a puncher and inoculated on the plate, and the culture was carried out in a 28°C constant temperature incubator, and the diameter of the pathogenic fungus colony was measured at a fixed time every day for 7 consecutive days, and the inhibition effect of different antagonistic strains on the growth of the pathogenic fungus was calculated. At the same time, sterile water was used as a control for the pathogenic fungus colony growth determination.

[0034] Inhibition rate (%) = (control colony diameter - treatment colony diameter) / control colony diameter x 100%.

[0035] It can be seen from Figure 4 that the fermentation liquor of the antagonistic strain F2 also has a significant inhibitory effect on the growth of the pathogenic fungus, and the inhibition rate reaches 41.7%.

[0036] 4. Identification of antagonistic strain F2 2 mL of the spore solution of the antagonistic strain F2 (spore concentration 1.0×0 7 cfu / mL) was inoculated in PDA medium, and the plate was grown in a 28°C constant temperature incubator until the plate was covered, and the genomic DNA of the strain was extracted with a genomic reagent kit. The genomic DNA was used as a template, and the ITS universal primer was used for PCR amplification in the ITS1 region.

[0037] The amplification product was subjected to 1% agarose gel electrophoresis test, the target band was obtained, the gel was purified and recovered, and the sequence was determined (the sequence is shown in the sequence table). The obtained sequence was subjected to BLAST comparison by NCBI database, and the sequences of the isolated antagonistic bacteria and model bacteria were subjected to phylogenetic analysis by MEGA7.0 software. According to the comparison results, the phylogenetic tree was constructed combined with the model strain with the highest similarity, and the results are shown in Figure 5 .

[0038] It was identified that the antagonistic strain F2 had high similarity with the genus of Pyropolyporus, Bjerkandera adusta , and the strain was determined as the genus of Pyropolyporus, and was named as F2. Bjerkandera adusta

[0039] 5. Pot experiment 5.1 Seedling culture The pepper seeds (No. YJ-199) in this example were provided by the Pepper Group of the Horticulture College of Hunan Agricultural University. An appropriate amount of pepper seeds was soaked in warm water at 50°C for 3 h, and after sufficient water absorption, the seeds were rapidly surface sterilized with 75% (v / v) ethanol for 30 s, sterilized with 1% (v / v) sodium hypochlorite for 20 min, and then washed with sterile water for 5 times. The cleaned seeds were evenly dispersed and laid on sterile Petri dishes containing 4 mL of sterile water with double layers of sterile filter paper inside, and germinated at 30°C for 48 h in the dark. After the seed germination was completed, the seeds with uniform germination were selected and transplanted into the seedling plug tray filled with sterile vermiculite, and cultured in a light incubator (light cycle 16 / 8 h). When the pepper seedlings grew to four leaves with a heart, the pepper seedlings with the same growth were selected for pot experiment.

[0040] 5.2 Inoculation The pot experiment was carried out in the glass greenhouse on the 6th floor of the 11th teaching building of Hunan Agricultural University, and the experiment was set up for 5 seasons. The treatments included: 1) Inoculation of pathogenic bacteria (control); 2) Inoculation of pathogenic bacteria and antagonistic strain F2; Each treatment was set up with 30 replicates. Each pot contained 3.0 kg of fresh soil and 1.0 g of compound fertilizer (N+P2O5+K2O) purchased from Huaiqiang Chemical Co., Ltd. in Hunan Shuangfeng Gardening and Agricultural Store, and 1 plant of pepper seedling with the same growth was transplanted into each pot. After 7 days of transplantation, the inoculation treatment was carried out by root irrigation. The inoculation amount of pathogenic bacteria was 1×10 7 cfu / g soil, and the inoculation amount of antagonistic strain F2 was 1×10 7 cfu / g soil. The pot experiment lasted for 2 months, and the growth of pepper plants was observed and the incidence was recorded every week. At the end of each season, the number of diseased pepper plants in each treatment was counted. ​

[0041] 5.3 Calculate the incidence of disease When the edge of the pepper plant leaves showed dirty brown spots or the base of the stem showed dark green waterlogged soft rot or typical symptoms such as sudden collapse, the plant was defined as a diseased pepper plant. The number of diseased plants in each treatment was observed and recorded regularly, and when the disease was stable (no new diseased plants for 2 consecutive weeks), the incidence of disease in each treatment was calculated (incidence of disease = number of diseased pepper plants / total number of pepper plants in treatment x 100%). The results are shown in Figure 6 .

[0042] As can be seen from Figure 5 , the antagonistic strain F2 has good biological control effect on pepper blight and can promote the growth of pepper plants.

[0043] 5.3 Determination of pathogen quantity At the end of the pot experiment, 5 pots of pepper plants were randomly selected from each treatment to collect soil, rhizosphere soil and root samples, and the pathogen quantity was determined. Specifically, 0.5g of soil sample or root was placed in a triangular flask containing 49.5mL of sterilized water and mixed well to prepare a soil suspension. 1mL of soil suspension was taken from the triangular flask and diluted to 10 -2 , 10 -3 , 10 -4 and 10 -5 times successively, and then spread on the medium.

[0044] 200μL of soil suspension dilution was accurately taken with a pipette and transferred to the pepper blight fungus selective medium under sterile conditions. The medium was evenly spread with a sterile spreader. Each soil sample in each treatment was set up with 5 replicates, and the number of colonies was counted after 48h of incubation in a 28℃ incubator in the dark. Finally, the number of pepper blight fungus in each gram of sample was calculated by the dilution spread formula, i.e. the number of pepper blight fungus colonies = (the number of colonies on the plate x 1000 / the volume of the plate x the dilution factor x the dilution factor of the suspension) / the water content of the soil. The final number of colonies was statistically analyzed by Log 10 , and the results are shown in Figure 7 .

[0045] As can be seen from Figure 7 , with the continuous cultivation of the antagonistic strain F2, the incidence of pepper blight and the number of pathogenic bacteria in the soil showed a significant downward trend. In the 5th season, compared with the control, the incidence of pepper blight decreased by 61.9%, and the number of pathogenic bacteria decreased from 1.23x10 7 cfu / g soil in the control group to 6.17x10 3 cfu / g soil in the treatment group inoculated with the antagonistic strain F2, with a nearly 10000-fold decrease in the number of pathogenic bacteria.

[0046] In summary, the antagonistic strain F2 screened by the application has the effect of efficiently preventing and controlling pepper blight, the strain has strong colonization ability in soil, long effective period and stable effect, can provide support for biological prevention and control of pepper blight and development of related biological pesticides, bacterial manure and biological organic fertilizer, and has important practical value for guaranteeing sustainable development of the pepper industry.

[0047] The foregoing description of specific exemplary embodiments of the application is intended to illustrate and exemplify the application. The description is not meant to limit the application to the precise forms disclosed, and obviously many modifications and variations are possible in light of the above teachings. The specific exemplary embodiments were chosen and described in order to explain the particular principles of the application, its practical application, and to enable others skilled in the art to understand the application for various embodiments with various modifications as are suited to the particular use contemplated. The scope of the application is intended to be defined by the claims and their equivalents.

Claims

1. An antagonistic bacterial strain F2, characterized in that, The taxonomic name of the antagonistic strain F2 is Antrodia xantha Bjerkandera adusta , which was preserved in Guangdong Microbial Culture Collection Center on August 23, 2024, with the preservation number of GDMCC No: 65051.

2. The use of the antagonistic strain F2 of claim 1 in the prevention and control of plant diseases.

3. Use according to claim 2, characterized in that, The plant disease includes pepper blight.

4. Use according to claim 3, characterized in that, The pathogen of the pepper blight is Phytophthora capsici.

5. A biopesticide, microbial fertilizer or bio-organic fertilizer, characterized by, The biopesticide, microbial fertilizer or bio-organic fertilizer comprises the antagonistic strain F2 of claim 1.

6. A method for efficiently preventing and controlling pepper bacterial wilt, characterized by, The method comprises the following steps: The antagonistic strain F2 of claim 1 is applied to the rhizosphere of pepper seedlings.

7. The method of claim 6, wherein, The antagonistic strain F2 concentration was 1.0 x 10 8 cfu / mL, and the inoculum was 1.0 x 10 8 cfu / g of soil.

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