Antagonistic bacterium ZJ11 capable of inhibiting, preventing and controlling amorphophallus konjac soft rot and application of antagonistic bacterium ZJ11

Abstract

The invention discloses an antagonistic bacterium ZJ11 capable of inhibiting, preventing and controlling amorphophallus konjac soft rot and application of the antagonistic bacterium ZJ11. The antagonistic bacterium ZJ11 is preserved in China Center for Type Culture Collection, the preservation number is CCTCC NO. M 2023720, the preservation address is Wuhan University, Wuhan, China, and the preservation time is May 10, 2023. The invention aims to construct a synergistic biological control system by providing a strain of efficient bacillus subtilis ZJ11 which is autonomously screened and identified and creatively and scientifically compounding the efficient bacillus subtilis ZJ11 with alginate oligosaccharide. The system not only pursues direct inhibition on pathogenic bacteria, but also focuses on realizing lasting, stable and efficient prevention and control on the soft rot of the konjak by stimulating internal disease-resistant physiological and biochemical response of the konjak, so as to thoroughly get rid of dependence on chemical pesticides and promote healthy and sustainable development of the konjak industry.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an antagonistic bacterium ZJ11 that can inhibit and control soft rot of konjac and its application. Background Technology

[0002] Konjac soft rot, a serious soil-borne disease caused by pathogens such as *Achromobacter xylose oxidizingus*, poses a persistent and significant threat to the konjac industry. This disease spreads rapidly, often causing rot of the konjac corms and stem base, and lodging of the plants. High incidence rates in the field can directly lead to total crop failure, making it a key obstacle to large-scale, standardized konjac production. Currently, agricultural production still heavily relies on chemical pesticides for the control of this disease. However, this conventional method has a series of inherent drawbacks that are difficult to overcome. Chemical control not only has limited effectiveness and a short duration of action, but also leads to soil and water pollution, pesticide residue safety risks in konjac products, and the risk of pathogen resistance induced by long-term use of single pesticides. All of these run counter to the requirements of green and sustainable agricultural development in our time.

[0003] Konjac soft rot is a soil-borne disease caused by a variety of pathogens, especially *Achromobacter xylose oxidizing bacteria* (…). Achromobacter xylosoxidans This disease is one of the main pathogens causing konjac disease. It severely impacts konjac growth, leading to reduced yield and deteriorated quality. Current control methods primarily rely on chemical pesticides. While these pesticides can effectively control the disease in the short term, long-term use can cause environmental pollution, pesticide residues, and increased pesticide resistance in pathogens. In recent years, with the rise of sustainable agriculture, biological control methods have gradually gained attention and become a research hotspot.

[0004] Against this backdrop, environmentally friendly biological control technologies are seen as an important alternative. Although some microbial agents have been tested for plant disease control, existing technologies still have significant shortcomings when targeting konjac soft rot. Most commercially available biocontrol strains are broad-spectrum products, lacking specificity against konjac soft rot and exhibiting unstable efficacy. Furthermore, many strains do not originate from the konjac rhizosphere environment, and their colonization ability, survival rate, and interaction efficiency with host plants in this specific ecological niche are often less than ideal. More importantly, existing biological control programs are mostly limited to the single application of biocontrol agents, resulting in a relatively simple mode of action. Their efficacy is easily constrained by complex field environmental conditions, making it difficult to achieve stable and efficient control, and they lack a systematic design to enhance the konjac plant's own immunity.

[0005] Currently, the control methods for konjac soft rot mainly focus on the use of chemical pesticides. These chemical pesticides achieve their control effect by inhibiting the growth of pathogens, but the negative impacts of their use are becoming increasingly apparent. In addition to environmental pollution and pesticide residues, the problem of pathogen resistance is also becoming increasingly serious, leading to a gradual decrease in control effectiveness. Furthermore, existing biological control methods mostly rely on broad-spectrum antimicrobial agents, lacking specificity and resulting in less than ideal effects. Summary of the Invention

[0006] The purpose of this invention is to provide an antagonistic bacterium ZJ11 that can inhibit and control konjac soft rot and its application. By providing a highly efficient Bacillus subtilis strain ZJ11 that was independently screened and identified, and creatively combining it with brown algae oligosaccharides, the aim is to construct a synergistic biological control system.

[0007] The above-mentioned technical objective of the present invention is achieved through the following technical solution: An antagonistic bacterium that can inhibit and control konjac soft rot ( Bacillus subtilis ZJ11, the strain is deposited at the China Center for Type Culture Collection, accession number CCTCC NO. M 2023720, deposit address: Wuhan University, Wuhan, China, deposit date: May 10, 2023.

[0008] More preferably, the strain has a strong antagonistic effect on the pathogen of konjac soft rot, *Achromobacter xylose oxidizing*, with an inhibition zone diameter of not less than 2.00 cm.

[0009] More preferably, the 16S rRNA gene sequence of the strain has at least 99% homology with SEQ ID NO:1.

[0010] More preferably, the genome of the strain contains gene clusters encoding fengycin, surfactantin, and bacillaene.

[0011] The present invention also provides a microbial preparation of ZJ11 antagonistic bacteria that can inhibit and control konjac soft rot, comprising live cells, spores or fermentation broth of the antagonistic bacteria ZJ11, and an agriculturally acceptable carrier.

[0012] More preferably, the effective viable count of the antagonistic bacterium ZJ11 in the preparation is not less than 1×10⁻⁶. 8 CFU / mL.

[0013] More preferably, the formulation further comprises fucoidan oligosaccharide, the concentration of which is 1-10 g / L, and the ratio of the effective viable count of the antagonistic bacterium ZJ11 to the mass concentration of the fucoidan oligosaccharide is (1×10⁻⁶ g / L). 8CFU: (1-10 g).

[0014] The present invention also provides the application of antagonistic bacteria ZJ11 or microbial preparations in inducing disease resistance in konjac, wherein the disease resistance is manifested by increasing the activity of peroxidase (POD), polyphenol oxidase (PPO) and catalase (CAT) in konjac plants.

[0015] The present invention also provides the application of antagonistic bacteria ZJ11 or microbial preparations in promoting the growth of konjac.

[0016] In summary, the present invention has the following beneficial effects: Firstly, this invention has highly efficient specialized antagonistic activity: the Bacillus subtilis ZJ11 obtained through screening exhibits strong specialized antagonistic activity against Achromobacter xylose oxidizing bacteria, the main pathogen of konjac soft rot, with an inhibition zone diameter as high as 2.13 cm, which is significantly better than other initially screened strains.

[0017] Secondly, this invention has excellent field control efficacy: In field trials of konjac continuous cropping fields, the application of ZJ11 bacterial solution can reduce the disease index by 37.98%, effectively inhibiting the occurrence and spread of the disease.

[0018] Thirdly, this invention has a synergistic effect of compounding: when ZJ11 is used in combination with alginate oligosaccharide (AOS), compared with the use of ZJ11 fungicide alone, the potted plant control efficacy increases from 39.96% to 45.68%, and the incidence rate is further reduced, showing a significant synergistic effect of "using fungi to suppress fungi" and "inducing resistance".

[0019] Fourth, this invention provides a green and environmentally friendly biological control solution that can replace or reduce the use of chemical pesticides, fundamentally solving prominent problems such as pesticide residues, environmental pollution, and pathogen resistance, and ensuring the quality, safety, and ecological sustainability of konjac products. Attached Figure Description

[0020] Figure 1 This is a diagram illustrating the antagonistic effects of different antagonistic bacteria against soft rot pathogens in this invention. Figure 2 These are morphological images of the ZJ11 strain in this invention: a: frontal morphology; b: back morphology; c: Gram staining microscopy results. Figure 3 This is the phylogenetic tree of the ZJ11 strain in this invention; Figure 4 This is a diagram showing the antibacterial ability of the ZJ11 strain against different pathogens in this invention; Figure 5 This is an image showing the effect of strain ZJ11 on detached konjac corms in this invention; Figure 6This is a diagram illustrating the effect of the ZJ11 strain on konjac plants in this invention. Figure 7 This is a graph showing the effect of different treatment methods on the POD activity of konjac in this invention; Figure 8 This is a graph showing the effect of different treatment methods on the PPO activity of konjac in this invention; Figure 9 This is a graph showing the effect of different treatment methods on the CAT activity of konjac in this invention; Figure 10 This is a graph showing the effect of different treatment methods on the TP content of konjac in this invention; Figure 11 This is a graph showing the effect of different treatment methods on the activity of konjac TF in this invention; Figure 12 This is a graph showing the effect of different treatment methods on the H2O2 content of konjac in this invention; Figure 13 This is a graph showing the effect of different treatment methods on the MDA content of konjac in this invention; Figure 14 This is a genomic circle diagram of the ZJ11 strain in this invention; Figure 15 This is a schematic diagram of the genome of secondary metabolites of strain ZJ11 in this invention; Figure 16 These are the incidence and disease index of konjac under different treatments in this invention; Figure 17 This is a comparison of the control effect of ZJ11 on konjac soft rot in the field. Left: Natural growth of konjac soft rot in the field; Right: Irrigated with Bacillus subtilis ZJ11. Detailed Implementation The present invention will be further described in detail below with reference to the accompanying drawings.

[0021] Example 1: An antagonistic bacterium capable of inhibiting and controlling konjac soft rot ( Bacillus subtilis ZJ11, the strain described, is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO. M 2023720, located at Wuhan University, Wuhan, China, on May 10, 2023. This strain exhibits antagonistic activity against *Achromobacter xylose-oxidizing*, the pathogen of konjac soft rot, with an inhibition zone diameter of not less than 2.00 cm. The 16S rRNA gene sequence of this strain shares at least 99% homology with SEQ ID NO:1. The genome of this strain contains gene clusters encoding fengycin, surfactantin, and bacillaene.

[0022] A microbial preparation containing the antagonistic bacterium ZJ11 capable of inhibiting and controlling soft rot disease in konjac, comprising live cells, spores, or fermentation broth of the antagonistic bacterium ZJ11, and an agriculturally acceptable carrier. The effective viable count of the antagonistic bacterium ZJ11 in the preparation is not less than 1 × 10⁻⁶. 8 The formulation, described in CFU / mL, further comprises fucoidan oligosaccharides at a concentration of 1-10 g / L. The ratio of the effective viable count of the antagonistic bacterium ZJ11 to the mass concentration of the fucoidan oligosaccharide is (1×10⁻⁶). 8 CFU: (1-10 g).

[0023] Example 2: Isolation and Identification of Antagonistic Bacteria I. Isolation of Antagonistic Bacteria 1. Materials 1.1 Test culture medium and reagents Beef extract peptone solid (NA) medium (Shanghai Bowu Biotechnology Co., Ltd.), beef extract peptone liquid (NB) medium, and MH semi-solid medium: acid-hydrolyzed casein 17.5g, beef meal 2g, soluble starch 1.5g, agar 12g.

[0024] Alginate oligosaccharide (AOS) was purchased from Qingdao Bozhihuili Biotechnology Co., Ltd.

[0025] 1.2 Test strains The konjac corms used are of the Amorphophallus konjac variety. Select corms that are free from mechanical damage, uniform in size, and at normal maturity. Soak them in a 0.2% sodium hypochlorite solution for 2 minutes, then rinse and air dry before use.

[0026] The konjac plants were of the flowering konjac variety, and a total of 50 plants were growing well and uniformly. They were transplanted to the experimental greenhouse for pot cultivation.

[0027] 2. Screening methods for antagonistic strains The inhibition zone experiment employed a double-layer culture medium method. The double-layer culture plate consisted of two layers. The bacteria to be screened were inoculated at three points around the center of a beef extract peptone plate, forming the lower layer. After 1 day of incubation, MH semi-solid medium containing a certain amount of pathogenic MY-G1 bacterial suspension was poured in. The double-layer plates were then incubated at 28°C for 24 hours to observe for the appearance of inhibition zones. The diameter of the inhibition zones was measured using the cross-hatching method, and the transparency of the inhibition zones was recorded. The antagonistic activity was assessed based on the size of the inhibition zones. The experiment was conducted in triplicate.

[0028] 3. Screening results of antagonistic bacteria against konjac soft rot Using *Achromobacter xylose-oxidizing* as the target bacterium, a two-layer culture medium method was employed. Based on the presence or absence of inhibition zones, 12 strains (22% of the tested strains) with significant antagonistic effects were preliminarily screened from the aforementioned culturable rhizosphere bacteria. These strains were ZJ10, ZJ15, ZB1, ZB11, ZB20, ZB7, HJ1, HJ5, HJ15, HJ14, ZJ11, and HB13.

[0029] The 12 antagonistic bacteria in the rhizosphere of Amorphophallus obtained from the initial screening were further screened, and 8 strains with relatively significant antagonistic effects were obtained. The antibacterial effects of the secondary screening are shown in Table 1. Figure 1 As observed in the table and figures, the inhibition zones of all eight bacterial strains were greater than 1.75 cm, with the largest being 2.13 cm. The largest inhibition zone diameter was observed in strain ZJ11, while the smallest was in strain HJ15, with a diameter of 1.75 cm. The transparency of the inhibition zones varied; those of strains ZJ11, HB13, and HJ1 were completely transparent, while those of ZB1, ZJ10, and ZJ15 were relatively clear. Among the eight antagonistic bacteria, ZJ11 showed the best effect against konjac soft rot.

[0030] Table 1. Antibacterial effect of antagonistic bacteria against xylose-oxidizing achromobacterium. II. Identification of antagonistic bacteria ZJ11 1. Materials Test strain: ZJ11, an antagonistic bacterium obtained through screening.

[0031] 2. Identification methods for antagonistic bacteria Methods for identifying the antagonistic bacteria obtained through screening: Morphological identification of bacteria and fungi was performed according to the methods of Zhu Yihui, Guo Xiaoyue, et al. PCR amplification was performed using universal primers ITS1 and ITS4 for fungal ITS sequences and universal primers 27F and 1492R for bacterial 16S DNA sequences. PCR products were sent to a sequencing company for sequencing. After obtaining the sequences, they were aligned using NCBI BLAST, and a phylogenetic tree was constructed using MEGA 7.0 software. Physiological and biochemical determinations were performed according to the methods in the *Handbook of Systematic Identification of Common Bacteria*.

[0032] 3. Identification results of ZJ11, an antagonistic bacterium against konjac soft rot. ZJ11, the most effective antagonistic bacterium against konjac soft rot among eight antagonistic bacteria strains, was identified for future research. Pure culture revealed that its single colonies were raised, milky white, opaque, moist, rounded, and had neat edges. Scanning electron microscopy showed that the cells were short, rod-shaped, non-flagellated, often in pairs or clusters, arranged at an angle, and were Gram-positive bacteria. Figure 2As shown in Table 2, its physiological and biochemical characteristics were determined, including the availability of sucrose and cellulose, and a positive VP test. Molecular identification confirmed that the 16S DNA sequence of strain ZJ11 was consistent with that in GenBank. Bacillus subtilis Homology is as high as 99%, such as Figure 3 As shown, based on morphological and physiological biochemical structural analysis, this strain was identified as Bacillus subtilis.

[0033] Table 2 Physiological and biochemical characteristics of antagonistic bacteria ZJ11 Example 3: Determination of the antibacterial spectrum of antagonistic bacteria ZJ11 1. Materials Broad-spectrum antibacterial pathogens tested: Stagonosporopsis vannaccii Grape stem blight pathogen ( Didymella glomerata Fusarium solani () Fusarium solani ), Fusarium proliferatum, Alternaria alternata, Fusarium oxysporum ( Fusarium proliferatum ), fine-particle-alternating spores ( Alternaria tenuissima ), black occulta ( Epicoccum nigrum ), Winter wheat thorn disc spores ( Colletotrichum liriopes All of the above strains were isolated, identified and preserved in the laboratory.

[0034] Antagonistic bacteria: Bacillus subtilis ZJ11 ( Bacillus subtilis ).

[0035] 2. Methods for determining the antibacterial ability of antagonistic bacteria against different pathogens. The inhibitory activity of antagonistic bacteria was determined by confronting and culturing antagonistic bacteria and various tested pathogens. Using an 8mm diameter sterilized pipette tip, bacterial plugs of each pathogen were placed in the center of a PDA plate. Antagonistic bacteria strain ZJ11 was streaked along three sides, while the control was left unstreaked. All plates were incubated at 28℃ until the control hyphae reached the edge of the plate. The distance between the antagonistic bacteria and the edge of the fungal hyphae was measured in the experimental group. The experiment was repeated three times, and the inhibition rate was calculated.

[0036] Inhibition rate = (Coronary diameter of control group - Coronary diameter of treatment group) / (Coronary diameter of control group - Diameter of inoculated block) × 100%.

[0037] 3. Results of the antibacterial ability test of antagonistic bacteria ZJ11 against different pathogens The experimental results are shown in Table 3 and Figure 4As shown, Bacillus subtilis ZJ11 exhibited inhibitory effects on all tested pathogens, with significant differences in the inhibitory effects, demonstrating that Bacillus subtilis ZJ11 has broad-spectrum antibacterial activity. Among the tested pathogens, it showed the strongest inhibitory effect against *Botrytis cinerea*, with an inhibition rate exceeding 77.41%. It also showed strong inhibitory effects against other pathogens, with inhibition rates all exceeding 60%.

[0038] Table 3. Antibacterial activity of Bacillus subtilis ZJ11 against different pathogens Example 4: Determination of the control effect of antagonistic bacteria ZJ11 on konjac soft rot 1. Control efficacy test of detached bulbs 1.1 Materials Test subject: Fresh konjac tubers, cut into pieces approximately 5cm × 5cm × 1cm.

[0039] Tested strains: pathogenic bacterium MY-G1, antagonistic bacterium ZJ11.

[0040] 1.2 Method for determining the control efficacy of fermentation broths with different ratios of antagonistic bacteria against soft rot of detached bulbs Select fresh konjac corms, soak them in 75% alcohol for 20 seconds, rinse with sterile water, and cut them into pieces approximately 5cm × 5cm × 1cm using a sterile knife. Create a forked indentation in the center of each konjac piece. Inject bacterial solution using a sterile syringe, and place the konjac pieces in a sterile petri dish lined with folded filter paper for moist incubation. The treatment setup is as follows: a: Blank control, only wounds are created, without any other treatment; b: Inject 1 ml of a mixture of pathogenic bacteria and ZJ11 solution (ratio 1:4); c: Inject 1 ml of a mixture of pathogenic bacteria and ZJ11 solution (ratio 1:3); d: Inject 1 ml of a mixture of pathogen and ZJ11 (ratio 1:2); e: Inject 1 ml of a mixture of pathogenic bacteria and ZJ11 solution (ratio 1:1); f: Inject 1 ml of pathogenic bacterial solution; Six replicates per treatment. Keep the area moist, observe and record the disease progression after 5-7 days.

[0041] The disease severity of konjac corms was graded according to the method of Dai Xuefeng et al., and the disease index was calculated as follows: Disease index = [∑ (disease grade value × number of disease grade plants)] / (highest disease grade value × number of plants surveyed) × 100 Prevention efficacy = (Control disease index - Treatment disease index) / Control disease index × 100 1.3 Results of the experiment on the action of antagonistic bacteria ZJ11 on konjac corms The soft rot pathogen MY-G1 and Bacillus subtilis ZJ11 were mixed in different ratios and inoculated into 1 ml of solution onto tuber slices. Two controls were established: no inoculation with any liquid and inoculation with sterile water. Four experimental groups with different inoculation ratios were formed. The results showed that... Figure 5 As shown in Table 4, tubers inoculated with sterile water ( Figure 5 b) Showing mild oxidative discoloration, tubers completely inoculated with MY-G1 bacterial solution ( Figure 5 f) This manifests as severe rot, with a disease index reaching 86.66. When the tuber is inoculated with MY-G1 and ZJ11 bacterial suspensions at a ratio of 1:1 ( Figure 5 e), the control efficacy was only 7.6%; when the ratio of MY-G1 and ZJ11 bacterial suspensions inoculated into the tubers was 1:2 ( Figure 5 d), the control efficacy was 23.09%. When the ratio of MY-G1 and ZJ11 bacterial suspensions inoculated on the tubers was 1:3 ( Figure 5 c), with a control efficacy of 42.30%. The results showed that Bacillus subtilis ZJ11 had a certain inhibitory effect on konjac soft rot, and the higher the proportion of ZJ11 bacterial solution, the better the inhibitory effect.

[0042] Table 4. Control efficacy of ZJ11 against soft rot of detached konjac corms 2. Potted plant efficacy test (and the effect of compounding with brown algae oligosaccharide AOS) 2.1 Materials Test subject: Healthy Amorphophallus konjac plant.

[0043] Test preparations: ZJ11 bacterial suspension (10^8 cfu / mL), alginate oligosaccharide (AOS) solution (5 g / L), and ZJ11 + AOS mixture.

[0044] 2.2. The effect of antagonistic bacteria on potted konjac soft rot Healthy Amorphophallus konjac plants growing under the same conditions were treated with the following root irrigation methods: a) ZJ11 + AOS solution (10⁸ CFU / mL, 5 g / L), b) ZJ11 bacterial suspension (10⁸ CFU / mL), c) AOS solution (5 g / L), and d) sterile water. Each pot of Amorphophallus konjac was irrigated with 100 mL of the solution, with three replicates per treatment. 24 hours after root irrigation, a small incision was made at the base of the stem near the corm of the Amorphophallus konjac plant, and 1 mL of pathogenic bacterial suspension (10⁸ CFU / mL) was injected via needle puncture. The plants were then allowed to grow naturally in a greenhouse for one week. The incidence and severity of the disease were observed and recorded, and the length of lesions was measured.

[0045] The length of lesions on konjac stems was graded according to the method of Cui Shuang et al.: Grade 0, no lesion spread at the needle puncture point; Grade 1, lesion length L≤1cm; Grade 3, 1cm<lesion length L≤3cm; Grade 5, 3cm<lesion length L≤5cm; Grade 7, lesion length L>5cm; Grade 9, stem is completely rotten.

[0046] The disease index and prevention efficacy are calculated as follows: Disease index = [∑ (disease grade value × number of disease grade plants)] / (highest disease grade value × number of plants surveyed) × 100; Prevention efficacy = (Control disease index - Treatment disease index) / Control disease index × 100.

[0047] 2.3 Results of the experiment on the effect of antagonistic bacteria ZJ11 on konjac plants The experiment involved root irrigation of the plants with sterile water, alginate oligosaccharide solution (AOS), Bacillus subtilis ZJ11 fermentation broth, and a mixture of Bacillus subtilis ZJ11 fermentation broth and alginate oligosaccharide nutrient solution. Twenty-four hours after root irrigation, pathogenic bacteria were injected into the base of the konjac plant stems using a needle inoculation method. Results were obtained after 5 days of growth. Figure 6 As shown, the results revealed that plants watered only with sterile water exhibited significantly more severe stem disease, with an incidence rate of 100% and a disease index of 76.36. The disease progression was also faster than in other treatments, with leaves turning yellow and wilting after infection. Plants watered with brown algae oligosaccharide nutrient solution had an incidence rate of 88.89%, a disease index of 73.19, and a control efficacy of only 4.03%. The other two groups of konjac plants, although also affected by Bacillus subtilis ZJ11 fermentation liquid, showed improvement in disease severity. Figure 6 (abc) However, the disease progression rate was slower in the group treated with ZJ11 fermentation liquid than in the group not treated with ZJ11. On day 5, the disease incidence rate of plants treated with only ZJ11 was 55.55%, and the disease index was 48.14. The disease incidence rate of plants treated with ZJ11+AOS was 44.44%, and the disease index was 40.74, as shown in Table 5. After about one week of continued cultivation, the lesions in the plants treated with ZJ11+AOS expanded slightly but then stopped expanding. The konjac plants treated with only ZJ11 continued to grow, but the rate of lesion expansion at the wound site was much lower than in the groups treated with sterile water and AOS nutrient solution. The stems of the plants treated with only sterile water completely rotted and collapsed. This indicates that the disease resistance of plants treated with both ZJ11 and AOS is greater than that of plants treated with only ZJ11 fermentation liquid, while the differences between the treatments treated with only AOS nutrient solution and sterile water are not significant.

[0048] Table 5. Control efficacy of ZJ11 against soft rot in potted plants of konjac. Example 5: Determination of changes in the activity of resistance-related enzymes induced by antagonistic bacteria ZJ11 in konjac. 1. Test konjac: Plants treated with the same potted control efficacy test.

[0049] The following indicators were measured: peroxidase (POD), polyphenol oxidase (PPO), catalase (CAT), malondialdehyde (MDA), hydrogen peroxide (H2O2), total flavonoids (TF), and total phenols (TP).

[0050] 2. Determine the changes in the activity of enzymes related to resistance induced by antagonistic bacteria in Amorphophallus konjac under different treatments.

[0051] The same root irrigation treatment and pathogen inoculation were performed as described in the potted plant efficacy test. Leaf samples were taken at five time points after inoculation: 0d (2h), 1d, 3d, 5d, and 7d. The activities and contents of enzymes and substances related to plant disease resistance, such as peroxidase (POD), polyphenol oxidase (PPO), catalase (CAT), malondialdehyde (MDA), hydrogen peroxide (H2O2), total flavonoids (TF), and total phenols (TP), were measured.

[0052] The activities of POD, PPO, and CAT, and the contents of TP, TF, H2O2, and MDA in leaf tissues were determined using kits, following the instructions provided by the respective kit manufacturers.

[0053] 3. Effects of antagonistic bacteria ZJ11 on the activity of resistance-related enzymes in Amorphophallus konjac (1) Changes in peroxidase (POD) activity Further analysis was conducted to determine the activity and content of resistance-related enzymes in konjac plants treated with Bacillus subtilis ZJ11 fermentation broth. Figure 7 It can be seen that the changes in peroxidase activity in the four different treatment groups showed a clear pattern. The activity first decreased, then increased, reaching a maximum before starting to decrease again. B subtilis can be used alone in groups, AOS, and B The subtilis combination group reached the highest enzyme activity on day 5 (123.97 min / g FW, 101.20 min / g FW), while the control group reached the maximum on day 3 (72.63 min / g FW).

[0054] (2) Changes in polyphenol oxidase (PPO) activity from Figure 8 As can be seen, the PPO activity in the four different treatment groups did not initially increase and then decrease. PPO activity increased with increasing disease severity, but began to decrease as the lesions on the konjac stems expanded. The polyphenol oxidase activity in the konjac plants gradually increased one day after inoculation, reaching its maximum value (963 min / g FW) on the third day, and then showed a downward trend. BSubtilis individual irrigation group, AOS and B The PPO activity in the subtilis mixed treatment group was significantly higher than that in the other two groups on days 3 and 5. On day 3 B Subtilis individual irrigation group, AOS and B The PPO activity of the subtilis mixed treatment groups showed significant differences.

[0055] (3) Changes in catalase (CAT) activity like Figure 9 As shown, the CAT activity in the antagonistic bacteria and AOS mixed treatment group was higher than that in other treatment groups. The enzyme activity in the four different treatment groups initially increased and then decreased. Specifically, the AOS group showed a decrease in activity on day 3 followed by a rebound. The maximum CAT activity in the control group occurred on day 3, while the CAT activity in the konjac konjac mixed treatment group reached its maximum on day 5, which was 1.14 times, 1.32 times, and 1.37 times higher than the other three groups, respectively.

[0056] (4) Changes in total phenol (TP) content The results of the TP content change are as follows Figure 10 From day 0 to 1, the TP content initially decreased, then increased after day 1, reaching its highest level in the antagonistic bacteria and AOS mixed treatment group at day 5. By day 7, the TP content in all four treatments had decreased. The TP content in the antagonistic bacteria and AOS mixed treatment group on day 5 was 265.62 mg / g FW, significantly different from the other three groups. On day 7, the TP content in the control group and the AOS-only treatment group decreased to 129.29 mg / g FW and 140.52 mg / g FW, respectively.

[0057] (5) Changes in total flavonoid (TF) content Depend on Figure 11 It was found that the differences among the four different treatment groups were significant after day 3. The total flavonoid content decreased first from day 0 to day 1, and then increased after day 1. On day 5, the total flavonoid content reached its highest in the antagonist-AOS mixed treatment group and the antagonist-only treatment group, with flavonoid content being 1.59 times and 1.31 times that of the AOS-only group, respectively; and 1.64 times and 1.35 times that of the control group, respectively.

[0058] (6) Changes in hydrogen peroxide (H2O2) content like Figure 12As shown, at 2 hours, the H2O2 content in all four treatment groups was at a low level, gradually increasing over time. Significant differences in H2O2 content were observed among the treatments, but the overall trend was roughly the same. Throughout the process, the H2O2 content in the mixed treatment group of antagonistic bacteria and AOS, and the antagonistic bacteria-only treatment group, was lower than that in the AOS-only group and the control group. The H2O2 content in the AOS-only treatment group and the control group reached its maximum on day 7 (20.21 μmol / g FW and 20.23 μmol / g FW, respectively), showing significant differences from the other two groups.

[0059] (7) Changes in malondialdehyde (MDA) content like Figure 13 As shown, the MDA content of konjac in the antagonistic bacteria and AOS mixed treatment group was generally low. The MDA content steadily increased, reaching its maximum on day 7; on day 7, the MDA content of the control group, the AOS-only treatment group, and the antagonistic bacteria-only treatment group were 1.60 times, 1.35 times, and 1.48 times higher than that of the antagonistic bacteria and AOS mixed treatment group, respectively. The results indicate that konjac plants infected with soft rot fungi can rapidly accumulate more malondialdehyde (MDA), a product of membrane lipid peroxidation, while antagonistic bacteria... B The combination of subtilis and AOS can effectively reduce the accumulation of malondialdehyde (MDA) and decrease the damage to konjac plants caused by soft rot disease.

[0060] Example 6: Whole genome sequencing of ZJ11 Whole-genome sequencing of Bacillus subtilis strain ZJ11 was performed to reveal its antagonistic and growth-promoting abilities at the molecular level.

[0061] 1. Materials and Methods: Bacillus subtilis strain ZJ11 was inoculated into LB liquid medium and cultured overnight at 28°C and 120 rpm. The cells were then collected by centrifugation at 4°C and 8000 rpm for 10 min, the supernatant was discarded, and the cells were washed three times with PBS buffer. The resulting bacterial cells, along with the centrifuge tube, were immersed in liquid nitrogen and frozen completely for 1 hour. After sealing with dry ice, the cells were sent to Shanghai Ling'en Biotechnology Co., Ltd. for whole-genome sequencing. Sequencing results were analyzed using the Ling'en Biotechnology Cloud Platform.

[0062] 2. Whole genome sequencing results: Whole-genome sequencing results showed that the chromosome size of strain ZJ11 was 4,118,343 bp, with an average GC content of 44.4%. The genome encoded a total of 4,805 genes, including 86 tRNA genes and 27 rRNA genes, of which 9 were 5S rRNA genes, 9 were 16S rRNA genes, and 9 were 23S rRNA genes. Functional annotations were performed using NR, Swiss-Prot, eggNOG, KEGG, GO, CARD, and CAZy, resulting in 4,523, 1,711, 3,736, 2,387, 4,364, 183, and 110 annotated genes, respectively. Figure 14 As shown.

[0063] 3. Analysis of the antibacterial and growth-promoting mechanism of Bacillus subtilis strain ZJ11: Secondary metabolites are the core effector factors for Bacillus to exert its biocontrol effects. They can regulate plant health through direct inhibition of harmful microorganisms, induction of plant systemic resistance, and enhancement of rhizosphere competition, thereby achieving disease control. Based on the antiSMASH prediction results (see Table 6), Bacillus amyloliquefaciens strain ZJ11 encodes a total of 12 secondary metabolite gene clusters, covering multiple core biocontrol-related types such as polyketide synthase (PKS), nonribosomal peptide synthase (NRPS), terpenes, and thioether peptides. This suggests that it has rich metabolic potential and strong biocontrol application prospects, providing a solid genomic foundation for its control of konjac soft rot and promotion of konjac growth.

[0064] Comprehensive gene cluster function analysis showed that, for example Figure 15 As shown, strain ZJ11 can directly target and kill or inhibit plant pathogens by synthesizing a group of synergistic antibacterial compounds, constructing a core antibacterial defense line: including 1-carbapenem-2-ene-3-carboxylic acid (carbapenem antibiotic) encoded by region 1, which exerts a broad-spectrum antibacterial effect by interfering with bacterial cell wall synthesis; fengycin (encoded by region 3) and surfactantin (encoded by region 6) form a synergistic antibacterial network, with the former mainly targeting fungal cell membranes and disrupting their structural stability, and the latter achieving broad-spectrum inhibition against bacteria, fungi, and mycoplasma, with their synergistic effect significantly enhancing the antibacterial range and efficacy; bacillaene (encoded by region 4) serves as a core antibacterial substance, targeting Gram-negative bacteria by inhibiting bacterial protein synthesis, while also having the function of disrupting biofilms, enhancing the strain's competitive advantage against pathogens; in addition, bacilysin (encoded by region 9) and subtilosin (encoded by region 10) are also involved. Subtilisin A further broadens the antibacterial spectrum. The former specifically inhibits fungal cell wall synthesis, while the latter achieves broad-spectrum control of Gram-positive / negative bacteria and anaerobic bacteria by disrupting cell membrane permeability.

[0065] Meanwhile, strain ZJ11 possesses highly efficient rhizosphere competition and colonization capabilities, ensuring the stable performance of its biocontrol function: bacillibactin encoded by region 11, as a catechol siderophore, can efficiently chelate Fe³⁺ from the environment. + It is then transported into the cells, where it competes for nutrients in the low-iron rhizosphere environment, significantly enhancing the survival and competitiveness of the strain.

[0066] Furthermore, strain ZJ11 possesses significant growth-promoting potential, achieving a synergistic effect of "control-promotion" through regulation of plant growth: multiple terpenoid-related gene clusters (regions 2, 5, and 12) jointly construct a growth-promoting metabolic network. Regions 2 and 5 may synthesize plant hormone precursors such as gibberellins or terpenoid signaling molecules, directly stimulating root and leaf development. Region 12, as a general terpenoid precursor synthesis cluster, provides the basis for the synthesis of various active terpenoids, indirectly ensuring the realization of growth-promoting functions. Notably, fengycin encoded by region 3, while inhibiting bacterial growth, can also induce systemic resistance in plants and regulate plant hormone pathways, further enhancing the growth-promoting effect.

[0067] Table 6 Functional analysis of ZJ11 secondary metabolite gene clusters Through a multi-dimensional synergistic mechanism of "core inhibition - rhizosphere competition - colonization protection - growth promotion," strain ZJ11 can not only achieve highly efficient control of plant pathogens but also significantly promote the healthy growth of host plants, demonstrating excellent potential as a green biocontrol agent. Based on the genome-wide secondary metabolite gene cluster prediction results of Bacillus amyloliquefaciens strain ZJ11, its potential mechanism of action in controlling konjac soft rot and promoting konjac growth is summarized as follows: A. Direct antibacterial mechanism: multi-level synergistic defense based on specific gene clusters.

[0068] The ZJ11 strain constructs a multi-target, synergistic direct antibacterial system through multiple gene clusters encoding highly effective antibacterial substances, precisely targeting the pathogen of konjac soft rot (Gynostemma pentaphyllum). Pectobacterium Gram-negative pathogens such as spp.

[0069] (1) Core bactericidal gene cluster and its products The strain possesses multiple key gene clusters that directly combat Gram-negative bacteria, forming a primary killing system against the pathogen causing soft rot in konjac. Region 4 synthesizes the antibacterial polyketide bacitracin, which exerts a potent killing effect by irreversibly inhibiting pathogen protein synthesis. Region 10 encodes subtilisin A, whose unique membrane-action mechanism effectively disrupts the integrity of the pathogen's cell membrane, leading to leakage of cell contents and cell death. The combined use of the products from these two gene clusters, targeting different mechanisms of protein synthesis inhibition and cell membrane disruption, significantly enhances the killing effect against the soft rot pathogen and helps reduce the risk of drug resistance.

[0070] (2) Synergistic effect of auxiliary antibacterial gene clusters In addition to the core bactericidal components, multiple gene clusters assist in antibacterial activity through different mechanisms, enriching the direct attack strategy library. Region 1 encodes 1-carbapenem-2-ene-3-carboxylic acid (carbapenem antibiotics), which exerts a broad-spectrum antibacterial effect by interfering with pathogen cell wall synthesis, further enhancing the inhibitory effect on soft rot pathogens; Region 3 encodes fenamidcin and Region 6 encodes surfactant, which form a synergistic antibacterial network. The former mainly targets fungal pathogens that may co-infect, while the latter achieves broad-spectrum inhibition of bacteria and fungi, creating comprehensive antibacterial protection for konjac; Region 9 encodes bacilysin, which acts on fungal pathogens in complex infections through a mechanism that specifically inhibits fungal cell wall synthesis, helping to improve the overall disease prevention effect.

[0071] (3) Iron competition repressor gene cluster Region 11 encodes the siderophores responsible for synthesizing catechol-type siderophore-carrying bacteria. The expression of this gene cluster enables the ZJ11 strain to have a super strong iron chelating ability in the low iron environment of the rhizosphere. By efficiently depriving harmful microorganisms such as konjac soft rot pathogens of the key nutrient element iron, it achieves strong indirect antibacterial effect from the perspective of nutrient competition, providing auxiliary guarantee for the direct antibacterial effect.

[0072] B. Mechanism of action in promoting konjac growth: direct stimulation and ecological regulation through the synergistic effect of multiple gene clusters.

[0073] The ZJ11 strain, through its metabolite gene clusters, achieves direct stimulation of konjac growth and indirect improvement of the rhizosphere microenvironment, forming a synergistic effect of "disease prevention" and "growth promotion," thus helping konjac grow healthily.

[0074] (1) Gene clusters that directly promote the synthesis of growth-promoting substances Multiple terpenoid-related synthetic gene clusters (Regions 2, 5, and 12) predicted in the genome collectively construct a direct growth-promoting metabolic network. Regions 2 and 5 are terpenoid synthetic gene clusters, whose products may act as plant hormone (such as gibberellin) analogs or signaling molecules, directly participating in the regulation of physiological processes such as root development and leaf growth in konjac. Region 12 is a terpenoid precursor synthetic gene cluster, capable of synthesizing universal precursors of various terpenoid active substances, providing a foundation for the synthesis of direct growth-promoting substances and synergistically promoting konjac plant growth. Furthermore, the fenamidcin encoded by Region 3, while inhibiting bacteria, can also induce systemic resistance in konjac and regulate plant hormone pathways, further enhancing the growth-promoting effect.

[0075] (2) Gene clusters for rhizosphere colonization, resistance induction and microecological regulation The synthesis of surfactants encoded by Region 6 is a key regulatory factor. Its products not only promote the colonization of the ZJ11 strain on the root surface of konjac, but also act as elicitors to induce systemic resistance in konjac, enhancing its resistance to diseases such as soft rot and ensuring healthy plant growth. More importantly, through the combined effects of antibacterial, competitive, and colonization mediated by multiple gene clusters including Regions 3, 4, 6, 9, 10, and 11, the ZJ11 strain can effectively reshape the rhizosphere microbiota of konjac, inhibit the proliferation of harmful pathogens, and create a healthier, more nutrient-absorbable microecological environment for the konjac root system, thus achieving an indirect but sustainable growth-promoting effect.

[0076] Example 7: Field efficacy test of antagonistic bacterium ZJ11 1. Materials Experimental location: Amorphophallus konjac planting base, continuous cropping field, Shede Village, Qiubei County, Wenshan City, Yunnan Province.

[0077] Test preparation: ZJ11 bacterial fermentation broth (concentration 1×10⁻⁶) 9 CFU / mL); 20% agricultural streptomycin soluble powder.

[0078] 2. Field control efficacy An efficacy test was conducted in a continuous cropping field of Amorphophallus titanum in Shede Village, Qiubei County, Wenshan City, Yunnan Province. Three treatments were set up: a) antagonistic bacteria treatment, b) sterile water treatment, and c) agricultural streptomycin treatment. Treatment a received 500 mL of the antibacterial solution per plant at a concentration of 10%. 9 CFU / mL, treatment b was irrigated with an equal volume of sterile water, repeated 3 times, with plots arranged completely randomly, each plot area 8m². 2One week before the onset of the disease, water the roots once, then every 15 days for a total of 3 waterings. After 30 days, investigate and calculate the incidence rate, disease index, and control efficacy. The grading standard for konjac soft rot is based on the grading standard of Wu Xu et al., as follows: Grade 0: No symptoms of disease, normal growth; Grade 1: Water-soaked lesions appear on the stems or tubers, with a lesion area <2cm, and the leaves begin to turn slightly yellow; Grade 2: Water-soaked lesions with an area of ​​2cm < lesion < 5cm, the leaves are moderately yellow and accompanied by water loss and wrinkling; Grade 3: Water-soaked lesions with an area of ​​5cm < lesion < 10cm, the leaves are severely dehydrated and shrunken, and some stems show slight lodging; Grade 4: The stems are completely rotten, the plants are lodged, and the tubers are rotten and smelly.

[0079] 3. Evaluation of the control efficacy of antagonistic bacterium ZJ11 against soft rot of Amorphophallus konjac in the field. like Figure 16 As shown, compared with the control group in the naturally grown konjac continuous cropping field, the disease incidence of konjac plants treated with antagonistic bacteria ZJ11 was significantly lower in the control group. The disease index of the control group was 61.98, and water-soaked lesions were visible on the konjac stems, with some plants lodging, indicating severe infection by the konjac soft rot pathogen. Figure 4-15 The disease index of the treatment group irrigated with antagonistic bacteria ZJ11 was 38.44, which was significantly lower than that of the control group (p<0.01), indicating a better control effect with a control efficacy of 37.98%. The results show that antagonistic bacteria ZJ11 has a significant control effect on konjac soft rot, and its control efficacy is close to or better than that of conventional chemical agents such as agricultural streptomycin. Therefore, it can be used as an effective strain for the biological control of konjac soft rot.

[0080] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. An antagonistic bacterium capable of inhibiting and controlling konjac soft rot ( Bacillus subtilis ZJ11, characterized in that: The strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO. M 2023720, located at Wuhan University, Wuhan, China, on May 10, 2023.

2. The antagonistic bacterium ZJ11 capable of inhibiting and controlling konjac soft rot according to claim 1, characterized in that: The strain exhibits strong antagonistic activity against *Achromobacter xylose oxidizing*, the pathogen causing soft rot of konjac, with an inhibition zone diameter of not less than 2.00 cm.

3. The antagonistic bacterium ZJ11 capable of inhibiting and controlling konjac soft rot according to claim 2, characterized in that: The 16S rRNA gene sequence of the strain has at least 99% homology with SEQ ID NO:1, therefore it was identified as... Bacillus subtilis .

4. The antagonistic bacterium ZJ11 capable of inhibiting and controlling konjac soft rot according to claim 3, characterized in that: The strain's whole genome contains gene clusters encoding fengycin, surfactantin, and bacillaene.

5. A microbial preparation of ZJ11 antagonistic bacteria capable of inhibiting and controlling konjac soft rot, comprising live cells, spores or fermentation broth of the antagonistic bacteria ZJ11 as described in claim 4, and an agriculturally acceptable carrier.

6. The ZJ11 antagonistic microbial preparation for inhibiting and controlling konjac soft rot according to claim 5, characterized in that: The effective viable count of the antagonistic bacterium ZJ11 in the preparation is not less than 1×10⁻⁶. 8 CFU / mL.

7. The ZJ11 antagonistic microbial preparation for inhibiting and controlling konjac soft rot according to claim 6, characterized in that: The formulation further comprises fucoidan oligosaccharides, wherein the concentration of the fucoidan oligosaccharides in the formulation is 1-10 g / L, and the ratio of the effective viable count of the antagonistic bacterium ZJ11 to the mass concentration of the fucoidan oligosaccharides is (1×10⁻⁶ g / L). 8 CFU: (1-10 g).

8. The application of the antagonistic bacterium ZJ11 according to any one of claims 1 to 4 or the microbial preparation according to claim 5 or 7 in inducing disease resistance in konjac, characterized in that, The disease resistance is manifested by increased activity of peroxidase (POD), polyphenol oxidase (PPO), and catalase (CAT) in konjac plants.

9. The application of the antagonistic bacteria ZJ11 according to any one of claims 1 to 4 or the microbial preparation according to claim 5 or 7 in promoting the growth of konjac.

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