Bacterial strain and fungicide for antagonizing peach branch blight pathogen and application of bacterial strain and fungicide

Abstract

The invention relates to a bacterial strain for antagonizing peach branch blight pathogen, a fungicide and application of the bacterial strain and the fungicide, and belongs to the technical field of agricultural microorganisms. The bacterial strain for antagonizing the peach branch blight pathogen is paenibacillus polymyxa JD9, and is preserved in the China General Microbiological Culture Collection Center on September 8, 2025, and the preservation number is CGMCC No. 35855. The JD9 strain is obtained by being separated from rhizosphere soil of a vegetable field, has a remarkable antagonistic effect on Diaporthe amygdali, and has broad-spectrum bacteriostatic activity on 10 plant pathogenic fungi such as peach monilinia, colletotrichum gloeosporioides and magnaporthe oryzae. The strain JD9 can be prepared into a microbial inoculum (the concentration is 1 * 10 < 8 > cfu / mL) through fermentation culture, is used for preventing and treating branch blight of peach fruits and branches, and has remarkable prevention and treatment effects. The biocontrol bacterium provided by the invention is green and safe, solves the problems of generation of drug resistance of germs and pesticide residues, environmental pollution and the like caused by excessive and nonstandard use of chemical agents, and has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of agricultural microbiology, and in particular to a strain, inoculant, and application of a fungus that antagonizes peach twig blight. Background Technology

[0002] Currently, the main method for controlling peach twig blight in production is chemical control, which mainly uses fungicides such as carbendazim, thiophanate-methyl, prochloraz, and difenoconazole. However, long-term and excessive irrational use of chemical pesticides can not only lead to drug resistance in pathogens, but also cause environmental pollution and damage to the ecological balance.

[0003] Therefore, green pest control in orchards is receiving increasing attention. Biological control uses beneficial microorganisms to suppress harmful organisms; this method is green and safe. Research has shown that Bacillus licheniformis (…) Bacillus licheniformis W10 and its antimicrobial protein can significantly disrupt the hyphal structure of peach twig blight fungus, causing problems such as hyphal deformity, cell protoplasm leakage, and cell wall damage, and also has good biocontrol potential against peach twig blight. Summary of the Invention

[0004] This invention addresses the need for existing biological control technologies for peach twig blight by providing a biocontrol bacterium that exhibits strong antagonistic activity against peach twig blight, enabling its control in agricultural production.

[0005] To address the aforementioned problems, this invention first provides a strain antagonistic to *Prunus truncatula*, characterized in that the strain is *Bacillus polymyxa* JD9, deposited on September 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35855, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as *Bacillus polymyxa*. Paenibacillus polymyxa .

[0006] Furthermore, the 16S rDNA sequence of the aforementioned Bacillus polymyxa JD9 is similar to... Paenibacillus polymyxa QH-G has a high degree of homology.

[0007] This invention also provides an antagonistic agent against peach twig blight, which is prepared by cultivating the above-mentioned Bacillus polymyxa JD9 through the following steps: Step 1: Inoculate a single colony of Bacillus polymyxa JD9 into NB medium and culture at 28℃ and 200 r / min for 12-16 h with shaking to obtain a bacterial suspension; Step 2: Adjust the concentration of the bacterial suspension obtained in Step 1 to 1×10⁻⁶ 8 A *Bacillus polymyxa* JD9 bacterial agent was prepared with cfu / mL and OD value = 1.0.

[0008] This invention further provides the application of the above-mentioned antagonistic fungal agent against peach twig blight in the control of peach twig blight pathogens, specifically for controlling the pathogen of peach twig blight. Diaporthe amygdali .

[0009] This invention also provides the application of the aforementioned *Bacillus polymyxa* in the preparation of broad-spectrum antifungal drugs, primarily used to control pathogenic fungi including *Prunus truncatula* (…). Diaporthe amygdali ), Peach brown rot fungus ( Monilinia fructicola ), Colloidal anthrax bacteria ( Colletotrichum gloeosporioides ), rice blast fungus ( Magnaporthe oryzae Fusarium graminearum ( ), Fusarium graminearum ), Staphylococcus aureus ( Botryosphaeria dothidea ), Apple black rot peel genus ( Valsa mali ), Botrytis cinerea ( Botrytis cinerea Alternaria ( Alternaria sp.), Rhizopus ( Rhizopus sp.).

[0010] Furthermore, the present invention also provides the application of the above-mentioned polymyxa JD9, which antagonizes peach twig blight, in the preparation of biopesticides or microbial fertilizers.

[0011] This invention has the following advantages: The *Bacillus polymyxa* JD9 bacterial agent of this invention is composed of *Bacillus polymyxa*, which was isolated from the rhizosphere soil of a vegetable field. This avoids the invasion of alien species and the environmental constraints on biocontrol bacteria in biological control, while also preventing environmental pollution and drug resistance problems caused by chemical agents. The *Bacillus polymyxa* strain JD9 of this invention can be prepared into a bacterial agent (concentration 1×10⁻⁶) through fermentation culture. 8 The (cfu / mL) method is used to control twig blight in peach fruits and branches. It has significant preventive and therapeutic effects, overcomes the problems of pesticide resistance and pesticide residues and environmental pollution caused by excessive or improper use of chemical agents, and has good application prospects. Attached Figure Description

[0012] Figure 1 The present invention is based on the polymyxa bacillus (Bacillus polymyxa). P. polymyxa The effect of JD9 strain bacterial suspension on the vegetative mycelium of *Prunus truncatus*. Figure 2 The present invention is based on the polymyxa bacillus (Bacillus polymyxa). P. polymyxa The JD9 strain inoculum exhibits a broad-spectrum inhibitory effect on the vegetative hyphae of various plant pathogenic fungi. Figure 3 For the present invention, the polymyxa bacillus ( P. polymyxa Colony morphology diagram of strain JD9; Figure 4 The present invention is based on the polymyxa bacillus (Bacillus polymyxa). P. polymyxa Gram staining image of strain JD9; Figure 5 The present invention is based on the polymyxa bacillus (Bacillus polymyxa). P. polymyxa ) JD9 strain phylogenetic tree; Figure 6 The present invention is based on the polymyxa bacillus (Bacillus polymyxa). P. polymyxa Growth curve of strain JD9; Figure 7 Polymyxobacterium ( P. polymyxa Detection of substances related to the antibacterial activity of strain JD9; Figure 8 Polymyxobacterium ( P. polymyxa The control effect of strain JD9 on detached peach fruit; Figure 9 Polymyxobacterium ( P. polymyxa The control effect of strain JD9 on detached peach branches. Detailed Implementation

[0013] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0014] First, prepare for implementation: The main reagents and instruments used in the following examples are: Green Taq Mix (NovaZ), deionized water, PCR instrument (BIO-RAD), constant temperature incubator (Ningbo Jiangnan Instrument Factory), full temperature incubator shaker (Shanghai Fuma Test Equipment Co., Ltd.), and ultra-micro spectrophotometer (Genova plus 198-1000nm).

[0015] All pathogens used below have been verified for accuracy using morphological and ITS sequence analysis.

[0016] The tested strain was *Prunus truncatus* (Prunus truncatus var. truncatus Diaporthe amygdali ), Peach brown rot fungus ( Monilinia fructicola ), Colloidal anthrax bacteria ( Colletotrichum gloeosporioides ), rice blast fungus ( Magnaporthe oryzae Fusarium graminearum ( ), Fusarium graminearum ), Staphylococcus aureus ( Botryosphaeria dothidea ), Apple black rot peel genus ( Valsa mali ), Botrytis cinerea ( Botrytis cinerea Alternaria ( Alternaria sp.), Rhizopus ( Rhizopus All sp.) were independently isolated, identified, and preserved by our laboratory.

[0017] W10 ( Bacillus licheniformis This is a biocontrol bacterium with good biocontrol effect that was previously screened in our laboratory, and it was used as a positive control in the in vitro experiments on peach fruit and peach branches.

[0018] Culture medium preparation: NA medium formula (5.0 g bacterial peptone, 1.0 g yeast extract, 3.0 g beef extract, 10.0 g sucrose, purified water to 1 L, pH adjusted to 6.8, agar 15.0 g); NB medium formula (5.0 g bacterial peptone, 1.0 g yeast extract, 3.0 g beef extract, 10.0 g sucrose, purified water to 1 L, pH adjusted to 6.8); PDA medium formula (200 g peeled potato, 20 g glucose, 15 g agar powder, purified water to 1 L). All were autoclaved at 121℃ for 30 min.

[0019] Example 1: Isolation, purification, and screening of Bacillus polymyxa JD9 strain Soil sampling and bacterial isolation and purification: Rhizosphere soil from 15 cm underground in a vegetable field in Jiangdu District, Yangzhou City, Jiangsu Province was collected. 10 g of the soil was weighed and placed in a 250 mL Erlenmeyer flask (pre-filled with sterile glass beads and 90 mL of sterile water). The flask was shaken at 28℃ and 180 r / min for 30 min to obtain a soil suspension. The soil suspension was then diluted to 10... -2 10 -3 10 -4 10 -5 100 µL of each sample was spread onto NA plates and incubated upside down at 28°C for 24 h. The resulting single colonies were purified three times by streaking on NA plates and placed in sterile 30% glycerol tubes to obtain the Bacillus polymyxa JD9 strain, which was then stored at -80°C for later use.

[0020] JD9 biocontrol bacteria antibacterial activity assay: Biocontrol strains were screened using the plate confrontation method. The screened biocontrol strains were inoculated into NB medium at 200 rpm and cultured with shaking at 28℃ for 12-16 h. The bacterial concentration was then adjusted to 1×10⁻⁶. 8The concentration of cfu / mL (OD=1.0) was used to obtain the JD9 bacterial agent for later use. One day in advance, a 2 mm mycelial cake of *Prunus truncatula* was inoculated in the center of a PDA plate of uniform thickness. Four 5 mm sterile filter paper discs were placed symmetrically at a distance of 25 mm from the pathogen. Five µL of JD9 bacterial agent was inoculated onto three of the discs, and five µL of sterile water was inoculated at the other disc as a control. The plates were incubated at 25°C for three cycles. When the colonies covered the entire plate, the size of the inhibition zone was measured using the cross-hatching method (inhibition zone width: 9, 7, 8, 7, 7, 7, 8, 8, 7 mm; inhibition diameter: 31, 31, 31, 30, 30, 29, 31, 33, 33). The results showed that strain JD9 had a good inhibitory effect on *Prunus truncatula*. Figure 1 With an inhibition rate of 70%, JD9 was selected for further research.

[0021] Example 2: Determination of broad-spectrum antibacterial activity of Bacillus polymyxa JD9 strain To further determine the antifungal activity and spectrum of the active strain JD9 against plant pathogenic fungi, the plate confrontation method was used to determine the antifungal effects of the strain against laboratory-preserved plant pathogenic fungi, including *Prunus truncatula*, *Prunus brown rot*, *Colletotrichum gloeosporioides*, *Bacillus oryzae*, *Fusarium graminearum*, *Botrytis cinerea*, *Oenococcus solani*, *Botrytis cinerea*, *Alternaria alternata*, and *Rhizopus*. The experiments showed that strain JD9 exhibited good broad-spectrum resistance to a variety of pathogenic fungi (see...). Figure 2 (Table 1) has a broad antibacterial spectrum, providing an important source of fungal agents for the preparation of broad-spectrum antifungal drugs.

[0022] The inhibition rate (%) was calculated as follows: (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100%

[0023] Example 3: Colony morphology observation and Gram staining of Bacillus polymyxa JD9 strain Colony morphology: Colonies were picked up with a sterile toothpick and streaked onto NA plates to observe the morphology of single colonies. Results showed that strain JD9 appeared milky white on NA medium, with slightly transparent edges, regular margins, a smooth surface, and raised areas. Figure 3 ).

[0024] The specific procedure for the Gram staining test of the bacterial strain is as follows: (1) Preparation of smear: First, add 1 drop of sterile water to a clean glass slide, spread the fresh bacterial solution on the glass slide, let it dry naturally, and then fix it with a flame.

[0025] (2) Staining steps: a. Primary staining: Add crystal violet staining solution to the fixed smear, stain for 1 min, and wash with water; b. Mordant: Add Gram's iodine solution, allow to act for 1 minute, then wash with water; c. Decolorization: Add decolorizing solution (95% ethanol), shake the slide until no more purple color comes off (about 20 seconds), then wash with water; d. Counterstaining: Add saxaul dye, counterstain for 1 min, rinse with water; blot dry with absorbent paper, and observe under an oil immersion microscope.

[0026] Gram-positive bacteria are stained purple, and Gram-negative bacteria are stained red. The results showed that strain JD9 stained purple, indicating it was a Gram-positive bacterium. Figure 4 ).

[0027] Example 4: Identification of Bacillus polymyxa JD9 strain Physiological and biochemical identification: Physiological and biochemical tests were performed on the antagonistic bacteria according to Bergey's Manual of Bacterial Identification and the Manual of Systematic Identification of Common Bacteria. The physiological and biochemical characteristics of strain JD9 are shown in Table 2.

[0028] (2) Molecular biological identification of antagonistic strain JD9: In order to further identify strain JD9, a single colony of JD9 was picked and inoculated into NB medium and cultured in a constant temperature shaker at 28℃ and 200 r / min for 12-16 h. DNA of antagonistic strain JD9 was extracted using a bacterial genomic DNA extraction kit according to its instructions.

[0029]

[0030] Using JD9 strain DNA as a template, the JD9 strain sequence was amplified using universal primers 16S rDNA (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'). The amplification system and conditions are detailed in Tables 3 and 4 below. Electrophoresis was used to detect the amplified fragment; the target fragment was approximately 1500 bp, and the bands were correct. The fragment was then sent to the company for sequencing. After the sequencing results were returned, the target sequence was compared using the BLAST function on the NCBI website, and a phylogenetic tree was constructed using MEGA 7.0. The phylogenetic tree results are as follows: Figure 5 The JD9 sequence of strain is similar to Paenibacillus polymyxa The sequence homology of QH-G was the highest, and strain JD9 was preliminarily identified as a polymyxin Bacillus. Paenibacillus polymyxa .

[0031]

[0032]

[0033] Example 5: Determination of the growth curve of Bacillus polymyxa JD9 strain JD9 was first purified by streaking on NA plates to obtain single colonies. Each single colony was then picked and placed in 15 mL of NA liquid medium and incubated at 28°C and 200 rpm in a shaker. OD values ​​were measured every 3 hours during the incubation period. 600 The OD value was maintained until the bacterial culture entered the plateau phase (stable OD value) or the decline phase (decreasing OD value). The experiment was conducted in triplicate. The growth curve of strain JD9 is shown below. Figure 6 As shown, it is in the logarithmic growth phase from 3 h to 12 h, in the plateau phase from 12 h to 24 h, and begins to decline after 24 h.

[0034] Example 6: Detection of genes related to antibacterial activity of Bacillus polymyxa JD9 strain Literature review identified the disease-resistance-related substances produced by *Bacillus polymyxa*, and the relevant genes producing these substances were also identified. Pre-designed primers were found in the literature (see Table 5). These primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. P. polymyxa Using JD9 DNA as a template and pre-designed primers, PCR amplification was performed on the coding genes and regulatory genes (PMXB, PMXC, PMXD, FUSA, PJT, CEL44C, CEL5A, and CEL5B) of antibiotics (polymyxin B, polymyxin C, polymyxin D, fusaricidins, β-glucanase, CEL44C-MAN26A, and cellulase), as well as the genes for key enzymes in IAA synthesis. The amplification system and conditions are detailed in Tables 6 and 7 below. 3 µl of the PCR amplification reaction mixture was subjected to electrophoresis for 20 min.

[0035] The genes encoding cel44c, PJT, PMXB, Cel5B, FusA, PMXC, and PMXD amplified specific bands matching the expected size, indicating that *Bacillus polymyxa* JD9 can produce genes related to antimicrobial compounds, enabling the production of antimicrobial substances and inhibiting the growth of plant pathogens. Furthermore, the amplified band of the key enzyme gene for IAA synthesis matched the size of the target product, suggesting the presence of the IAA gene in *Bacillus polymyxa* JD9, and the potential production of IAA during JD9 growth. Figure 7 .

[0036]

[0037]

[0038]

[0039] Example 7: Efficacy of Bacillus polymyxa JD inoculum against detached peach fruit and peach branch twig blight Based on the above experimental results, this embodiment demonstrates that JD9 bacterial suspension has a biological control effect on peach twig blight. Chemical control of diseases mainly involves two effects: the first is prevention, where pesticides are sprayed before the disease manifests; the second is treatment, where pesticides are sprayed after the disease has developed to inhibit its further damage to the host. Therefore, pathogenicity experiments were conducted on peach fruit and branches using these two types of effects, as follows: Harvest eight-tenths ripe peaches (variety: "Lakeview Honey Dew") of uniform shape and size from healthy peach trees, rinse the surface of the peaches with tap water, disinfect with 75% alcohol for 30 seconds, rinse repeatedly with sterile water 3 times, and air dry them in a sterile operating table for later use. (1) Prevention effect: Spray the pre-treated peaches with antagonistic bacteria JD9 bacterial solution (OD 600 =1.0), after drying, spray again (repeated 3 times), place on the operating table to dry; 24 h later, use a sterile needle to make wounds on the surface of the peach fruit, and inoculate with a 5 mm diameter peach branch blight pathogen ZN32 fungal cake. With sterile water as a control, repeat 3 times, culture at 25℃ with alternating light and dark for 7 days, measure the diameter of the lesion and take pictures to record. (2) Treatment effect: Select three relatively far parts in the middle of the dried peach fruit surface, make three tight wounds in each part with a sterile needle, and inoculate with a 5 mm diameter peach branch blight pathogen ZN32 fungal cake; 24 h later, remove the fungal cake, spray the antagonistic bacteria JD9 bacterial solution (OD value = 1.0) on the surface of the peach fruit, and spray JD9 bacterial solution again after drying (repeated 3 times). With sterile water as a control, repeat 3 times, culture at 25℃ with alternating light and dark for 7 days, measure the diameter of the lesion and take pictures to record.

[0040] Fresh peach branches (variety: Lakeview Honey Dew) were taken from healthy peach trees. The branches were cut with uniform thickness and length using sterilized pruning shears, washed with tap water, disinfected with 75% alcohol for 30 seconds, and rinsed repeatedly with sterile water 3 times. The branches were then placed in a sterile laminar flow hood to dry for later use. (1) Prevention effect: The surface of the dried branches was sprayed with antagonistic bacteria JD9 bacterial solution, dried, and then sprayed again (repeated 3 times). After 24 hours, a wound was made in the middle of the branch with a sterile needle and inoculated with ZN32 bacterial cake of uniform size. Sterile water was used as a control. The branches were cultured at 25℃ for 7 days, the length of the lesions was measured and photographed, and the experiment was repeated 3 times. (2) Treatment effect: A wound of uniform depth was made in the middle of the sterilized branches and inoculated with 5 mm ZN32 bacterial cake. After 24 hours, the bacterial cake on the surface of the branches was removed, and antagonistic bacteria JD9 bacterial solution was sprayed on the surface. After drying, the branches were sprayed again (repeated 3 times). Sterile water was used as a control. After culturing at 25°C under alternating light and dark conditions for 7 days, the diameter of the lesions was measured and photographed. The experiment was repeated three times.

[0041] The efficacy of antagonistic bacteria can be calculated using the following formula: Control efficacy (%) = (Diameter of lesions in control group - Diameter of lesions in experimental group) / Diameter of lesions in control group × 100% Experimental results showed that JD9 bacterial solution had a good control effect on detached peach twig blight. Specifically, in terms of both prevention and treatment, the disease severity was significantly lower compared to the control group, with an inhibition rate of 60%. Figure 8 AF); the preventive effect is better than the therapeutic effect, with an inhibition rate of 63% ( Figure 8 C); The biological control effect is similar to that of Bacillus licheniformis strain W10.

[0042] In the control of peach branches, the diameter of lesions on branches treated with JD9 bacterial solution was significantly reduced, with an inhibition rate of 66%, and the preventive and curative effects were both higher than those of Bacillus licheniformis strain W10. Figure 9 (AF), indicating that JD9 bacterial suspension has a good inhibitory effect on the disease development of peach twig blight on branches. In conclusion, JD9 bacterial suspension has good application prospects in the field of biological control of peach twig blight.

[0043] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions of the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain antagonistic to *Prunus truncatus*, characterized in that, The strain is Bacillus polymyxa JD9, which was deposited at the China General Microbiological Culture Collection Center on September 8, 2025, with accession number CGMCC No. 35855.

2. The strain antagonizing peach twig blight according to claim 1, characterized in that, The 16S rDNA sequence of the polymyxa bacillus JD9 is similar to... Paenibacillus polymyxa QH-G has a high degree of homology.

3. A fungal agent antagonizing *Prunus truncatus*, characterized in that, The polymyxa bacillus JD9 described in claim 1 or 2 was prepared by cultivating it through the following steps: Step 1: Inoculate a single colony of Bacillus polymyxa JD9 into NB medium and culture at 28℃ and 200 r / min for 12-16 h with shaking to obtain a bacterial suspension; Step 2: Adjust the concentration of the bacterial suspension obtained in Step 1 to 1×10⁻⁶ 8 A bacterial agent of Bacillus polymyxa JD9 was prepared with cfu / mL and OD value = 1.

0.

4. The application of the fungal agent according to claim 3 in the control of peach twig blight pathogen, characterized in that, The pathogen used to control peach twig blight is Diaporthe amygdali .

5. The application according to claim 4, characterized in that, The inoculum of Bacillus polymyxa JD9 showed a growth inhibition rate of 70% against multiple pathogens using the plate confrontation method.

6. The use of the fungal agent described in claim 3 in the preparation of a broad-spectrum antifungal drug, characterized in that, The pathogenic fungi used for prevention and control include *Prunus truncatus* (…). Diaporthe amygdali ), Peach brown rot fungus ( Monilinia fructicola ), Colloidal anthrax bacteria ( Colletotrichum gloeosporioides ), rice blast fungus ( Magnaporthe oryzae Fusarium graminearum ( ), Fusarium graminearum ), Staphylococcus aureus ( Botryosphaeria dothidea ), Apple black rot peel genus ( Valsa mali ), Botrytis cinerea ( Botrytis cinerea Alternaria ( Alternaria sp.), Rhizopus ( Rhizopus sp.).

7. The application of the strain according to claim 1 or 2 in the preparation of biopesticides or microbial fertilizers, characterized in that, The strain is Polymyxin Bacillus JD9.

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