Application of microbacterium oxydans XZT347 in prevention and treatment of plant diseases

Abstract

The invention belongs to the technical field of fermentation engineering and microbial disease prevention and control, and particularly relates to application of microbacterium oxydans XZT347 in prevention and control of plant diseases. Experimental results show that the infection rates of the Microbacterium oxydans XZT347 to pepper root rot bacteria, cucumber target leaf spot bacteria, eggplant fusarium wilt bacteria, tomato gray mold bacteria and tea anthracnose bacteria are 10.0%, 13.3%, 20.0%, 6.7% and 10.0% respectively, and the morbidity of pepper root rot, cucumber target leaf spot, eggplant fusarium wilt, tomato gray mold and tea anthracnose is remarkably reduced.

Description

Technical Field

[0001] This invention belongs to the field of fermentation engineering and microbial disease control technology, specifically involving the application of Microbacterium oxysporum XZT347 in the control of plant diseases. Background Technology

[0002] Fusarium solani is the pathogen of pepper root rot, belonging to the family Russulaceae and the genus Fusarium. This fungus can survive in the soil for over 10 years, overwintering as chlamydospores, mycelium, or sclerotia in diseased plant debris. The overwintering fungus spreads the following year via rainwater, invading through wounds on the rhizome and roots. Conidia produced from infected parts spread further with rainwater. Cucumber target spot disease is caused by Corynespora cassiicola, and is particularly severe in protected cultivation areas such as overwintering greenhouses, winter-spring greenhouses, and spring greenhouses. Cucumber target spot disease severely impacts cucumber yield and quality, typically causing a 20% reduction, and in severe cases, a 60-70% reduction. Eggplant wilt (Fusarium wilt) is a major disease in eggplant production caused by the soil-borne pathogen *Fusarium oxysporum* f. sp. *melongenae*, severely affecting eggplant yield and quality. Tomato gray mold has become a major disease in protected tomato production in my country. Gray mold is caused by the fungus *Botrytis cinerea* Pers. ex Fr. (a type of Deuteromycete). Tea anthracnose is a major leaf disease of tea plants, occurring in major tea-producing provinces such as Zhejiang, Fujian, and Guizhou. This disease primarily infects mature leaves, but can also occur on young and old leaves. After infection, lesions appear on the leaves, affecting photosynthesis, causing premature leaf drop, weakening plant growth, and impacting tea yield and quality. Tea anthracnose can be caused by *Colletotrichum camelliae*.

[0003] There are no reports in the current technology on whether *Oxidative Microbacterium* can antagonize pathogens such as pepper root rot (*Fusarium solani*), cucumber target spot (*Corynespora cassiicola*), eggplant wilt (*Fusarium oxysporum* f. sp. melongenae), tomato gray mold (*Botrytis cinerea* Pers. ex Fr.), and tea anthracnose (*Colletotrichum camelliae*). Summary of the Invention

[0004] To address the issue of obtaining a safe biological pesticide for controlling root rot in chili peppers, target spot disease in cucumbers, wilt disease in eggplants, gray mold in tomatoes, and anthracnose in tea, this invention provides a strain of Microbacterium oxydans XZT347 and its applications.

[0005] This invention discloses a strain of Microbacterium oxydans XZT347, which was deposited at the China Center for Type Culture Collection on July 16, 2025, with accession number CCTCC M 20252623, at Wuhan University, Wuhan, China.

[0006] This invention discloses the application of Microbacterium oxydans XZT347 or a biopesticide containing Microbacterium oxydans XZT347 in inhibiting the growth of plant pathogens. The plant pathogens include at least one of Fusarium solani (pepper root rot fungus), Corynesporacassiicola (cucumber target spot fungus), Fusarium oxysporum f. sp. melongenae (eggplant wilt fungus), Botrytis cinerea Pers. ex Fr. (tomato gray mold fungus), and Colletotrichum camelliae (tea anthracnose fungus). The Microbacterium oxydans XZT347 was deposited at the China Center for Type Culture Collection on July 16, 2025, with accession number CCTCC M 20251623.

[0007] This invention also discloses the application of Microbacterium oxydans XZT347 or a biopesticide containing Microbacterium oxydans XZT347 in the preparation of a pesticide to inhibit the growth of plant pathogens, wherein the plant pathogens include at least one of Fusarium solani (root rot of pepper), Corynesporacassiicola (target spot of cucumber), Fusarium oxysporum f. sp. melongenae (wilt of eggplant), Botrytis cinerea Pers. ex Fr. (gray mold of tomato), and Colletotrichum camelliae (anthracnose of tea); wherein Microbacterium oxydans XZT347 was deposited at the China Center for Type Culture Collection on July 16, 2025, with accession number CCTCC M 20251623.

[0008] This invention discloses the application of Microbacterium oxydans XZT347 or biological pesticides containing Microbacterium oxydans XZT347 in the preparation of products for controlling root rot of pepper, target spot of cucumber, wilt of eggplant, gray mold of tomato and anthracnose of tea. The Microbacterium oxydans XZT347 was deposited at the China Center for Type Culture Collection on July 16, 2025, with accession number CCTCC M20251623.

[0009] A method for preparing a biopesticide containing Microbacterium oxydans XZT347 includes the following steps:

[0010] (1) Activation of strain XZT347: Microbacterium oxydans XZT347 was inoculated on LB solid medium and cultured at 30-32℃ for 2-3 days to obtain activated Microbacterium oxydans XZT347;

[0011] The formulation of the LB solid medium is as follows: tryptone 9-13 g / L, yeast extract 5-6 g / L, sodium chloride 9-11 g / L, agar powder 14-16 g / L, dissolved in water, and finally adjusted to 1000 mL, pH 7.0±0.3, and autoclaved at 121℃ for 30 min.

[0012] (2) Fermentation: The seed culture of Microbacterium oxydans XZT347 was inoculated into the fermentation medium in the fermentation tank at an inoculation amount of 1-5% of the fermentation medium mass. The culture was carried out in a shaker at 180-250 rpm for 24-48 hours at 30-35℃ to obtain a biological pesticide containing Microbacterium oxydans XZT347.

[0013] The fermentation medium is formulated as follows: tryptone 9-13 g / L, yeast extract 5-6 g / L, sodium chloride 9-11 g / L, 500-700 nmol / L PQQ, dissolved in water, and finally adjusted to 1000 mL, pH 7.0 ± 0.3, and autoclaved at 121℃ for 30 min.

[0014] Preferably, the concentration of Microbacterium oxydans XZT347 in the biopesticide is 6.2-7.6 × 10⁻⁶. 9 ×10 9 cfu / ml.

[0015] The beneficial effects of this invention are:

[0016] This invention provides a strain of Microbacterium oxydans XZT347. Experiments have shown that Microbacterium oxydans XZT347 has good antagonistic effects against Fusarium solani (root rot fungus of pepper), Corynespora cassiicola (target spot fungus of cucumber), Fusarium oxysporum f. sp. melongenae (wilt fungus of eggplant), Botrytis cinerea Pers. ex Fr. (gray mold fungus of tomato), and Colletotrichum camelliae (anthracnose fungus of tea). Attached Figure Description

[0017] Figure 1 The inhibitory effect of Microbacterium oxydans XZT347 on the root rot pathogen of pepper.

[0018] Figure 2 The inhibitory effect of Microbacterium oxydans XZT347 on target spot pathogens of cucumber.

[0019] Figure 3The inhibitory effect of Microbacterium oxydans XZT347 on Fusarium wilt pathogens of eggplant was investigated.

[0020] Figure 4 The inhibitory effect of Microbacterium oxydans XZT347 on tomato gray mold was investigated.

[0021] Figure 5 The inhibitory effect of Microbacterium oxydans XZT347 on anthrax bacteria in tea leaves. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0023] This invention provides a strain of Microbacterium oxydans XZT347, which was deposited at the China Center for Type Culture Collection on July 16, 2025, at Wuhan University, Wuhan, China, with accession number CCTCC M 20251623.

[0024] Example 1: Identification, purification, and preservation of Microbacterium oxydans strain XZT347

[0025] (1) Identification

[0026] The strain of this invention underwent 16S rRNA gene amplification. The PCR product was sequenced, and the resulting gene sequence (the nucleotide sequence of the 16S rRNA gene is shown in SEQ ID NO.1) was compared and analyzed using NCBI to clarify the taxonomic position of the strain. The sequencing results were then compared using BLAST on the NCBI website to determine the taxonomic position of closely related bacterial strains. The comparison results showed that the strain was 100% related to *Microbacterium oxydans*, and it was named *Microbacterium oxydans* XZT347.

[0027] SEQ ID NO.1

[0028]

[0029] (2) Purification and preservation of bacterial strains

[0030] The streak plating method was used: the isolated *Microbacterium oxydans* XZT347 was streaked on LB agar plates and incubated upside down in a 30°C biochemical incubator for at least 24 hours. Single colonies were picked and labeled XZT347. *Microbacterium oxydans* XZT347 strain was inoculated into LB liquid medium and incubated at 30°C and 220 rpm in a shaker for 48 hours. Then, 2 mL of the fermentation broth of *Microbacterium oxydans* XZT347 strain was mixed with 2 mL of 50% sterile glycerol, gently shaken, and stored at -80°C for long-term preservation.

[0031] Example 2: Preparation of a biopesticide containing Microbacterium oxydans XZT347

[0032] The specific preparation process is as follows:

[0033] (1) Activation of strain XZT347: Microbacterium oxydans XZT347 was inoculated on LB solid medium and cultured at 30-32℃ for 2-3 days to obtain activated Microbacterium oxydans XZT347;

[0034] The formulation of the LB solid medium is as follows: tryptone 9-13 g / L, yeast extract 5-6 g / L, sodium chloride 9-11 g / L, agar powder 14-16 g / L, dissolved in water, and finally adjusted to 1000 mL, pH 7.0±0.3, and autoclaved at 121℃ for 30 min.

[0035] (2) Fermentation: The seed culture of Microbacterium oxydans XZT347 was inoculated into the fermentation medium in the fermentation tank at an inoculation amount of 1-5% of the fermentation medium mass. The culture was carried out in a shaker at 180-250 rpm for 24-48 hours at 30-35℃ to obtain a biological pesticide containing Microbacterium oxydans XZT347.

[0036] The fermentation medium is formulated as follows: tryptone 9-13 g / L, yeast extract 5-6 g / L, sodium chloride 9-11 g / L, 500-700 nmol / L PQQ, dissolved in water, and finally adjusted to 1000 mL, pH 7.0 ± 0.3, and autoclaved at 121℃ for 30 min.

[0037] Example 3: Microbacterium oxydans XZT347 plate confrontation experiment

[0038] Experimental method: The plate confrontation method was used to determine the inhibition rate of *Microbacterium oxysporum* against fungal pathogens. Pepper root rot (Fusarium solani), cucumber target spot (Corynespora cassiicola), eggplant wilt (Fusarium oxysporum f. sp. melongenae), tomato gray mold (Botrytis cinerea Pers. ex Fr.), and tea anthracnose (Colletotrichum camelliae) were inoculated in the center of PDA plates. The test strain XZT347 was inoculated 2.5 cm from the center using a cross-cross method. The control group consisted of no inoculation with the antagonistic strain. The plates were incubated at 28°C. When the uninoculated antagonistic strains of pepper root rot (Fusarium solani), cucumber target spot (Corynespora cassiicola), eggplant wilt (Fusarium oxysporum f. sp. melongenae), tomato gray mold (Botrytis cinerea Pers. ex Fr.), and tea anthracnose (Colletotrichum camelliae) reached the edge of the plate, the colony diameter was measured. Each treatment was repeated three times.

[0039] The formula for calculating the antibacterial rate is as follows:

[0040] Fungal inhibition rate (%) = (Control colony diameter - Treated colony diameter) / Control colony diameter × 100%

[0041] The experimental results are shown in Table 1: The Microbacterium oxydans XZT347 of this application has good antagonistic effects on pepper root rot (Fusarium solani), cucumber target spot (Corynespora cassiicola), eggplant wilt (Fusarium oxysporum f. sp. melongenae), tomato gray mold (Botrytis cinerea Pers. ex Fr.), and tea anthracnose (Colletotrichum camelliae).

[0042] Table 1. Inhibitory effects of *Microbacterium oxysporum* on *Potatoxa Root Rot*, *Cucumber Target Spot*, *Eggplant Fusarium*, *Tomato Gray Mold*, and *Tea Anthracnose*.

[0043] Pathogen Name Antibacterial rate ± standard deviation (%) Pepper root rot fungus (Fusarium solani) 66.14±6.41 Cucumber target spot disease pathogen (Corynespora cassiicola) 73.85±4.58 Fusarium oxysporum f. sp. melongenae is the pathogen that causes eggplant wilt. 63.43±4.26 Tomato gray mold (Botrytis cinerea Pers. ex Fr.) 84.37±1.83 Tea anthracnose fungus (Colletotrichum camelliae) 70.48±1.37

[0044] Example 4

[0045] The method of Example 2 was used to obtain a sample containing 6.5 × 10⁻⁶. 9 A biological pesticide containing Microbacterium oxydans (CFU / ml) XZT347 was diluted 150 times and applied as a root drench and spray to peppers, cucumbers, eggplants, tomatoes, and tea plants inoculated with the corresponding pathogens.

[0046] The specific method for inoculating leaves with pathogens is as follows: Sixty pepper plants were selected and divided into one treatment group and one control group, with 30 pepper plants in each group. Five wounds were made on each leaf using a needle. For the treatment group, 10 μL of *Fusarium solani* (the root rot pathogen of pepper) was applied to the wounds, while for the control group, 10 μL of water was applied. Similarly, 60 cucumber plants were selected and divided into one treatment group and one control group, with 30 cucumber plants in each group. Five wounds were made on each cucumber plant using a needle. For the treatment group, 10 μL of *Corynespora cassiicola* (the target spot pathogen of cucumber) was applied to the wounds, while for the control group, 10 μL of water was applied. Sixty eggplant plants were selected and divided into one treatment group and one control group, with 30 eggplant plants in each group. Five wounds were made on each leaf using a needle. The treatment group received 10 μL of *Fusarium oxysporum* f. sp. melongenae at the wound sites, while the control group received 10 μL of water. Similarly, 60 tomato plants were selected and divided into one treatment group and one control group, with 30 tomato plants in each group. Five wounds were made on each leaf using a needle. The treatment group received 10 μL of *Botrytiscinerea* Pers. ex Fr. at the wound sites, while the control group received 10 μL of water. Sixty tea plants were selected and divided into one treatment group and one control group, with 30 tea plants in each group. The leaves were pricked with a needle to create wounds, with five wounds on each leaf. 10 μL of tea anthracnose bacteria (Colletotrichum camelliae) was dripped into the wounds of the treatment group, while 10 μL of water was dripped into the control group.

[0047] Five days before inoculation with the pathogen, the above-mentioned peppers, cucumbers, eggplants, tomatoes, and tea leaves were treated with a biological pesticide, Microbacterium oxydans XZT347, by root irrigation (40 ml / plant). The infection rate was counted 15 days later. When leaf lesions appeared, the plants were called infected plants. The results are shown in Table 2.

[0048] Table 2. Infection rate of each plant after treatment with Microbacterium oxysporum XZT347.

[0049] deal with Infection rate (%) Chili processing group 10.0 Chili pepper control group 70.0 Cucumber treatment group 13.3 cucumber control group 66.7 Eggplant processing group 20.0 Eggplant control group 83.3 Tomato processing 6.7 Tomato control group 76.7 Tea processing 10.0 Tea control group 90.0

[0050] Table 2 shows that the biopesticide treatment containing Microbacterium oxydans XZT347 resulted in infection rates of Fusarium solani (pepper root rot), Corynespora cassiicola (cucumber target spot), Fusarium oxysporum f. sp. melongenae (eggplant wilt), Botrytiscinerea Pers. ex Fr. (tomato gray mold), and Colletotrichum camelliae (tea anthracnose) of 10.0%, 13.3%, 20.0%, 6.7%, and 10.0%, respectively, significantly reducing the incidence of pepper root rot, cucumber target spot, eggplant wilt, tomato gray mold, and tea anthracnose.

Claims

1. The application of Microbacterium oxydans XZT347 or a biopesticide containing Microbacterium oxydans XZT347 in inhibiting the growth of plant pathogens, characterized in that, The plant pathogens include at least one of the following: *Fusarium solani* (root rot fungus of pepper), *Corynespora cassiicola* (target spot fungus of cucumber), *Fusarium oxysporum f. sp. melongenae* (wilt fungus of eggplant), *Botrytis cinerea Pers. ex Fr.* (gray mold fungus of tomato), and *Colletotrichum camelliae* (anthrax fungus of tea); the *Microbacterium oxydans* XZT347 was deposited at the China Center for Type Culture Collection on July 16, 2025, with accession number CCTCC M 20251623.

2. The application of Microbacterium oxydans XZT347 or a biopesticide containing Microbacterium oxydans XZT347 in the preparation of pesticides that inhibit the growth of plant pathogens, characterized in that, The plant pathogens include at least one of the following: *Fusarium solani* (root rot fungus of pepper), *Corynespora cassiicola* (target spot fungus of cucumber), *Fusarium oxysporum f. sp. melongenae* (wilt fungus of eggplant), *Botrytiscinerea Pers. ex Fr.* (gray mold of tomato), and *Colletotrichum camelliae* (anthracnose fungus of tea); the *Microbacterium oxydans* XZT347 was deposited at the China Center for Type Culture Collection on July 16, 2025, with accession number CCTCC M 20251623.

3. The application of Microbacterium oxydans XZT347 or biological pesticides containing Microbacterium oxydans XZT347 in the preparation of products for controlling root rot of pepper, target spot of cucumber, wilt of eggplant, gray mold of tomato, and anthracnose of tea, characterized in that, The Microbacterium oxydans XZT347 was deposited at the China Center for Type Culture Collection on July 16, 2025, with accession number CCTCC M 20251623.

4. The application according to any one of claims 1-3, characterized in that, The preparation method of the biological pesticide includes the following steps: (1) Activation of strain XZT347: Microbacterium oxydans XZT347 was inoculated on LB solid medium and cultured at 30-32℃ for 2-3 days to obtain activated Microbacterium oxydans XZT347; The formulation of the LB solid medium is as follows: tryptone 9-13 g / L, yeast extract 5-6 g / L, sodium chloride 9-11 g / L, agar powder 14-16 g / L, dissolved in water, and finally adjusted to 1000 mL, pH 7.0±0.3, and autoclaved at 121℃ for 30 min. (2) Fermentation: The seed culture of Microbacterium oxydans XZT347 was inoculated into the fermentation medium in the fermenter at an inoculation amount of 1-5% of the fermentation medium mass. The culture was carried out in a shaker at 180-250 rpm for 24-48 hours at 30-35℃ to obtain a microbial agent containing Microbacterium oxydans XZT347. The fermentation medium is formulated as follows: tryptone 9-13 g / L, yeast extract 5-6 g / L, sodium chloride 9-11 g / L, 500-700 nmol / L PQQ, dissolved in water, and finally adjusted to 1000 mL, pH 7.0 ± 0.3, and autoclaved at 121℃ for 30 min.

5. The application according to any one of claims 1-3, characterized in that, The concentration of Microbacterium oxydans XZT347 in the biopesticide is 6.2-7.6 × 10⁻⁶. 9 cfu / ml.

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