Xanthomonas anhuica, bacterial agent and application thereof in prevention and treatment of potato scab

Abstract

This invention discloses a strain of *Flavobacterium huinanense*, an inoculant, and its application in the control of potato scab, belonging to the field of green control technology for potato scab. This invention isolates a strain of *Flavobacterium huinanense* from the soil surface of potatoes. Flavobacterium anhuiense R10, which has a significant antagonistic effect on *Streptomyces bozzolani*, the pathogen of potato scab. *Flavobacterium huinanense* ( Flavobacterium anhuiense The fermentation broth of R10, when used for root irrigation of potatoes, can significantly reduce the incidence and severity index of potato scab.

Description

Technical Field

[0001] This invention relates to the field of green control technology for potato scab, specifically to a strain of Flavobacterium huinanense, an inoculant, and its application in the control of potato scab. Background Technology

[0002] Potato scab (PCS) is a major disease affecting potato-growing areas, primarily caused by pathogenic Streptomyces spp. through the secretion of thaxtomin, a plant toxin. These toxins specifically inhibit cellulose biosynthesis in plant cell walls, leading to the formation of corky lesions of varying sizes on the tuber surface (Bignell et al., 2010), severely reducing its market value and commercial viability.

[0003] Effective control of potato scab is a complex systemic challenge. Current control strategies mainly include agricultural control, chemical control, and biological control. Traditional agricultural control methods are time-consuming, costly in soil amendment, and difficult to completely eliminate soil pathogens. While chemical pesticides have achieved some success in controlling potato scab (Al-Mughrabi et al., 2016), they cause environmental pollution and harm human health. Therefore, biological control, which is both environmentally friendly and effective, has become an ideal approach to control soil-borne diseases (He et al., 2024).

[0004] The rhizosphere is the most active interface for soil-plant root-microbe interactions and a crucial pathway for soil pathogens to invade crop roots. Plant root-associated soil microbiota constitute a core defense system for plant health (Yang et al., 2023; Zhou et al., 2023), constructing barriers against pathogen invasion through spatial competition and nutrient antagonism (Wei et al., 2020; Singh et al., 2025). Therefore, the breakthrough point for reducing soil biological barriers lies in the rhizosphere, and the regulation of the rhizosphere soil microbiome has become one of the most promising means for the green development transformation of agriculture.

[0005] Anhui Flavobacterium ( Anhui Flavobacterium This is a new species of Flavobacterium isolated from vegetable garden soil in Anhui Province (“Isolation and Polyphasic Taxonomy of Three New Species of Flavobacterium”, Master’s Thesis, Nanjing Agricultural University, 2008). However, research on the function of Flavobacterium huianense is still extremely scarce, and there are very few reports on Flavobacterium huianense in existing technologies. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of this invention is to provide a strain of Flavobacterium huinanense and its application in the prevention and control of potato scab.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a strain of Flavobacterium huinanense ( Anhui Flavobacterium Strain R10 was deposited on January 12, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China 430072, China; its accession number is CCTCC NO: M2026069; and its classification name is... Anhui Flavobacterium R10.

[0009] The present invention is based on the Flavobacterium huishen ( Anhui Flavobacterium Compared with existing Flavobacterium strains, R10 has the following characteristics:

[0010] (1) It has the functions of nitrogen fixation, phosphorus solubilization and potassium solubilization; at the same time, it also has the function of nitrate and nitrite reduction, which can convert easily leached nitrate into microbial biomass, temporarily fix nitrogen in the soil, and reduce nitrogen loss.

[0011] (2) For Streptomyces bozzoli ( Bottrop Streptomyces Fusarium solani () Fusarium solani ), Horsetail ( F. equiseti ), Fusarium oxysporum ( F. fujikuroi Fusarium graminearum ( ), F. graminearum ) and Fusarium moniliformes ( F. moniliforme It has significant antagonistic effects on various pathogens, including , and has broad-spectrum antibacterial activity.

[0012] (3) It has salt and alkali tolerance characteristics; the strain can tolerate a maximum salt concentration of 1% NaCl and a maximum alkali concentration of pH=9.

[0013] In a second aspect, the present invention provides a microbial agent containing the aforementioned Flavobacterium huinanense (Flavobacterium huinanense). Anhui Flavobacterium R10.

[0014] Preferably, the bacterial agent contains Flavobacterium huinanense ( Anhui Flavobacterium R10 exists in the form of cultured live bacteria, bacterial suspension, or fermentation broth.

[0015] Furthermore, the fermentation broth is prepared by the following method:

[0016] Anhui Flavobacterium ( Anhui Flavobacterium R10 was inoculated into R2A liquid medium and cultured at 28℃ and 180 rpm for 24-36 h with constant temperature shaking.

[0017] In a third aspect, the present invention provides the above-mentioned Flavobacterium huinanense ( Anhui Flavobacterium R10 or the inoculant can be used in the following (1) or (2):

[0018] (1) Prevention and control of potato scab;

[0019] (2) Prepare agents for the prevention and treatment of potato scab disease.

[0020] In the above application, the potato scab is caused by Streptomyces bozzolani (… Bottrop Streptomyces Caused by ).

[0021] A fourth aspect of the present invention provides a method for preventing and controlling potato scab, comprising the following steps:

[0022] Once the potatoes enter the tuber enlargement stage, the roots of the potatoes are treated with the aforementioned microbial agent.

[0023] Preferably, the root drenching treatment is performed twice, with a time interval of 7 days between the two root drenching treatments.

[0024] Preferably, the application rate for each root dredging treatment is 200 mL per plant.

[0025] The beneficial effects of this invention are:

[0026] This invention isolated a strain of Flavobacterium huinanense from potato topsoil. Flavobacterium anhuiense R10, which has a significant antagonistic effect on *Streptomyces bozzolani*, the pathogen of potato scab. *Flavobacterium huinanense* ( Anhui Flavobacterium The fermentation broth of R10, when used for root irrigation of potatoes, can significantly reduce the incidence and severity index of potato scab. Attached Figure Description

[0027] Figure 1 Antagonistic test of fermentation broth of strain R10 against Streptomyces bozzoli. Figure A shows the culture medium control; Figure B shows the fermentation broth inoculated with Flavobacterium R10.

[0028] Figure 2 Single colony morphology of strain R10

[0029] Figure 3 Gram staining results of strain R10.

[0030] Figure 4 Evolutionary clade diagram of strain R10.

[0031] Figure 5 The results of the inhibition of strain R10 against various fungal pathogens are shown in the figure. A represents the treatment group and B represents the control group.

[0032] Figure 6 The results of the salt tolerance test of strain R10 are shown in the figure. In the figure, A shows the growth of strain R10 under the condition of 0.5% NaCl, and B shows the growth of strain R10 under the condition of 1% NaCl.

[0033] Figure 7 Results of the alkali resistance test of strain R10; In the figure, A is the growth of strain R10 at pH = 6.8; B is the growth of strain R10 at pH = 7; C is the growth of strain R10 at pH = 8; D is the growth of strain R10 at pH = 9.

[0034] Figure 8 : Potato scab disease diagram (A), disease severity (B), and disease index (C) for each treatment group in Experiment Example 1. Detailed Implementation

[0035] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0036] To enable those skilled in the art to more clearly understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments. If specific experimental conditions are not specified in the embodiments, they are generally based on conventional conditions or conditions recommended by the reagent company; the reagents, consumables, etc. used in the following embodiments, unless otherwise specified, can be obtained commercially. Wherein:

[0037] The pathogen used: Streptomyces bozzoli ( Bottrop Streptomyces Fusarium solani () Fusarium solani ), Horsetail ( F. equiseti ), Fusarium oxysporum ( F. fujikuroi Fusarium graminearum ( ), F. graminearum ) and Fusarium moniliformes ( F. moniliforme All of these are pathogens already existing in current technology, provided by the Microbial Resource Preservation Center of Shandong Agricultural University.

[0038] R2A liquid medium: peptone 0.5 g / L; acid-hydrolyzed casein 0.5 g / L; yeast extract 0.5 g / L; soluble starch 0.5 g / L; dipotassium hydrogen phosphate 0.3 g / L; magnesium sulfate 0.1 g / L; sodium pyruvate 0.3 g / L; glucose 0.5 g / L; pH 7.2±0.2.

[0039] R2A solid medium: Add 15.0 g / L of agar to R2A liquid medium.

[0040] SNB liquid medium: 15g sucrose, 15g glycerol, 40g peptone, 0.5g potassium dihydrogen phosphate, 0.5g sodium chloride, 0.5g magnesium sulfate, 0.5g calcium carbonate, and bring the volume to 1L with sterile distilled water.

[0041] LB, TSB, and PDA solid media are all commercially available media.

[0042] Example 1: Isolation and purification of strains

[0043] Weigh 1g of potato surface soil into a sterile 15mL centrifuge tube, add 10mL of sterile water, and vortex to thoroughly mix the soil. Take 1mL of the soil suspension and add it to 10mL of sterile water to prepare 10... -1 Soil dilution solution, take 1 mL of 10 -1 Soil dilution solution was added to 10 mL of sterile water to prepare 10 -2 Soil dilution solution, and so on, to prepare 10 -3 10 -4 10 -5 10 -6 10 -7 A soil dilution solution of a certain concentration. Take 10... -3 10 -4 10 -5 10 -6 The diluted soil solution was spread onto LB, TSB, R2A, and PDA solid culture media and incubated at 28°C.

[0044] Single colonies of different morphologies were isolated and purified. These single colonies were inoculated into LB, TSB, R2A, and PDA media and cultured at 28℃ and 180 rpm for 24 h to obtain the fermentation broth. 200 mL of *Streptomyces bozzoli* fermentation broth was evenly spread onto Gao's No. 1 solid medium. A hole was punched in the center of the medium, and 200 mL of the fermentation broth was added to each hole. The medium was cultured at 28℃ for 4-7 days, and the presence and size of inhibition zones were observed. Finally, a strain with good antagonistic effect was selected. Figure 1 It was named R10 and stored in a glycerol tube.

[0045] This invention selects four culture media with different nutrient compositions and selectivity for strain screening, enabling targeted recovery of microbial groups with different physiological characteristics. This maximizes the coverage of the inherent microbial diversity in the soil environment and overcomes the selectivity bias of a single culture medium on the microbial community. Specifically:

[0046] LB and TSB media are nutrient-rich media, suitable for isolating rapidly growing bacteria, and are beneficial for obtaining common and rapidly growing microbial resources.

[0047] R2A medium is a low-nutrient medium that simulates natural environmental conditions. It can effectively promote the recovery and growth of oligotrophic bacteria, slow-growing or rare strains, and helps to isolate strains that are inhibited in nutrient-rich media.

[0048] PDA medium is suitable for the isolation and culture of fungi and yeasts, and it also supports the growth of certain actinomycetes, thereby expanding the range of specific groups of eukaryotic and prokaryotic microorganisms that can be obtained.

[0049] By employing a multi-culture medium parallel screening strategy, the likelihood of isolating strains with special metabolic functions, producing novel active substances, or having specific application value can be significantly increased, providing abundant strain resources for subsequent development and application.

[0050] Example 2: Identification of strain R10

[0051] 1. Morphological and physiological-biochemical identification of strain R10:

[0052] Strain R10 colonies are round, with regular, intact yellow edges, a moist, smooth, slightly raised surface, and are opaque. Figure 2 Gram staining was negative (G-). Figure 3 ).

[0053] Partial physiological and biochemical identification of strain R10 was performed, and the results are shown in Table 1.

[0054] Table 1: Identification results of physiological and biochemical properties of strain R10

[0055]

[0056] Note: "+" represents positive; "-" represents negative.

[0057] The results showed that strain R10 possesses starch hydrolysis activity; it can perform nitrogen fixation, phosphorus solubilization and potassium solubilization, but it does not have the ability to produce siderophores; the strain also has nitrate and nitrite reduction functions; its catalase and urease tests were positive; and the VP (Voges-Proskauer) test was negative.

[0058] 2. Molecular biological identification of strain R10:

[0059] Primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3', SEQ ID NO.1) and 1492R (5'-TACGGYTACCTTGTTAYGACTT-3', SEQ ID NO.2) were used to amplify the 16S rRNA sequence of strain R10. The amplification system and amplification program are shown in Tables 2 and 3, respectively.

[0060] Table 2: PCR amplification system

[0061]

[0062] Table 3: PCR amplification program

[0063]

[0064] The obtained PCR products were detected by 1% agarose gel electrophoresis. After confirming the specific bands of the expected size, the corresponding PCR products were sent to the company for sequencing. The results are shown in SEQ ID NO.3. The sequencing results were compared with the NCBI database, and it was found that this strain had the highest similarity to *Flavobacterium huinanense*. Therefore, strain R10 was identified as *Flavobacterium huinanense*. Similar strains were selected and their 16S rRNA gene sequences were downloaded. Phylogenetic analysis was performed using MEGA 11 software. A phylogenetic tree was constructed using the neighbor-joining method, with a bootstrap value of 1000 (…). Figure 4 ).

[0065] Based on the results of morphological, physiological and biochemical, and molecular biological identification of the strain, strain R10 was identified as *Flavobacterium huinanense*. Anhui Flavobacterium (and carried out biological preservation of the patented technology, with the preservation information as follows:)

[0066] Reference biological material (strain): R10;

[0067] Suggested category naming: Anhui Flavobacterium R10;

[0068] Accession number: CCTCC NO: M2026069.

[0069] Example 3: Flavobacterium huinanense ( Anhui Flavobacterium R10 plate broad-spectrum antagonism experiment

[0070] 1. Preparation of fermentation broth for Flavobacterium huinanense R10 strain:

[0071] Flavobacterium huinanense R10 was removed from the -80℃ freezer and activated and revived on R2A solid medium plates using the streak method. A single colony was picked and inoculated into 100 mL of R2A liquid medium and cultured for 24 h in a constant temperature shaking incubator at 28℃ and 180 rpm to obtain the Flavobacterium huinanense R10 seed culture. A 1% inoculum was then inoculated into R2A liquid medium and cultured for 24 h in a constant temperature shaking incubator at 28℃ and 180 rpm to obtain the fermentation broth of Flavobacterium huinanense R10.

[0072] 2. Preparation of fermentation broth for pathogenic bacteria strains:

[0073] The pathogenic fungi (Fusarium solani, Fusarium equisetifolium, Fusarium truncatum, Fusarium graminearum, and Fusarium moniliforme) were removed from a -80℃ freezer and activated and revived on PDA solid medium plates using the streak method. The picked mycelia were inoculated into 100 mL of PDA liquid medium and cultured in a constant temperature shaking incubator at 28℃ and 180 rpm for 4-5 days to obtain the pathogenic fungal seed culture. A 1% inoculum was then inoculated into PDA liquid medium and cultured in a constant temperature shaking incubator at 28℃ and 180 rpm for 4-5 days to obtain the fermentation broth of the pathogenic fungal strain.

[0074] 3. Plate antagonism test:

[0075] 100 μL of the bacterial fermentation broth was spread onto a PDA solid medium plate to obtain a pathogenic bacterial plate. The antagonistic ability of Flavobacterium R10 against the pathogenic fungus was determined using the plate confrontation method. The pathogenic fungal plate was perforated using a punch to obtain pathogenic fungal discs. The discs were placed upside down on a PDA solid plate, and biocontrol bacteria were streaked 1 cm away from the pathogenic fungal discs. The plates were then incubated statically for 4-5 days.

[0076] The results are as follows Figure 5 As shown, the results indicate that biocontrol bacteria R10 can significantly inhibit Fusarium solani (…). Fusarium solani ), Horsetail ( F. equiseti ), Fusarium oxysporum ( F. fujikuroi Fusarium graminearum ( ), F. graminearum ) and Fusarium moniliformes ( F. moniliforme ) growth.

[0077] Example 4: Flavobacterium huinanense ( Anhui Flavobacterium Salt and alkali resistance of R10 were investigated.

[0078] 1. Salt tolerance test:

[0079] Anhui Flavobacterium ( Anhui Flavobacterium R10 was inoculated onto TSB solid medium plates containing different NaCl concentrations, and the growth of the strain was observed.

[0080] The results are as follows Figure 6 As shown, the results indicate that: Anhui Flavobacterium ( Anhui Flavobacterium R10 has a maximum salt concentration tolerance of 1% NaCl.

[0081] 2. Alkali resistance test:

[0082] Anhui Flavobacterium ( Anhui Flavobacterium R10 was inoculated onto TSB solid medium plates at different pH values, and the growth of the strain was observed.

[0083] The results are as follows Figure 7 As shown, the results indicate that: Anhui Flavobacterium ( Anhui Flavobacterium R10 has a maximum alkali resistance concentration of pH=9.

[0084] Example 5: Flavobacterium huinanense Anhui Flavobacterium Field efficacy trials of R10

[0085] 1. Test method:

[0086] The plant material used in the field trial was the Dutch Fifteen potato, and the trial site was the Zaozhuang Academy of Agricultural Sciences.

[0087] Healthy seed potatoes were selected as the experimental subjects. The surface of the seed potatoes was disinfected with potassium permanganate, and the potatoes were cut into pieces using a sterile knife to ensure that each piece had an eye. Sprouting treatment was carried out under dark conditions, and the potatoes were planted in the field after sprouting small sprouts of 2-3 cm.

[0088] There are a total of 3 processing groups, as detailed below:

[0089] Group P: During the potato bulking stage, inoculate with 200 mL of Streptomyces bozzoli fermentation broth; inoculate again with 200 mL of Streptomyces bozzoli fermentation broth seven days later.

[0090] Group R10: During the potato bulking stage, 200 mL of Streptomyces bozzoli fermentation broth was inoculated; seven days later, 200 mL of Streptomyces bozzoli fermentation broth was inoculated again, along with 200 mL of strain R10 fermentation broth; seven days later, 200 mL of strain R10 fermentation broth was inoculated again.

[0091] Group CK: Potato roots were treated with an equal amount of pure water to replace the fermentation broth of Streptomyces bozzoli and strain R10.

[0092] in:

[0093] The fermentation broth of strain R10 was prepared by the following method:

[0094] Flavobacterium huinanense R10 was removed from the -80℃ freezer and activated and revived on R2A solid medium plates using the streak method. A single colony was picked and inoculated into 100 mL of R2A liquid medium and cultured for 24 h in a constant temperature shaking incubator at 28℃ and 180 rpm to obtain the Flavobacterium huinanense R10 seed culture. A 1% inoculum was then inoculated into R2A liquid medium and cultured for 24 h in a constant temperature shaking incubator at 28℃ and 180 rpm to obtain the fermentation broth of strain R10.

[0095] The fermentation broth of *Streptomyces bozzoli* was prepared by the following method:

[0096] *Streptomyces borzoides* was removed from the -80°C freezer and activated and revived using the streak plating method on Gao's No. 1 solid medium plates. Single colonies were picked and inoculated into 100 ml of SNB liquid medium and cultured for 5 days in a constant temperature shaking incubator at 28°C and 180 rpm to obtain the pathogen seed culture. A 1% inoculum was then inoculated into SNB liquid medium and cultured for 5 days in a constant temperature shaking incubator at 28°C and 180 rpm to obtain the *Streptomyces borzoides* fermentation broth.

[0097] After the potatoes matured, they were harvested in the field, and the disease severity and disease index of each treatment group were recorded.

[0098] Nine different grades, from 1 to 9, were used to characterize the severity of potato scab disease. The percentages of potato lesion coverage corresponding to grades 1-9 were 0%, 0.1-0.8%, 0.9-2.8%, 2.9-7.9%, 8.0-18.0%, 18.1-34.0%, 34.1-55.0%, 55.1-77.0%, and 77.1-100%, respectively.

[0099]

[0100] 2. Test Results:

[0101] The incidence and disease index of potato scab in potatoes under different treatments are as follows: Figure 8 As shown, the results indicate that: Anhui Flavobacterium Anhui Flavobacterium R10 can significantly reduce the incidence and severity index of potato scab.

[0102] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A strain of Flavobacterium huinanense ( Flavobacterium anhuiense R10, characterized in that, The accession number is CCTCCNO: M2026069.

2. A microbial agent, characterized in that, The bacterial agent contains the Anhui Flavobacterium as described in claim 1 ( Flavobacterium anhuiense R10.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent contains Flavobacterium huisiensis (Anhui Flavobacterium) Flavobacterium anhuiense R10 exists in the form of cultured live bacteria, bacterial suspension, or fermentation broth.

4. The microbial agent according to claim 3, characterized in that, The fermentation broth is prepared by the following method: Anhui Flavobacterium ( Flavobacterium anhuiense R10 was inoculated in R2A liquid medium and cultured at 28℃ with constant temperature shaking at 180rpm for 24-36 h.

5. The Anhui Flavobacterium as described in claim 1 ( Flavobacterium anhuiense R10 is used in the following (1) or (2): (1) Prevention and control of potato scab; (2) Preparation of agents for the prevention and control of potato scab; The potato scab disease is caused by Streptomyces bozzola (… Streptomyces bottropensis Caused by ).

6. The use of the microbial agent according to claim 2 in either (1) or (2) below: (1) Prevention and control of potato scab; (2) Preparation of agents for the prevention and control of potato scab; The potato scab disease is caused by Streptomyces bozzola (… Streptomyces bottropensis Caused by ).

7. The application according to claim 6, characterized in that, The methods for preventing and controlling potato scab are as follows: Once the potatoes have entered the tuber enlargement stage, the root irrigation treatment of the potatoes is carried out using the inoculum agent described in claim 2.

8. The application according to claim 7, characterized in that, The root drenching treatment was performed twice, with a 7-day interval between the two treatments; the application rate for each treatment was 200 mL per plant.

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