Bacillus velezensis and application thereof in prevention and treatment of fruit tree branch and trunk diseases

Abstract

The invention belongs to the technical field of microorganisms, and discloses a bacillus velezensis XJKS20, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.34361, and the preservation date is April 25, 2025. The bacillus velezensis XJKS20 has the advantages that the preservation number is CGMCC No.34361; the strain has prevention, treatment and induction effects on apple tree canker, and the prevention and treatment effects are better than the induction effect.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology and relates to a strain of Bacillus belye and its application in the prevention and control of diseases on fruit tree branches and trunks. Background Technology

[0002] Apple canker, also known as apple bark rot, is a fruit tree disease caused by one or more fungi. In recent years, with the development of molecular biology and omics sequencing technologies, studies based on molecular data and morphological characteristics have identified an increasing number of pathogens causing apple canker. Among them, fungi of the genus *Cytospora* can infect more than 100 species of woody plants, including some important crops. Currently, apple canker has become a major and persistent problem hindering the healthy and sustainable development of the industry.

[0003] Traditional methods for controlling apple tree canker include scraping the bark, applying pesticides, sawing off dead branches, bridging grafts, and removing diseased trees. However, these methods are not only labor-intensive, costly, and difficult to implement, but also leave significant pesticide residues, are inefficient, and have poor efficacy. Furthermore, these methods can cause further damage to the tree and even reduce the plant's resistance, thus facilitating the further spread and infection of the pathogen. Especially when the pathogen enters the phloem or xylem of the trunk and lateral branches, scraping off lesions and applying pesticides become even less effective in controlling the infection. This has led to the increasing prevalence and spread of apple tree canker in Xinjiang, causing devastating damage to the local apple industry.

[0004] Biological control is a technique that utilizes beneficial microorganisms and their metabolites to control plant diseases. Compared with traditional pesticide control, this method is environmentally friendly, non-toxic, highly efficient, and can provide sustained protection within the host, making it one of the best options for controlling apple tree canker. In recent years, extensive research has been conducted both domestically and internationally on the biological control of apple tree canker. Existing studies have shown that different beneficial microorganisms and their metabolites have demonstrated great potential in inhibiting the pathogen of apple tree canker. For example, Valetti et al. (2022) isolated seven Trichoderma strains from the rhizosphere soil of healthy apple trees in Argentina and found that they all inhibited the growth of mycelia of Valsa ceratosperma, the causative agent of apple tree canker, and had good protective and curative effects against apple tree canker. Therefore, how to develop better microbial agents for more effective control of apple tree diseases has always been an urgent technical problem to be solved. Summary of the Invention

[0005] This invention addresses the technical problem of ineffective control of apple tree diseases by providing a strain of Bacillus belye XJKS20, which has preventive, curative, and inducing effects on apple tree canker, with the preventive and curative effects being better than the inducing effect.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a strain of Bacillus velezensis XJKS20, which is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No.34361, deposit date: April 25, 2025.

[0008] The 16S rDNA sequence of the Bacillus belyssus XJKS20 is shown in SEQ ID NO.1.

[0009] This invention provides a biological agent containing the fermentation broth of Bacillus vesiculosus XJKS20.

[0010] This invention provides the application of the above-mentioned Bacillus berreatus XJKS20 in inhibiting plant pathogens, including: Cytospora mali (apple rot fungus), Botryosphaeria dothidea (walnut dry rot fungus), Diaporthe eres (magnolia branch blight fungus), Fusarium pseudograminearum (wheat stem base rot fungus), Alternaria sp. (garlic leaf blight fungus), Fusarium sp. (garlic root and stem rot fungus), Bipolarismaydis (corn small spot fungus), Fusarium oxysporum (wheat root rot fungus), Corynesporacassiicola (strawberry leaf spot fungus), Botrytis cinerea (tomato gray mold), Corynesporacassiicola (cucumber leaf spot fungus), and Rhizoctonia solani (wheat sheath blight fungus).

[0011] The present invention also provides the application of the above-mentioned Bacillus belye XJKS20 in the prevention and control of apple tree diseases caused by plant pathogens, wherein the plant pathogens include: *Bacillus maculatus* and *Botrytis cinerea*.

[0012] The present invention also provides the application of the above-mentioned Bacillus belye XJKS20 in the prevention and control of diseases of apple leaves and branches caused by plant pathogens, wherein the plant pathogens include: *Bacillus maculatus* and *Botrytis cinerea*.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] The present invention relates to Bacillus velezensis XJKS20, which exhibits preventive, curative, and inducing effects against apple tree canker, with the preventive and curative effects being better than the inducing effect. Furthermore, the antibacterial spectrum of this endophytic bacterium was determined, revealing that it also demonstrates good antagonistic effects against 12 pathogenic fungi isolated from fruit trees, wheat, and garlic. Attached Figure Description

[0015] Figure 1 The colony morphology of strain XJKS20 grown on the culture medium.

[0016] Figure 2 This is a Gram-positive trait of strain XJKS20.

[0017] Figure 3 Phylogenetic tree of the 16S rDNA gene of strain XJKS20.

[0018] Figure 4 Screening for strain XJKS20.

[0019] Figure 5 The study investigated the inhibitory effect of fermentation filtrate of strain XJKS20 on *Cytospora maculata*.

[0020] Figure 6 The study investigated the preventive, inducing, and therapeutic effects of strain XJKS20 on detached leaves of apple tree rot pathogen.

[0021] Figure 7 The study investigated the preventive, inducing, and therapeutic effects of strain XJKS20 on detached branches of apple tree rot fungus.

[0022] Figure 8 The study investigated the preventive, inducing, and therapeutic effects of strain XJKS20 on detached branches of apple tree ring rot fungus.

[0023] Figure 9 The study investigated the preventive, inducing, and therapeutic effects of strain XJKS20 on live apple tree rot pathogens.

[0024] Figure 10 The antibacterial effect of strain XJKS20 against a variety of pathogens. Detailed Implementation

[0025] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the test methods in the following embodiments are conventional methods.

[0026] Example 1: Isolation and Identification of Strains

[0027] (1) Isolation and purification of strains

[0028] Healthy apple branches were collected from an apple orchard in Zepu County, Kashgar Prefecture, Xinjiang Uygur Autonomous Region. After the bark tissue was washed clean with tap water, it was cut into pieces approximately 5mm thick. 2 The treated bark tissue was surface-sterilized in a sterile operating table: it was immersed in 75% ethanol for 3 minutes, followed by immersion in 1% sodium hypochlorite solution for 1 minute. It was then rinsed three times with sterile distilled water and thoroughly dried with sterile filter paper. The treated tissue was inoculated onto LBA plates and incubated at 28°C for 2–3 days. Endophytic bacteria growing on the plates were purified by streak picking, with two consecutive streak purifications. The purified tissue was then stored at -80°C for further use.

[0029] (2) Morphological identification

[0030] Based on the colony morphology, cell morphology, Gram staining characteristics, and spore staining characteristics of the strain, relevant physiological and biochemical identification tests are conducted.

[0031] Strawberry strain XJKS20 was streaked onto LBA plates and incubated in the dark at 28°C for 48 hours. Morphological characteristics such as colony edge, size, color, and transparency were then observed.

[0032] When strain XJKS20 was cultured on LBA medium, the following characteristics were observed: In the early stages of culture, the colony surface was smooth and the edges were regular. With prolonged culture time, the colonies gradually became nearly round, approximately 3-5 mm in diameter, with irregular edges, wrinkled surfaces, and an overall milky white, opaque appearance (see...). Figure 1 Under an optical microscope, XJKS20 bacteria appear as short rods, staining purple with Gram stain, indicating they are Gram-positive bacteria (see...). Figure 2 ).

[0033] Physiological and biochemical identification was mainly based on the "Handbook of Systematic Identification of Common Bacteria". The identification results are shown in Table 1. The physiological and biochemical characteristics of strain XJKS20 are consistent with the biochemical metabolic characteristics of Bacillus and are consistent with the Bacillus velezensis type strain.

[0034] Table 1 Results of physiological and biochemical characteristics determination of strain XJKS20

[0035]

[0036]

[0037] (3) Molecular biological identification

[0038] Total DNA extracted from strain XJKS20 was used as a template, and then PCR amplification was performed using universal 16S rDNA primers (27F: 5'AGAGTTTGATCMTGGCTCAG-3', 1492R: 5'-GGTTACCTTGTTACGACTT-3'). The PCR reaction volume was 20 μL, including 10 μL of 2×NG PCR Master Mix, 1 μL each of forward and reverse primers, 1 μL of DNA template, and 7 μL of ddH2O. The PCR program was 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 54℃ annealing for 30 s, 72℃ extension for 1 min, for 30 cycles, and a final extension at 72℃ for 5 min. The PCR products were purified and sequenced at Shanghai Sangon Biotech Co., Ltd. DNA sequences were compared with the NCBI database. 16S rDNA sequences of highly homologous type or representative strains were selected as references. Closely related outgroups were chosen, and a phylogenetic tree was constructed using MEGAv.11 with the maximum likelihood method to preliminarily determine the taxonomic position of strain XJKS20. Results are attached. Figure 3

[0039] (4) Screening of antagonistic strains of apple rot pathogen

[0040] Using *Cytospora mali* as the target strain, the antagonistic ability of isolated endophytic bacteria was determined using the plate confrontation method. *Cytospora mali* was inoculated in the center of a PDA plate, and endophytic bacteria were inoculated 3 cm from the edge of the mycelial cake. A five-point confrontation method was used for initial screening, and the antagonistic activity of the endophytic bacteria against *Cytospora mali* was measured. After 3 days, the inhibition rate of the endophytic bacteria was calculated, and strains with strong antagonistic ability were selected for later use. PDA plates inoculated only with the pathogen served as a control. Based on the initial screening results, strains with inhibitory effects were selected and further screened using the plate confrontation method. The growth of the inhibited target strains was restricted, the hyphae became sparse, and the hyphal color changed. Among them, strain XJKS12 showed the best inhibitory effect against *Cytospora mali*, with an inhibition rate of 82.1%. Figure 4 ).

[0041] Inhibition rate (%) = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group × 100%.

[0042] (5) Activity detection of fermentation filtrate of strain XJKS20

[0043] Single colonies of strain XJKS20 were picked and inoculated into a 250 mL Erlenmeyer flask containing 50 mL of LB liquid medium. The flask was incubated on a shaker at 37 °C and 200 rpm for 20 h to obtain the fermentation broth of strain XJKS20. The fermentation broth was transferred to a 2 mL centrifuge tube and centrifuged at 4 °C and 12000 rpm for 20 min. The supernatant was collected and filtered through a 0.22 μm bacterial filter to obtain the sterile fermentation filtrate of strain XJKS20. Sterile water was added to the bacterial cells after removing the supernatant, and the mixture was thoroughly mixed by pipetting. The concentration was then measured and adjusted to a final concentration of 10. 8 CFU / mL was used to obtain the fermentation broth of strain XJKS20. The sterile fermentation filtrate was transferred to PDA medium at approximately 50℃ at concentrations of 6% and 10%, mixed thoroughly, and poured into petri dishes. Using a PDA plate without sterile fermentation filtrate as a control, 5 mm diameter *Cyclocarya maculata* mycelial cakes were inoculated and incubated at 25℃ for 2 days. The colony diameter was observed and measured. The results showed that ( Figure 5 The fermentation filtrate of XJKS20 showed an inhibitory effect on *Cyclocarya maculata*, with significant differences in inhibitory effects among different concentrations of fermentation filtrate. At a concentration of 10%, the target strain's growth diameter was 16±1 mm, with an inhibition rate of 61.9%.

[0044] Example 3: In vitro control efficacy of fermentation broth of strain XJKS20 against apple tree canker.

[0045] The biocontrol effect of fermentation broth of strain XJKS20 on *Cyclocarya maculata* was tested on detached leaves and branches of crabapple.

[0046] Leaf inoculation: Healthy and uniformly sized fresh crabapple leaves were selected. The leaves were surface-sterilized with 75% ethanol (v / v), rinsed thoroughly with sterile water, and then pricked six times with a sterile insect needle (0.5 mm diameter) at the center of the leaf, from the midrib to the leaf margin. Three treatments were set up: 1) Preventive treatment: 30 μL of XJKS20 fermentation broth was added to the wound of the detached leaf, and the mixture was incubated at 25℃ for 24 hours. Then, a 5 mm diameter *Cyclocarya maculata* mycelium block was inoculated into the wound. 2) Induction treatment: 30 μL of XJKS20 fermentation broth was added to the wound of the detached leaf, and the mixture was incubated at 25℃ for 24 hours. Then, a sterile insect needle (0.5 mm diameter) was used to prick the wound six times at a distance of 3 cm from the inoculation point, from the midrib to the leaf margin, and a 5 mm diameter *Cyclocarya maculata* mycelium block was inoculated into the new wound. 3) Treatment: Inoculate the wound with a 5mm diameter *Carya carnata* mycelium block and incubate at 25℃ for 24 hours. Remove the mycelium block and add 30μL of strain XJKS20 fermentation broth to the wound. Place the inoculated leaves on a plastic tray with absorbent cotton soaked in sterile water at the bottom. Seal the tray with plastic wrap to maintain high humidity. After 3 days of incubation, measure the lesion length and calculate the relative efficacy.

[0047] Branch inoculation: Fresh one-year-old apple tree branches were selected. The surface was first disinfected with 75% ethanol (v / v), then washed with sterile water. Several 15cm long branch segments were cut, and both ends were sealed with paraffin wax to prevent excessive moisture loss. A 5mm diameter hole punch, sterilized at high temperature, was used to create a wound on each detached branch. Three treatments were set up in the experiment: 1) Preventative treatment: A wound was created in the center of the detached branch using a sterile hole punch. 30μL of XJKS20 fermentation broth was added to the wound. After incubation at 25℃ for 24 hours, a 5mm diameter *Cyclocarya maculata* mycelium block was inoculated onto the wound. 2) Induction treatment: A wound was created 3 cm from the center of the detached branch using a sterile punch. 30 μL of XJKS20 fermentation broth was added to the wound. After incubation at 25°C for 24 hours, another wound was created in the center of the detached branch using a sterile punch. A 5 mm diameter *Cyclocarya maculata* mycelial block was inoculated into the new wound. 3) Treatment treatment: A wound was created in the center of the detached branch using a sterile punch. A 5 mm diameter *Cyclocarya maculata* mycelial block was inoculated into the wound. After incubation at 25°C for 24 hours, the mycelial cake was removed. 30 μL of XJKS20 fermentation broth was added to the wound. The mycelial cake was inoculated into the wound of the detached branch. A control was prepared by inoculating a 5 mm diameter *Cyclocarya maculata* mycelial block with LB liquid medium. Cover the mycelium block with absorbent cotton soaked in sterile water to keep it moist. Place the inoculated detached branches in a plastic tray (the bottom of the tray is covered with absorbent cotton soaked in sterile water). Cover the plastic tray tightly with plastic wrap to ensure that the tray always maintains a high humidity environment. Then place the tray in an artificial climate chamber at 25°C with alternating light and dark for 12h / 12h. After 7 days of cultivation, remove the plastic wrap, observe and record the disease condition, and measure the length of the lesions.

[0048] Control efficacy (%) = (Longevity of lesions in control group - Longevity of lesions in treatment group) / Longevity of lesions in control group × 100%.

[0049] The results show ( Figure 6-7 The strain XJKS20 significantly inhibited the infection and lesion expansion of the target strain. Compared with the control group, the control efficacy of the treatment group inoculated with XJKS20 first and then inoculated with *Cytosporum malaccensis* was 52.54% and 71.06% for detached leaves and branches, respectively; the control efficacy of the treatment group inoculated with XJKS20 first and then inoculated with *Cytosporum malaccensis* at a certain distance was 50.72% and 67.65% for detached leaves and branches, respectively; and the control efficacy of the treatment group inoculated with *Cytosporum malaccensis* first and then inoculated with XJKS20 was 13.16% and 43.41% for detached leaves and branches, respectively. This indicates that strain XJKS20 has certain preventive, induction, and therapeutic effects, and the preventive and induction effects are better than the therapeutic effects.

[0050] Example 4: In vitro control efficacy of fermentation broth of strain XJKS20 against apple tree ring rot

[0051] Apple ring rot is mainly caused by two pathogens: *Botryosphaeria dothidea* and *B. kuwatsukai*. This example uses *Botryosphaeria dothidea* as an example. The biocontrol effect of the fermentation broth of strain XJKS20 on apple ring rot pathogens was tested on detached apple tree branches. The experimental procedure followed the branch inoculation method in Example 3.

[0052] The results show that ( Figure 8 The strain XJKS20 significantly inhibited the infection and lesion expansion of *Staphylococcus aureus*, the causal agent of apple ring rot. Compared with the control group, the relative efficacy of the treatment group inoculated with XJKS20 first and then *Staphylococcus aureus* was 74.05%; the relative efficacy of the treatment group inoculated with XJKS20 first and then *Staphylococcus aureus* at a certain distance was 32.14%; and the relative efficacy of the treatment group inoculated with *Staphylococcus aureus* first and then XJKS20 was 13.16%. This indicates that strain XJKS20 has certain preventive, induction, and therapeutic effects, with the preventive effect being superior to the induction and therapeutic effects.

[0053] Example 5: Pathogenicity of living apple trees

[0054] The concentration of strain XJKS20 was 10 on apple trees on the Longzihu campus of Henan Agricultural University. 8 Experiment on the in vivo control efficacy of CFU / mL fermentation broth against apple tree rot disease.

[0055] Select healthy apple tree branches of relatively uniform thickness. First, disinfect their surface with 75% ethanol (v / v), then rinse with sterile water. Create wounds on the branches using a sterile, high-temperature sterilized 5mm diameter punch. Three treatments were set up in the experiment: 1) Preventative treatment: Create wounds on the branches using a sterile punch, add 30μL of strain XJKS20 fermentation broth to the wounds, allow the branches to dry, then wrap the wounds with plastic wrap. After 24 hours, inoculate the wounds with 5mm diameter *Cyclocarya maculata* mycelium blocks, cover the mycelium blocks with sterile water-soaked absorbent cotton, and wrap with plastic wrap to keep them moist. A control was prepared by inoculating 5mm diameter *Cyclocarya maculata* mycelium blocks but adding LB liquid medium to the wounds. 2) The induction treatment was as follows: a wound was made on the branch using a sterile punch, and 30 μL of the fermentation broth of strain XJKS20 was dripped onto the wound. After the branch dried, the wound was wrapped with plastic wrap. After 24 hours, a new wound was made 3 cm away from the original wound using a sterile punch. A 5 mm diameter block of *Cyclocarya maculata* was inoculated into the new wound. Sterile water-soaked defatted cotton was placed on the block, and the block was wrapped with plastic wrap to keep it moist. A control was prepared by inoculating a 5 mm diameter block of *Cyclocarya maculata* with LB liquid medium dripped onto the wound. 3) The therapeutic treatment involved creating wounds on branches using a sterile punch, inoculating the wounds with 5mm diameter *Cyclocarya maculata* mycelial blocks, covering the mycelial blocks with sterile water-soaked absorbent cotton, and wrapping them with plastic wrap to keep them moist. After 24 hours, the mycelial blocks were removed, and 30μL of *Cyclocarya maculata* strain XJKS20 fermentation broth was dripped into the wounds. After the branches dried, the wounds were wrapped with plastic wrap. A control was prepared by dripping LB liquid medium into the wounds but inoculating them with 5mm diameter *Cyclocarya maculata* mycelial blocks. Each treatment was replicated four times. After 7 days of moist cultivation, the length of lesions was measured, and the relative efficacy was calculated.

[0056] Figure 9 The study investigated the preventive, induction, and therapeutic effects of strain XJKS20 against live *Pseudomonas aeruginosa*, the causal agent of apple tree rot. It was found that strain XJKS20 significantly inhibited the infection and lesion expansion of the target strain. Compared to the control group, the control efficacy of the treatment group inoculated with XJKS20 before inoculation with *Pseudomonas aeruginosa* was 62.39% for detached branches; the induction efficacy of the treatment group inoculated with XJKS20 at a certain distance before inoculation with *Pseudomonas aeruginosa* was 46.21% for detached branches; and the therapeutic efficacy of the treatment group inoculated with *Pseudomonas aeruginosa* before inoculation with XJKS20 was 28.42%. This indicates that strain XJKS20 has certain preventive, induction, and therapeutic effects, with the preventive and induction effects being superior to the therapeutic effects.

[0057] Example 6: Determination of the antibacterial spectrum of strain XJKS20

[0058] The plate confrontation method was used to evaluate the antagonistic effect of strain XJKS20 against 11 plant pathogenic fungi (all tested strains were provided by the Institute of Mycology, College of Plant Protection, Henan Agricultural University): *Cytospora mali* (apple rot), *Botryosphaeria dothidea* (walnut dry rot), *Diaporthe eres* (magnolia branch blight), *Fusarium pseudograminearum* (wheat stem base rot), *Alternaria sp.* (garlic leaf blight), *Fusarium sp.* (garlic root and stem rot), *Bipolaris maydis* (corn small spot), *Fusarium oxysporum* (wheat root rot), *Corynespora cassiicola* (strawberry leaf spot), *Botrytiscinerea* (tomato gray mold), *Corynespora cassiicola* (cucumber leaf spot), and *Rhizoctonia solani* (wheat sheath blight). A 5mm diameter fungal block of the tested pathogen was inoculated in the center of a PDA plate. A single colony of strain XJKS20 was then picked up with a sterilized pipette tip and inoculated along 3cm lines at 3cm intervals on either side of the pathogen. Fungal discs were also inoculated separately on the PDA plate as a blank control. After inoculation, the plates were incubated at 25℃. Once the control group had completely covered the PDA plate, the diameter of the fungal colonies in both the control and treatment groups was measured, and the inhibition rate was calculated. Figure 10 It can be seen that strain XJKS20 exhibited certain antibacterial effects against all tested strains. Among them, it showed better antibacterial effects against apple tree rot, magnolia branch blight, garlic root and stem rot, corn leaf spot, and wheat sheath blight, all exceeding 60%.

[0059] The embodiments described above are merely preferred embodiments of the present invention and are only used to explain the present invention. They are not intended to limit the scope of the present invention. For those skilled in the art, other implementation methods can be easily made by substitution or modification based on the technical content disclosed in this specification. Therefore, all changes and improvements made on the principle of the present invention should be included within the scope of the patent application of the present invention.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis XJKS20, characterized in that, Deposited at: China General Microbiological Culture Collection Center, accession number: CGMCC No.34361, deposit date: April 25, 2025.

2. The Bacillus belye according to claim 1, characterized in that, The 16S rDNA sequence of the Bacillus belyssus XJKS20 is shown in SEQ ID NO.

1.

3. A biological agent, characterized in that, Fermentation broth containing Bacillus belye XJKS20 as described in claim 1 or 2.

4. The application of Bacillus belye XJKS20 according to claim 1 in inhibiting plant pathogens, characterized in that, The plant pathogens include: *Phyllostachys emblica* (Apple rot pathogen). Cytospora mali Walnut dry rot pathogen ( Botryosphaeria dothidea Magnolia twig blight fungus ( Diaporthe eres ), wheat stem rot fungus ( Fusarium pseudograminearum ), garlic leaf blight pathogen ( Alternaria sp.), garlic root and stem decay fungus ( Fusarium sp.), corn leaf blight fungus ( Bipolaris maydis ), wheat root rot pathogen ( Fusarium oxysporum ), strawberry leaf spot fungus ( Corynespora cassiicola ), tomato gray mold ( Botrytis cinerea ), Cucumber leaf spot pathogen ( Corynespora cassiicola Wheat sheath blight pathogen ( Rhizoctonia solani ).

5. The application of Bacillus belye XJKS20 as described in claim 1 in the prevention and control of apple tree diseases caused by plant pathogens, characterized in that, The plant pathogens include: *Malus maculatus* and *Staphylococcus aureus*.

6. The application of Bacillus belye XJKS20 as described in claim 1 in the prevention and control of leaf and branch diseases in apple trees caused by plant pathogens, characterized in that, The plant pathogens include: *Malus maculatus* and *Staphylococcus aureus*.

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