Aeromonas veronii and its application in preventing and treating pepper anthracnose

Abstract

The application discloses a priestia megaterium and application thereof in prevention and treatment of pepper anthracnose, and belongs to the technical field of plant disease biocontrol strains.The priestia megaterium (Priestia megaterium) GR046 has been preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: M 20241112.A priestia megaterium is obtained through isolation and screening, and it is found through experiments that the priestia megaterium has an inhibiting effect on Sphaeropsis sclerotivorus, which causes pepper anthracnose, and through a prevention and treatment experiment, it is found that, compared with 100% of the incidence rate of a control group, the incidence rate of a GR046 treatment group is significantly reduced, and is only 28.33+ / -2.89%, and the lesion diameter is only 7.37+ / -0.91 mm, which is equivalent to the prevention and treatment effect of a commonly used bactericide, prochloraz.The priestia megaterium provided by the application has great potential to be developed into a commercialized live biocontrol preparation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant disease biocontrol strains, and particularly relates to a Pseudomonas putida and application thereof in prevention and treatment of pepper anthracnose. BACKGROUND

[0002] Pepper (Capscium) is an annual or limited perennial plant of Solanaceae. Pepper plays an important role in health care, medicine, beauty and other industries, and most importantly, plays an important role in food and seasoning. Due to the characteristic of pungency, pepper is widely used for seasoning of various dishes to add flavor. Because it is rich in vitamins, carotene and capsaicin and other components, and a variety of trace elements beneficial to physical and mental health, it has become a kind of pollution-free vegetable that is deeply loved by the public.

[0003] However, with the continuous expansion of pepper planting area in China, the problem of pepper diseases is becoming increasingly prominent. Among the pepper diseases, virus disease, root rot, anthracnose, blight, and bacterial wilt are the main types of diseases. Among them, pepper anthracnose has become a particularly noteworthy one among these diseases due to its serious impact on pepper production. The main anthracnose that harms pepper production in China is pepper anthracnose (Colletotrichum Capsici), Colletotrichum Gloeosporioides, Colletotrichum scovillei and Colletotrichum acutatum. When pepper is infected by Colletotrichum scovillei, the initial infected site will be concave downward, forming an oblong or irregular lesion, and then the diseased part will become black-brown, forming irregular concentric rings on the surface, and the surrounding tissue will appear shriveling. When the disease is serious, small black spots will appear, and orange arthrospore groups will grow in a humid environment.

[0004] At present, the prevention and treatment of pepper anthracnose is mainly chemical control and biological control. In chemical control, the use of chemical agents to prevent and treat pepper anthracnose is currently adopted. However, the traditional agents such as carbendazim, thiophanate-methyl, mancozeb and the like have not very ideal prevention and treatment effect on pepper anthracnose, and the fungicides relying on a single chemical component will cause the pathogenic bacteria to develop drug resistance, thereby increasing the difficulty of disease management. Therefore, the researches at home and abroad start from screening high-efficiency prevention and treatment agents to provide scientific basis for preventing and treating pepper anthracnose. Biological control is to inhibit the growth of pathogenic bacteria through the metabolic products and excretion substances generated by biocontrol bacteria, so as to reduce the harm of diseases to plants. Studies have shown that biological control has the characteristics of green, safety and high efficiency, and is one of the most ideal prevention and treatment methods. Therefore, biological control has occupied a prominent position in the field of pepper disease prevention and treatment research and has become a research hotspot at present. In the application of biological control, some specific Bacillus strains have been proved to have significant effect. It is found by Yuan Chenhong et al. that the fermentation liquid and fermentation supernatant of Bacillus licheniformis BL-24 have the prevention and treatment effect equivalent to that of chemical agent carbendazim, which provides a new idea for the biological control of tobacco anthracnose. It is found by Yin Xiaodong et al. through the study on the growth-promoting indicators of Priestia megaterium ACCC 02979 that the fermentation liquid and dilution liquid of the strain have the promoting effect on the germination of wheat. These studies further prove the wide application prospect of Bacillus in the field of biological control. It is of more important significance to further screen the biocontrol strain resources with better effect. SUMMARY

[0005] The purpose of the present application is to provide a Priestia megaterium and its application in preventing and treating pepper anthracnose, so as to solve the problems existing in the prior art. A Priestia megaterium GR046 is isolated and screened in the present application, which can prevent and treat pepper anthracnose caused by Colletotrichum scovillei and has great potential to be developed into a commercialized live biocontrol preparation.

[0006] In order to achieve the above purpose, the present application provides the following solutions.

[0007] The present application provides a Priestia megaterium GR046, the preservation number of the Priestia megaterium is CCTCC NO: M 20241112, the preservation date is May 30, 2024, the preservation unit is China Center for Type Culture Collection (CCTCC), and the preservation address is Wuhan University, China.

[0008] The present application also provides a microbial agent containing the Priestia megaterium GR046 or a bacterial suspension thereof.

[0009] Further, the live bacterial concentration of the microbial agent is 1x10 7~1x10 9 CFU / mL.

[0010] The application also provides a use of the above-mentioned P. macrocephalum GR046 or the above-mentioned microbial inoculant in resisting S. scovillei.

[0011] The application also provides a use of the above-mentioned P. macrocephalum GR046 or the above-mentioned microbial inoculant in preventing and treating plant diseases, wherein the plant diseases include pepper anthracnose caused by S. scovillei.

[0012] Further, the P. macrocephalum GR046 plays a role in resisting S. scovillei or preventing and treating plant diseases caused by S. scovillei by destroying hyphae of S. scovillei.

[0013] The application also provides a method for preventing and treating pepper anthracnose caused by S. scovillei, comprising the following steps:

[0014] treating pepper fruits with the above-mentioned P. macrocephalum GR046 or a bacterial suspension thereof;

[0015] The bacterial suspension has a viable bacterial concentration of 1x10 7 ~1x10 9 CFU / mL.

[0016] Further, the method for treating pepper fruits comprises inoculating a bacterial suspension of the P. macrocephalum GR046 with a concentration of 1x10 8 CFU / mL on the surface of the pepper fruits.

[0017] The application also provides a biocontrol preparation for preventing and treating pepper anthracnose caused by S. scovillei, wherein the biocontrol preparation comprises the above-mentioned P. macrocephalum GR046 or a bacterial suspension thereof.

[0018] The application discloses the following technical effects:

[0019] The application successfully isolates and screens a P. macrocephalum GR046, which exhibits a good prevention and treatment effect on pepper anthracnose caused by S. scovillei, and provides a new strain resource for biological prevention and treatment of pepper anthracnose. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to make the technical solutions in the embodiments of the present application or the prior art clearer, the accompanying drawings needed in the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description only need to be some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0021] Figure 1 Figure is the morphological characteristics map of GR046; A: colony morphology; B: gram staining; C: scanning electron microscope map;

[0022] Figure 2 Figure is the gel electrophoresis map (A) and phylogenetic tree (B) of GR046;

[0023] Figure 3 Figure is the enzyme production ability result map of GR046; A: protease production medium; B: iron carrier production medium;

[0024] Figure 4 Figure is the control effect of GR046 on pepper fruits; A: control effect of different treatment groups on pepper fruits; B: disease incidence statistical chart of different treatment groups; C: lesion diameter statistical chart of different treatment groups; Control: control group; GR046: GR046 treatment group; 0.01% Prochloraz: prochloraz treatment group;

[0025] Figure 5 Figure is the influence of GR046 on pepper C. scovillei mycelium morphology; Control: control group; GR046: GR046 treatment group; C. scovillei: C. scovillei treatment group; C. scovillei / GR046: GR046+C. scovillei treatment group;

[0026] Figure 6 Figure is the colonization of GR046 at the wound of pepper fruits;

[0027] Figure 7 Figure is the influence of GR046 on the antioxidant enzyme activity of pepper; A: POD; B: CAT; CK: control group; C. scovillei: C. scovillei treatment group; GR046: GR046 treatment group; C. scovillei+GR046: GR046+C. scovillei treatment group;

[0028] Figure 8 Figure is the influence of GR046 on the weight loss of pepper; Control: control group; GR046: GR046 treatment group. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present application will now be described in detail, with reference to the drawings, which are not to be construed as limiting the application, but rather as illustrating certain aspects, features and embodiments of the application.

[0030] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of the term "about" in the present disclosure is intended to mean that quantities, dimensions, and other specified values need not be exact, but can be approximations. Unless otherwise stated, the use of "about" in the present disclosure is intended to mean that quantities, dimensions, and other specified values need not be exact, but can be approximations. In the present disclosure, the use of "at least" followed by a list of one or more items suggests that among other things, each individual item in the list can be present alone, and that in addition, any combination of two or more items of the list can be present. In the present disclosure, the use of "or" in the cases of "A or B" means a de novo choice of A or B; in the cases of "at least one of A or B"; "A or B" means at least one of A and at least one of B. In the present disclosure, the use of "and / or," will be understood to include any and all permutations of one or more of the associated listed items. In the present disclosure, the term "one or more" and "at least one" for one or more of a particular listing of items means that at least one of any one item in the list is present and

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not construed as an admission that it is prior art with respect to the present application.

[0032] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof, and it is to be understood that all such modifications and variations warrant the patentable subject matter under the patent laws. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples given are exemplary only and are not intended to be limiting.

[0033] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0034] The C. scovillei strain used in the following examples was preserved in the Hunan Provincial Key Laboratory of Plant Disease and Pest Biology and Control, College of Plant Protection, Hunan Agricultural University, which has been disclosed in the literature (Qiu Z L, Liu SD, Li XG, et al. Identification and mechanism characterization of Streptomyces griseoaurantiacus XQ-29 with biocontrol ability against pepper southern blight caused by Sclerotium rolfsii [J]. Pesticide Biochemistry and Physiology, 2024, 202. DOI: 10.1016 / j.pestbp.2024.105956.).

[0035] The culture medium used in the following examples:

[0036] (1) PDA culture medium: potato 200 g, glucose 20 g, agar powder 20 g, deionized water to 1 L, 121 ℃, sterilization for 30 min.

[0037] (2) PDB culture medium: potato 200 g, glucose 20 g, deionized water to 1 L, 121 ℃, sterilization for 30 min.

[0038] (3) NA culture medium: peptone 10 g, beef extract 3 g, sodium chloride 5 g, agar powder 15 g, deionized water to 1 L, 121 ℃, sterilization for 30 min.

[0039] (4) NB culture medium: peptone 10 g, beef extract 3 g, sodium chloride 5 g, deionized water to 1 L, 121 ℃, sterilization for 30 min.

[0040] (5) Protease production medium

[0041] A: 4% (w / v) water agar (4 g agar powder dissolved in 100 mL distilled water), high pressure sterilization at 121 ℃ for 20 min;

[0042] B: 4% (w / v) skim milk powder (4 g skim milk powder dissolved in 100 mL distilled water), the skim milk powder was weighed and added to 100 mL 55 ℃ sterile water in the workbench, and it was prepared immediately before use.

[0043] Mix A and B well to make plates ready for use.

[0044] (6) Iron carrier production medium

[0045] Solution A:

[0046] Solvent 1: 1.35 mg of ferric chloride hexahydrate was dissolved in 5 mL of 10 mmol / L hydrochloric acid;

[0047] Solvent 2: 30.25 mg of chrome azurol (CAS) was dissolved in 25 mL of deionized water;

[0048] Solvent 3: 36.4 g of cetyltrimethylammonium bromide (CTAB) was dissolved in 20 mL of deionized water;

[0049] After mixing Solvent 1 and Solvent 2 evenly, slowly add Solvent 3 and mix well.

[0050] Solution B:

[0051] 0.5 g of ammonium chloride, 0.15 g of potassium dihydrogen phosphate and 0.25 g of sodium chloride were dissolved in 375 mL of deionized water, and then 15.12 g of piperazine-14-disulfonic acid (PIPES) was added to the solution, and the pH value was adjusted to 6.8 with 50% potassium hydroxide solution, 9 g of agar powder was added, and deionized water was added to 400 mL.

[0052] Solution C:

[0053] 1 g of mannitol, 1 g of glucose, 0.5 mg of sodium molybdate dihydrate, 5.5 mg of calcium chloride, 6 mg of zinc sulfate heptahydrate, 0.7 g of boric acid, 246 mg of magnesium sulfate heptahydrate, 0.02 mg of copper sulfate pentahydrate and 0.635 mg of manganese sulfate monohydrate were dissolved in deionized water to 25 mL.

[0054] Solution D:

[0055] 0.15 g of casein amino acid was dissolved in 15 mL of deionized water, and slowly added 50% potassium hydroxide to dissolve it.

[0056] Solution A, B, C and D were respectively 1 × 10 5 Pa sterilization for 25 min, when the medium cooled to about 50℃, solution C and D were poured into solution B, and finally solution A was added, and the plate was mixed well and inverted in a sterile environment.

[0057] In the following examples, the data of the experimental results were arranged by Microsoft Excel, the statistics and analysis of the data were carried out by SPSS 20.0, the difference significance test was carried out by Duncan's new complex difference method, the graph was drawn by GraphPad Prism 8.0 software, and MEGA7.0 was used to construct the phylogenetic tree.

[0058] Example 1 Identification of Pristinamycetinresistens GR046

[0059] The application obtains an antagonistic bacterium with a prevention and treatment effect on C. scovillei from rhizosphere soil of healthy pepper in Changsha, Hunan, and the bacterium is identified as Pristinamycetin giganteus and named as GR046 through morphological observation and molecular biology identification.

[0060] 1 Morphological characteristic identification of Pristinamycetin giganteus GR046

[0061] The colony of GR046 is observed and identified according to the Common Bacteria System Identification Manual.

[0062] Scanning electron microscope observation: GR046 is inoculated in NB medium, and cultured at 28℃ and 180r / min for 2d; the bacterial liquid is diluted by 10000 times with sterile water, 50ul of the diluted liquid is dropped on a cover glass. The sample is fixed with 2.5% glutaraldehyde, replaced with isoamyl acetate, and dried in the natural environment, and then gold spraying treatment is performed, and the morphological characteristics of the GR046 strain are observed by scanning electron microscope. The results are shown in Figure 1 .

[0063] From Figure 1 It can be observed from the medium A that the single colony of GR046 is round, the color of the colony is opaque light yellow, the texture is mucilaginous, the surface is slightly raised, the edge is neat, and the colony is easy to pick. Figure 1 The result of gram staining of medium B shows that the cells of GR046 are purple short rods under an oil immersion lens, and the strain is determined as a gram-positive bacterium. It can be directly observed from the scanning electron microscope (SEM) that the GR046 is rod-shaped and oval, the cell surface is not smooth, and there are gullies and depressions, forming a certain texture structure (medium C). Figure 1

[0064] 2 Molecular biology identification of Pristinamycetin giganteus GR046

[0065] The genomic DNA of the streak culture GR046 single colony is extracted by CTAB method, and the DNA is used as a template, and the universal primer pair of bacterial 16S rDNA (forward primer 16S-27F: 5'-AGAGTTTGATCCTGGCTCAG-3', reverse primer 16S-1492R: 5'-GGTTACCTTGTTACGACTT-3') is used for PCR amplification.

[0066] ​PCR amplification reaction system: template DNA 1 μL, 16S-27F 1 μL, 16S-1492R 1 μL, 2x Fine Taq PCR SuperMix 12.5 μL, and ddH2O 9.5 μL. The PCR reaction program is set as follows: 95℃ pre-denaturation for 5 min; 35 cycles of 95℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 1 min 30 s; final 72℃ extension for 5 min, and 4℃ storage.

[0067] After the reaction was completed, the PCR product was subjected to 1% agarose gel electrophoresis for 30 min using DL2000Plus as the DNA marker and 5 μL of sample loading. The results showed a specific band of about 1500 bp (Fig. 2A). Figure 2 The PCR product was sent to Beijing Qikong Biotechnology Co., Ltd. Changsha Branch for sequencing analysis. The sequencing results were subjected to BLAST homology comparison through the GenBank database, and sequences with high similarity were screened and downloaded. MEGA7.0 software was used for analysis by the neighbor-joining method to construct a phylogenetic tree. Figure 2 As shown in Fig. 2B, GR046 had the highest homology with Bacillus megaterium strain mxH12 (GenBank accession number: AF311995.1), and belonged to the same branch as Bacillus megaterium strain OSR43 (GenBank accession number: KJ155812.1) and Priestia megaterium strain KMT-8 (GenBank accession number: MT626661.1). Based on the morphological characteristics and molecular biological identification of the strain, GR046 was identified as Priestia megaterium.

[0068] The above-mentioned Priestia megaterium GR046 has been preserved, and its preservation number is CCTCC NO: M 20241112, the preservation date is May 30, 2024, the preservation unit is China Center for Type Culture Collection (CCTCC), and the preservation address is Wuhan University, China.

[0069] Example 2 Determination of the enzyme production capacity of Priestia megaterium GR046

[0070] A 5 mm diameter circular sterilized filter paper was placed in the center of the siderophore-producing and protease-producing medium plate, 10 μL of GR046 bacterial liquid cultured at 28℃ for 2 days on a 180 r / min shaking table was dropped, and it was placed in a 26℃ incubator for 3-7 days. Whether transparent circles were produced was observed.

[0071] The results are shown in Table 1.Figure 3 As shown, after inoculation of GR046 in protease-producing medium and siderophore-producing medium, both of them appeared significant transparent circle: in protease-producing medium, after 3d culture, the transparent circle diameter produced by GR046 was 2.8cm; in siderophore-producing medium, after 7d culture, the transparent circle diameter produced by GR046 reached 4.6cm. The results showed that GR046 had the ability to produce protease and siderophore.

[0072] Example 3 The preventive effect of Pantoea agglomerans GR046 on pepper fruits

[0073] Dip GR046 single colony in NB medium, culture in 28℃ 180r / min shaker for 2d, centrifuge at 6000g for 5min, collect the precipitate, use UV-visible spectrophotometer, with sterilized distilled water as control, prepare 1×10 8 CFU / mL of GR046 bacterial suspension;

[0074] Put S. sclerotiorum cake in PDB medium, culture in 28℃ 180r / min shaker for 2d, filter spore solution, suspend in sterile water, prepare 1×10 6 CFU / mL of S. sclerotiorum spore suspension;

[0075] Weigh 0.01g of prochloraz, add 1mL of N,N-dimethylisopropanolamine and 1mL of sterile water, then dilute with sterile water to prepare 0.01% prochloraz.

[0076] Pick fresh and healthy peppers of the same size from the market, treat the surface with 75% alcohol, place them in a transparent preservation box, use a sterile needle to evenly prick 3 wounds on the surface of each pepper, and divide them into the following groups for treatment: ① control group: inoculate 1×10 6 CFU / mL of S. sclerotiorum spore suspension; ② GR046 treatment group: inoculate GR046 bacterial suspension (1×10 8 CFU / mL), and then inoculate the same concentration of S. sclerotiorum spore suspension after 24h; ③ prochloraz treatment group: inoculate 0.01% prochloraz, dry it, and then inoculate S. sclerotiorum spore suspension. Each inoculation is 20μL dropped into the wounds of the peppers, and 10 peppers are set for each treatment. Place them in a 28℃ incubator for constant temperature culture, and pay attention to keep the humidity of the preservation box during the period. Observe the disease incidence of peppers on the 2nd, 4th, and 6th day, record the number of diseased peppers and the disease incidence of each pepper, measure the lesion diameter by cross method, and calculate the disease incidence.

[0077] From Figure 4As can be seen, 2d after inoculation, there was no significant difference among the three groups, and none of them got sick. 4d after inoculation, most of the control group showed symptoms of disease, and the fruit inoculation site showed a concave shape with a brown periphery. The disease developed rapidly, and the incidence rate continued to rise to 81.67±2.89%, and the lesion diameter was about 11.03±1.27mm. The pepper fruit in the GR046 treatment group was significantly less sick, with only a few concave shapes and normal tissue color. The incidence rate was 13.33±5.77%, and the lesion size was 2.30±0.20mm. The incidence rate of the propanil treatment group was 11.67±7.63%, and the lesion size was 2.73±0.12mm. 6d after inoculation, the anthracnose lesion of the control group was concave inward, with an elliptical shape and a diameter of up to 16.90±1.21mm. The central part produced orange spore masses, and the incidence rate reached 100%. The GR046 treatment group also had some disease, with an increase in concave shapes, a lesion diameter of 7.37±0.91mm, and an incidence rate of 28.33±2.89%. The incidence rate of the propanil treatment group was 25.00±5.00%, and the lesion concave area was slightly larger than that of the GR046 treatment group, about 8.17±1.26mm. Overall, the GR046 bacterial suspension with a concentration of 1×10 8 CFU / mL and 0.01% propanil had similar effects on peppers, and GR046 had a certain effect on the prevention and control of pepper anthracnose.

[0078] Example 4 Effect of P. macrocephalum GR046 on the mycelial morphology of C. scovilii

[0079] Peppers were treated and grouped as follows: ① control group: inoculated with sterile water; ② GR046 treatment group: inoculated with the GR046 bacterial suspension in Example 3; ③ C. scovilii treatment group: inoculated with the C. scovilii spore suspension in Example 3; ④ GR046+C. scovilii treatment group: inoculated with the GR046 bacterial suspension in Example 3, and then inoculated with the C. scovilii spore suspension in Example 3 after 24h. Each treatment had 2 peppers, and the samples were taken at 96h, with the tissue size controlled at 1-3mm 3 , and placed in a 1.5mL centrifuge tube containing an appropriate amount of 2.5% glutaraldehyde. The mycelial morphology of C. scovilii was observed according to the scanning electron microscope method in Example 1.

[0080] As can be seen from the scanning electron microscope (SEM), the strain GR046 on the surface of the inoculated GR046 bacterial suspension of the pepper is rod-shaped, long oval, rough surface, and the spores have depressions; the pepper hyphae inoculated with S. sclerotiorum are relatively uniform in thickness, and the surface is relatively rough; from the pepper tissue inoculated with GR046 + S. sclerotiorum, it can be seen that GR046 adheres to S. sclerotiorum, S. sclerotiorum shrinks and becomes small, and gaps appear on the surface, indicating that GR046 destroys the hyphae of S. sclerotiorum, causing it to appear deformed and dissolved.

[0081] Example 5 Colonization of P. megaspermatum GR046 on the wound of pepper fruit

[0082] Use a sterile needle to evenly puncture 3 wounds on each pepper skin, and drop 20 μL of GR046 bacterial suspension in Example 3 on the wound. On the 0th, 1st, 2nd, 3rd, 4th, 5th, and 6th days, cut 1 cm diameter pepper tissue at the wound with a sterile knife, and put it into a 2 mL centrifuge tube containing 1 mL of sterile water, and shake for 10 min. Dilution plate method was used, and the sample was diluted by 10 -3 , 10 -4 , 10 -5 gradient, and plated on NA plates. Each gradient was plated on 3 plates, and incubated in a 26°C incubator for 24 h before counting. Each treatment was repeated 3 times, and the results were expressed as the number of colonies per plate.

[0083] From Figure 6 it can be seen that the colonization of GR046 on the wound of pepper fruit showed a trend of first increasing and then tending to be stable. Immediately after inoculation with GR046 bacterial suspension, dilution and plating showed about 10 5 CFU colonies colonized per wound; 1 day after inoculation, it can be seen that a large number of colonies have gathered at the wound, and the number of colonies colonized per wound is close to 10 7 CFU, which indicates that strain GR046 can quickly colonize and multiply in large numbers on the surface of the wound of pepper fruit; by the 4th day after inoculation, the density of GR046 at the wound reached a maximum value of about 10 8 CFU colonies colonized, and the number of colonies colonized began to stabilize thereafter.

[0084] Example 6 Determination of defensive enzyme activity of pepper

[0085] Peppers were grouped as in Example 4, and each treatment had 3 peppers. Sampling was performed at 0, 24, 48, and 72 h, and 9 peppers were cut at each time point for each group, with 0.1 g per tube.

[0086] POD activity assay: The POD activity of chili peppers was determined using a peroxidase (POD) activity assay kit from Beijing Solarbio Science & Technology Co., Ltd. For each treatment, 0.1 g of chili pepper tissue was weighed, 1 mL of extraction buffer was added, and the mixture was homogenized on ice. The homogenate was centrifuged at 8000 g for 10 min at 4℃, and the supernatant was placed on ice. In a 1 mL glass cuvette, 15 μL of sample, 270 μL of distilled water, 520 μL of reagent one, 130 μL of reagent two, and 135 μL of reagent three were added sequentially. The mixture was immediately mixed and the absorbance was recorded at 470 nm for 30 s (A1) and 1 min 30 s (A2). The result was calculated as ΔA = A2 - A1.

[0087] CAT activity assay: The CAT activity of chili peppers was determined using a catalase (CAT) activity assay kit from Beijing Solarbio Science & Technology Co., Ltd. For each treatment, 0.1 g of chili pepper tissue was weighed, 1 mL of extraction buffer was added, and the mixture was homogenized on ice. The homogenate was centrifuged at 8000 g for 10 min at 4℃, and the supernatant was placed on ice. 1 mL of CAT detection working solution was added to a 1 mL quartz cuvette, followed by 35 μL of sample, and mixed for 5 s. The initial absorbance A1 at 240 nm and the absorbance A2 after 1 min were measured immediately at room temperature. ΔA = A1 - A2 was calculated.

[0088] CAT activity is expressed as CAT activity per kilogram of capsicum protein (U / kg), and POD activity is expressed as enzyme activity per kilogram of capsicum tissue (U / kg). All measurements were performed in triplicate, and each sample was collected from three biological replicates.

[0089] from Figure 7 As shown in Figure A, the POD activity in the control group remained at a low level throughout the experiment. Compared with the control group, the GR046 treatment group significantly induced an upregulation of POD activity; the Scoville's anthrax treatment group showed no significant difference from the control group. The GR046 + Scoville's anthrax treatment group showed a further significant increase in POD activity, reaching a peak at 72 h, which was much higher than that of either strain alone.

[0090] from Figure 7 As shown in Figure B, the CAT activity in the control group did not change significantly throughout the experiment. Compared with the control group, the Scoville's anthrax treatment group slightly increased CAT activity, but the overall difference was not significant. In the GR046 + Scoville's anthrax treatment group, the CAT activity was significantly increased and at comparable levels to the GR046 treatment within 0-48 hours, reaching a peak at 48 hours. However, at 72 hours, the CAT activity of both groups decreased significantly.

[0091] Example 7: Effect of *Priestella megaterium* GR046 on weight loss in chili peppers

[0092] Fresh and healthy chilies were selected and divided into two groups. One group was soaked with distilled water and the other group was soaked with GR046 bacterial suspension in Example 3. The soaking time for both groups was 10 minutes and 10 chilies were selected for each treatment. After drying, the chilies were placed in transparent preservation boxes and weighed every three days until the chilies began to rot. The weight data was recorded in detail.

[0093] From Figure 8 As can be seen, the weight loss of both groups showed an upward trend throughout the experiment. In the first nine days of weighing, the weight loss of both groups was relatively slow, and the difference in weight loss rate was not large. The control group lost a little more. From the twelfth day of weighing, the weight loss of the control group was significantly higher than that of the GR046 treatment group, and the gap gradually became obvious. This shows that the chilies treated with GR046 will slow down their metabolism, thereby slowing down the weight loss of the chilies.

[0094] The above-described examples are only to describe the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. Use of P. megaspora GR046 against S. scotivora, characterized in that, The preservation number of the P. megasperma GR046 is CCTCC NO: M 20241112.

2. Use of P. macrocephalum GR046 for controlling plant diseases, characterized in that, The plant disease is pepper anthracnose caused by C. scoville; The preservation number of the P. megasperma GR046 is CCTCC NO: M 20241112.

3. Use according to claim 1 or 2, characterized in that, The P. megasperma GR046 exerts an effect of resisting C. scoville or preventing the plant disease caused by C. scoville by destroying the mycelium of C. scoville.

4. A method of controlling pepper anthracnose caused by Colletotrichum scovillei, characterized by, The method comprises the following steps: The pepper fruits are treated with the P. megasperma GR046 or the bacterial suspension thereof according to claim 1; The concentration of viable bacteria in the bacterial suspension is 1 x 10 7 ~ 1 x 10 9 CFU / mL.

5. The method of claim 4, wherein, The method of treating pepper fruits comprises: inoculating a bacterial suspension of Pristinamycetin-Resistant Bacterium GR046 at a concentration of 1 x 10 8 CFU / mL onto the surface of pepper fruits.

6. A biocontrol agent for controlling pepper anthracnose caused by Colletotrichum scovillei, characterized by, The biocontrol preparation comprises the P. megasperma GR046 or the bacterial suspension thereof according to claim 1.

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