Bacillus velezensis B31 and its application

Abstract

The present invention discloses a Bacillus velezensis strain B31, which is deposited in the General Microbiology Center of the China National Committee for the Collection of Microorganisms, and its deposit number is CGMCC No. 24599. The present invention also discloses a microbial agent containing B31, and their uses. The strain B31 of the present invention has a high preventive effect. The preventive effect on wilt diseases such as tomato wilt is 87.53%-97.12%; secondly, it has a wide spectrum of prevention and control, and has a high preventive effect on wilt diseases such as tomato wilt, watermelon wilt, and cucumber wilt caused by Fusarium oxysporum. In addition, the strain B31 of the present invention has strong specialization, good long-lasting efficacy, is not easy to develop drug resistance, and is environmentally friendly.

Description

Technical Field

[0001] The invention belongs to the field of biocontrol microorganisms, and particularly relates to Bacillus velezensis, and also relates to a microbial agent produced by utilizing the bacterium, and uses thereof. Background Art

[0002] Fusarium oxysporum is a soil-borne pathogenic fungus with a worldwide distribution. It has a very wide host range and can cause wilt diseases in more than 100 plant species, including melons, Solanaceae, bananas, cotton, legumes, and flowers. Continuous cropping for many years will lead to increasingly severe diseases, causing huge losses to the production of economic crops such as melons.

[0003] At present, production mainly relies on soil fumigation, grafting technology and chemical agents to control wilt disease of vegetable plants. However, these control methods have the following problems: (1) Soil fumigation not only destroys the balance of soil microbial flora, but also causes serious soil and ecological environment pollution. (2) Although the use of grafting technology to control watermelon and cucumber wilt has a certain effect, it directly affects the quality and taste of the fruit, and this method is time-consuming and labor-intensive. (3) The use of chemical pesticides for control is prone to drug resistance and environmental pollution. The use of biocontrol microorganisms for biological control of plant diseases has received more and more attention due to its advantages such as high prevention efficiency, long-lasting drug effect, strong specialization, low resistance to drug resistance and environmental friendliness.

[0004] At present, the biocontrol microorganisms used to prevent and control wilt caused by Fusarium oxysporum mainly include Trichoderma fungi such as Trichoderma harzianum (Zhang Heqing et al. Sichuan Agricultural Science and Technology. 2020 (05): 35-37), and Bacillus bacteria such as Bacillus polymyxa, Bacillus megaterium, Bacillus subtilis, and Bacillus amyloliquefaciens. Their control efficiency against wilt is generally 60%-79%. Since the types of Fusarium oxysporum present in different ecological environments and different crop conditions are different, it is necessary to continuously screen strains that can prevent and control Fusarium oxysporum under corresponding environmental conditions in order to solve the problem in a targeted manner. Bacillus is a dominant species in the natural environment. Because it can produce a variety of antibacterial active substances (Li Baoqing et al. Chinese Agricultural Science. 2010, 43 (017): 3547-3554), and can form stress-resistant and heat-resistant spores, it is beneficial to product development and shelf life extension. It has a good safety evaluation and is easy to scale up production. Therefore, it has become an important resource for the development of microbial fungicides.

[0005] Fusaric acid (5-butyl-2-pyridinecarboxylic acid) is a non-specific toxin secreted by Fusarium oxysporum. It can increase the permeability of host plant cell membranes, reduce the content of mitochondrial reactive oxygen species in host cells, hinder ATP synthesis, cause plant metabolic disorders, and cause plant wilt and death (Zhang Jiacheng et al. Grassland Science. 2021, 38(08): 1513-1524). Fusaric acid can also inhibit the growth and metabolism of various microorganisms, including beneficial microorganisms (Bacon CW et al. Journal of applied microbiology. 2006, 100(1): 185-194; Raza W, et al. Plant Pathology. 2015, 64(5): 1041-1052). Therefore, the Fusarium oxysporum, which is present in large quantities in the rhizosphere of plants, inhibits the growth and colonization of rhizosphere biocontrol bacteria by producing fusaric acid (Chen Qiaohuan et al. Chinese Journal of Experimental Traditional Chinese Medicine. 2021, 27(11):7), especially the growth and colonization of Bacillus (Guo Qinggang et al. Acta Phytophylacica Sinica. 2013, 40(1):45-50), thereby reducing the biocontrol effect of biocontrol bacteria. Therefore, screening Bacillus that are both tolerant to fusaric acid and have strong antagonistic activity against Fusarium oxysporum will have the potential to become biocontrol bacteria for controlling crop wilt, and thus be used as an effective means to control plant wilt caused by Fusarium oxysporum. Summary of the Invention

[0006] In order to solve the problem of plant wilt caused by Fusarium oxysporum, the present invention aims to provide a Bacillus Velezii B31.

[0007] Another object of the present invention is to provide a microbial agent containing the above-mentioned Bacillus Velezii B31.

[0008] The third object of the present invention is to provide uses of the above-mentioned Bacillus Velezii B31 and its bacterial agent.

[0009] To achieve the above objectives, the present invention adopts the following technical solutions.

[0010] The invention discloses a Bacillus velezensis strain B31, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, and its deposit number is CGMCC No. 24599.

[0011] The present invention also provides the use of the Bacillus velezensis strain B31 in preventing and treating tomato wilt, watermelon wilt or cucumber wilt.

[0012] The present invention also provides a microbial agent, which contains the above-mentioned Bacillus Velezii strain B31.

[0013] The present invention also provides application of the microbial agent in preventing and treating tomato wilt, watermelon wilt or cucumber wilt.

[0014] The pathogen of tomato wilt is Fusarium oxysporumf.sp.lycopersici Snyder et Hansen.

[0015] The pathogen of watermelon wilt disease is Fusarium oxysporum f.sp. Hiveum (EF Smith) Wollen.

[0016] The pathogen of cucumber wilt is Fusarium oxysporum (Schl.) F. sp cucumerinum Owen.

[0017] A genetically engineered bacterium, wherein the starting strain of the genetically engineered bacterium is the above-mentioned Bacillus Velez strain B31, and the genetically engineered bacterium has the function of preventing and treating tomato wilt, watermelon wilt or cucumber wilt.

[0018] Compared with the prior art, the present invention has the following beneficial technical effects: (1) High prevention effect. The prevention effect on tomato wilt is 97.12%, the prevention effect on watermelon wilt is 92.09%, and the prevention effect on cucumber wilt is 87.53%. (2) It has a wide spectrum of prevention and control, and has a high prevention effect on tomato wilt, watermelon wilt, cucumber wilt and other wilt diseases caused by Fusarium oxysporum. (3) The strain B31 of the present invention is a strain screened after high-temperature treatment at 80°C. It is resistant to high temperatures and can form stress-resistant and heat-resistant spores, which is beneficial to product development and shelf life extension. (4) The strain of the present invention has strong specialization, good long-lasting efficacy, is not easy to develop drug resistance, and is environmentally friendly.

[0019] Biological preservation: The Bacillus velezensis strain B31 of the present invention is a sample collected by the inventor from healthy tomato plants and their soil in the diseased greenhouse in Feixiang County, Handan City, Hebei Province in March 2021, and obtained by screening in an 80°C water bath. It was deposited in the General Microbiology Center of the China Culture Collection Administration on March 28, 2022, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; its preservation number is CGMCC No. 24599. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the electrophoresis pattern of PCR amplification of the 16S rDNA gene of strain B31.

[0021] Figure 2 Phylogenetic tree of strain B31 constructed based on 16S rDNA gene sequence.

[0022] Figure 3 This is the electrophoresis pattern of PCR amplification of the gyrB gene of strain B31.

[0023] Figure 4 This is the phylogenetic tree of strain B31 constructed based on the gyrB gene sequence.

[0024] Figure 5 This is a bar chart of tomato yield treated with B31 strain. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to specific examples, which however do not limit the scope of protection of the present invention.

[0026] Example 1 Screening of biocontrol microorganisms that are resistant to fusaric acid and have antagonistic effects on Fusarium oxysporum

[0027] Proceed as follows:

[0028] (1) In the area where cucumber, tomato and watermelon wilt disease occurred, a total of 48 soil samples were collected from the rhizosphere of healthy plants. Each soil sample weighed 1 g and was added to a conical flask containing 10 mL of distilled water. The soil samples were shaken at 30°C and 180 rpm for 30 minutes, then taken out and allowed to stand at room temperature for 30 minutes. Then 1 mL of the supernatant was taken and placed at 80°C for 15 minutes, and cooled at room temperature to obtain soil suspensions of Bacillus in the form of spores.

[0029] (2) Dilute the soil suspension obtained in step (1) 100-fold with distilled water; take 100 μL of the diluted soil suspension and evenly spread it on LB medium containing 20 μg / mL fusaric acid (LB medium prepared according to conventional methods), and culture it at 37°C for 12 hours. Select the vigorously growing colonies, which are the fusaric acid-tolerant Bacillus.

[0030] (2) Antibacterial test: The target bacteria were Fusarium oxysporum f.sp. lycopersici FQ143, Fusarium oxysporum f.sp. Niveum XG0010 and Fusarium oxysporum f.sp. cucumerinum FOC1-2-11, pathogens of tomato, watermelon and cucumber wilt, respectively, which were stored in the Plant Disease Biological Control Laboratory of the Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences. The pathogens were inoculated on PDA plates in advance and cultured at a constant temperature of 25°C until the mycelium on the plate covered the entire culture dish. Then, a 6 mm diameter bacterial block was punched out with a hole punch and inoculated on the center of a new pre-poured PDA plate with a diameter of 9 cm. The above-screened bacteria resistant to fusaric acid were inoculated at a distance of 2.5 cm and cultured in an incubator at 25° C. for 5 days to screen bacteria resistant to fusaric acid with inhibition zones of more than 0.5 cm in three antibacterial activity tests.

[0031] Results: 68 biocontrol strains were screened out from 500 microorganisms, which had antibacterial effects on the three pathogens (inhibition zone > 0.5 cm) and were resistant to fusaric acid.

[0032] Example 2 Preparation of biocontrol bacteria fermentation liquid

[0033] The 68 biocontrol bacteria obtained in Example 1 were inoculated into 5 mL of liquid LB medium, shaken and cultured at 180 rpm and 37°C for 12 h, and then transferred to 200 mL of LB liquid medium at a ratio of 1%, shaken and cultured at 180 rpm and 37°C for 48 h to obtain fermentation broths of different strains. The bacterial cell concentration (CFU / mL) in the fermentation broth was calculated by the plate count method.

[0034] Example 3 Screening test of biocontrol strains with preventive effect on cucumber wilt

[0035] Proceed as follows:

[0036] (1) Cucumber (Zhongnong No. 6, highly susceptible to cucumber wilt) seed germination and seedling cultivation: Soak the cucumber seeds in 55°C warm water for 30 minutes, rinse with clean water, and then soak in clean water for 8 hours. After soaking, wrap them with wet gauze, germinate them in a 25°C incubator overnight, and then grow them in vermiculite.

[0037] (2) Application of biocontrol bacteria fermentation liquid: When the cucumbers grew two leaves and one heart, the first application of biocontrol bacteria (biocontrol bacteria fermentation liquid prepared in Example 2) was applied, with 3 mL of the bacterial liquid applied to the roots of each seedling.

[0038] (3) Preparation of pathogen FOC 1-2-11 (cucumber-specific type) bacterial solution and soil mixing: Use a cork punch to punch out 6 mm diameter Fusarium oxysporum fungus blocks and inoculate them into PDB liquid culture medium. Cultivate at 25°C and 180 rpm for 5 days. Count the spores after filtering through sterile gauze. Mix 1 L of pathogen bacterial solution with 20 kg of sterile soil in a soil mixer to a final concentration of 5.0 × 10 5 Spore / Ksutsu.

[0039] (4) Transplanting: Transplant the seedlings 3 days after the transplantation. During the transplanting process, align the roots of the cucumber seedlings, cut off the fibrous roots with scissors (leave two centimeters), and do root wounding treatment. Then transplant them into small flower pots, and set up three replicates for each treatment.

[0040] (5) Second application of biocontrol bacteria: Three days after transplanting, apply the second application of biocontrol bacteria (the biocontrol bacteria fermentation liquid prepared in Example 2) with the same usage and dosage as the first application of biocontrol bacteria. Water and observe on time, and investigate the disease condition after 10 days.

[0041] Grading standards for cucumber wilt at the seedling stage (refer to the standards reported by Zhou Hongmei, Mao Aijun, etc. for disease investigation).

[0042] Disease index = ∑ (number of diseased plants at each level × representative value of each level) / (total number of plants surveyed × highest representative value) × 100 (the same below).

[0043] Control effect (%) = [(control disease index - treatment disease index) / control disease index] × 100 (the same below).

[0044] (6) Select strains with a biocontrol effect of more than 60% for rescreening, and finally select 5 strains of biocontrol bacteria with high control effect.

[0045] The results showed that among the 68 antagonistic bacteria tolerant to fusarium acid, one strain, B31, had an initial screening efficacy of 87.53% against cucumber wilt and a secondary screening efficacy of 85.72%, indicating that strain B31 has a high and stable control efficacy against cucumber wilt.

[0046] Example 4 Screening test of biocontrol bacteria with preventive effect on tomato wilt

[0047] Proceed as follows:

[0048] (1) Germination and seedling cultivation of tomato seeds (Nami, highly susceptible to tomato wilt): Rinse the tomato seeds with clean water, wrap them with wet gauze after cleaning, germinate them in a 25℃ incubator overnight, and then grow them in vermiculite.

[0049] (2) Application of biocontrol bacteria fermentation liquid: When the tomato has two leaves and one heart, apply the first application of biocontrol bacteria (biocontrol bacteria fermentation liquid prepared in Example 2). Dilute 150 mL of the fermentation liquid by half and pour it into the seedling tray.

[0050] (3) Preparation of pathogen FQ 143 (tomato-specific type) bacterial suspension: Use a cork punch to punch out 6 mm diameter Fusarium oxysporum fungus pieces, inoculate them into PDB medium, and shake at 25°C and 180 rpm for 5 days. After filtering through sterile gauze, count the spores at a concentration of 4 × 10 7 spores / ml.

[0051] (4) Transplanting: Transplant the seedlings 3 days after the transplantation. During the transplanting process, align the roots of the tomato seedlings, cut off the fibrous roots with scissors (leave two centimeters), and do root wounding treatment. Soak the pathogen solution below the roots for 60 minutes, and then transplant them into flower pots. Set up three replicates for each treatment.

[0052] Water and observe on time, checking the condition for the first time after about 7 days and the second time after two weeks.

[0053] Grading standards for tomato wilt at the seedling stage (refer to the standards reported by Bai Minzhan, Zheng Guibin, etc. for disease investigation).

[0054] Results showed that strain B31, among 68 strains of antagonistic bacteria tolerant to fusarium acid, had a 7-day control effect on tomato wilt of 97.12% and a 15-day control effect of 100%, indicating that strain B31 had a good control effect on tomato wilt.

[0055] Example 5 Screening test of biocontrol strains with preventive effect on watermelon wilt

[0056] Proceed as follows:

[0057] (1) Germination and seedling cultivation of watermelon seeds (Zaojia 84-24, highly susceptible to watermelon wilt): Soak the watermelon seeds in 55°C warm water for 30 minutes, stirring constantly, rinse with clean water, and then soak in clean water for 8 hours. After soaking, wrap them with wet gauze, germinate them in a 25°C incubator overnight, and then grow them in vermiculite.

[0058] (2) Application of biocontrol bacteria fermentation liquid: When the watermelon has two leaves and one heart, the first application of biocontrol bacteria (biocontrol bacteria fermentation liquid prepared in Example 2) is applied, and 3 mL of the bacterial liquid is applied to the root of each seedling.

[0059] (3) Preparation of pathogen XG0010 (watermelon-specific type) bacterial solution and soil mixing: Use a cork punch to punch out 6 mm diameter Fusarium oxysporum fungus blocks, inoculate them into PDB medium, and shake at 25°C and 180 rpm for 5 days. Count the spores after filtering through sterile gauze. Mix 1 L of bacterial solution with 20 kg of sterile soil in a soil mixer to a final concentration of 5.0 × 10 5 Spore / gram soil.

[0060] (4) Transplanting: Transplant the seedlings 3 days after the sterilization. During the transplanting process, align the roots of the watermelon seedlings, cut off the fibrous roots with scissors (leave two centimeters), and do root wounding treatment. Then transplant them into flower pots. Set three replicates for each treatment.

[0061] (4) Second application of biocontrol bacteria: Apply the second application of biocontrol bacteria (biocontrol bacteria fermentation liquid prepared in Example 2) 3 days after transplanting the seedlings. The usage and dosage are the same as the first application of biocontrol bacteria. Water and observe on time. Investigate the disease condition after 10 days.

[0062] Grading standards for watermelon wilt at the seedling stage (refer to the standards reported by Yan Wen, Wang Xiqing, etc. for disease investigation).

[0063] The results showed that among the 68 antagonistic bacteria tolerant to fusarium acid, strain B31 had a 7-day control effect on watermelon wilt of 92.09% and a 15-day control effect of 84.26%, respectively, indicating that B31 had a high control effect on watermelon wilt.

[0064] Example 6 Classification and Identification of the Selected Strain B31 of the Present Invention

[0065] Proceed as follows:

[0066] The genome of strain B31 was extracted using a modified CTAB method. Universal primers for the 16S rDNA and gyrB gene sequences were used to amplify B31. The universal primers for the 16S rDNA sequence were 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; and 1492R: 5'-CTACGGCTACCTTGTTACGA-3'. Universal primers for the gyrB gene were gyrB-F: 5'-TTGRCGGHRGYGGHTATAAAGT-3', and gyrB-R: 5'-TCCDCCSTCAGARTCWCCCTC-3'. PCR products were analyzed by electrophoresis on 1% agarose gels and sequenced by Shanghai Sangon Biotechnology Co., Ltd. The resulting sequences are listed in the sequence listing (SEQ ID No. 1 and SEQ ID No. 2). Homology analysis and multiple sequence alignment were performed in the NCBI GenBank database, and a phylogenetic tree was constructed using MEGA software based on the alignment results.

[0067] Results The 16S rDNA gene of strain B31 amplified by PCR was a DNA fragment of 1515 bp in size (see Figure 1 ); the gyrB gene of the B31 strain obtained by PCR amplification is a DNA molecule fragment of 984 bp in size (see Figure 2 ). BLAST comparison results showed that the 16SrDNA gene sequence of strain B31 had the highest similarity with the 16SrDNA gene sequence of Bacillus velez, which was 99.93%; the gyrB gene sequence of strain B31 had the highest similarity with the gyrB gene sequence of Bacillus velez, which was 97.56%. Combined with the phylogenetic tree (see Figure 2 and Figure 4) It can be seen that the B31 strain belongs to the Bacillus species of the genus Bacillus, and is different from any known Bacillus strain, and is a new Bacillus strain.

[0068] Example 7B31 Indoor test on the efficacy of fermentation broth, bacterial suspension and supernatant against tomato wilt

[0069] Proceed as follows:

[0070] (1) Tomato seed germination and seedling cultivation: Rinse the tomato seeds with clean water, wrap them with wet gauze after cleaning, germinate them in a 25℃ incubator overnight, and then grow them in vermiculite.

[0071] (2) Preparation of B31 biocontrol bacteria fermentation broth, cells, and supernatant: The biocontrol bacteria were inoculated into 5 mL of liquid LB medium and cultured with shaking at 180 rpm and 37°C for 12 h. The culture was then transferred to 300 mL of liquid LB medium at a 1% ratio and cultured with shaking at 180 rpm and 37°C for 48 h to obtain a fermentation broth. The cell concentration (CFU / mL) in the fermentation broth was calculated using the plate count method. 150 mL of the biocontrol bacteria fermentation broth was centrifuged, the cells were resuspended, and the supernatant and the cells were each fixed to 150 mL.

[0072] (3) Application of B31 fermentation liquid, bacteria and supernatant: When the tomato has two leaves and one heart, dilute 150 ml of fermentation liquid, bacteria and supernatant by half and pour it into the seedling tray to facilitate the full colonization of B31 strains or absorption of active ingredients such as antibacterial substances by the roots.

[0073] (4) Preparation of pathogenic bacteria liquid: Use a cork punch to punch out 6 mm diameter Fusarium oxysporum bacteria, inoculate into PDB medium, shake at 25°C and 180 rpm for 5 days, filter through sterile gauze, and count the spores. The concentration is 4.5×10 7 spores / ml.

[0074] (5) Transplanting seedlings: Transplant seedlings 3 days after the inoculation of the antibacterial fermentation liquid, bacteria and supernatant. During the transplanting process, align the roots of the tomato seedlings, cut off the fibrous roots with scissors (leave two centimeters), and do root wounding treatment. Soak the pathogen liquid below the roots for 60 minutes, and then transplant them into flower pots.

[0075] (6) Water and observe the plant regularly, and check the plant's condition after about 10 days. Check the plant's condition again after about 20 days.

[0076] Results (see Table 1) showed that the control efficacy of the fermentation broth, bacterial suspension and sterile supernatant of strain B31 against tomato wilt was 90.01%, 89.73% and 62.03% respectively, indicating that B31 had a high control efficacy against tomato wilt.

[0077] Table 1 Control results of B31 bacterial suspension, sterile supernatant and fermentation liquid on tomato wilt

[0078] Serial number deal with Disease Index Prevention effect (%) 1 CK 73.15±24.25a 2 B31 bacterial suspension 7.51±11.64b 89.73 3 B31 fermentation broth 7.31±3.46b 90.01 4 B31 sterile supernatant 27.78±3.46b 62.03

[0079] Note: Data in the table are mean ± standard error. Different letters after the data in the same column indicate significant differences at the P < 0.05 level using Duncan's new multiple range test.

[0080] Example 8 Field control effect and yield increase test of strain B31 of the present invention on tomato wilt

[0081] Proceed as follows:

[0082] The field efficacy of B31 against Fusarium wilt was evaluated in greenhouse plots in Dingxing County, Baoding City, Hebei Province, where Fusarium wilt is a serious disease. The tomato variety was Provence. Before transplanting, 20 mL of the biocontrol agent B31 was applied to each seedling hole. Transplantation was carried out 12 hours after treatment. After transplanting, B31 was applied via drip irrigation (concentration of 100 million CFU / mL) at a rate of 500 L / mu. Each treatment consisted of five rows with four replicates. A water treatment served as a control. The incidence of Fusarium wilt was assessed during the flowering and fruiting stages of the tomatoes. Yield measurements were taken at the end of the growing season to compare tomato yield and red fruit percentage in the B31-treated and untreated plots.

[0083] The grading standard for tomato wilt at maturity is as follows: Level 0: healthy plants, no diseased leaves, and normal growth; Level 1: less than a quarter of the leaves on the plant are yellowing or wilting; Level 2: more than a quarter and less than one-half of the leaves on the plant are yellowing or wilting and drooping; Level 3: more than one-half and less than three-quarters of the leaves on the plant are yellowing or wilting and drooping; Level 4: less than three-quarters of the leaves are yellowing or wilting and drooping, or the entire plant is severely wilted and dies.

[0084] Results The disease index of tomato wilt in the area treated with strain B31 of the present invention was 13.28, while that in the control area was 54.27. The control efficacy of strain B31 on tomato wilt in the field was 75.53%. Figure 5 ), tomato yield increased by 17.23%, and the tomato fruit turned from green to red, with the red fruit rate increasing by 35.60%. This indicates that strain B31 has a high field efficacy against tomato wilt, greatly increasing tomato yield and significantly increasing the red fruit rate.

Claims

1. A Bacillus velezinoffii ( Bacillus velezensis ) strain B31, deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with the deposit number being CGMCC No.24599.

2. Use of the Bacillus Velez strain B31 according to claim 1 in preventing and treating tomato wilt, watermelon wilt or cucumber wilt; wherein the pathogen of tomato wilt is Fusarium oxysporum tomato-specific; the pathogen of watermelon wilt is Fusarium oxysporum watermelon-specific; and the pathogen of cucumber wilt is Fusarium oxysporum cucumber-specific.

3. A microbial agent, characterized in that: The microbial agent contains the Bacillus Velez subtilis strain B31 according to claim 1.

4. Use of the microbial agent according to claim 3 for preventing and controlling tomato wilt, watermelon wilt or cucumber wilt; wherein the pathogen of tomato wilt is Fusarium oxysporum tomato-specific; the pathogen of watermelon wilt is Fusarium oxysporum watermelon-specific; and the pathogen of cucumber wilt is Fusarium oxysporum cucumber-specific.

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