Bacterium strain antagonizing soft rot of xanthosoma sagittifolium, biocontrol agent and application thereof

Abstract

This invention belongs to the field of gene technology and discloses a bacterial strain B11 antagonistic to soft rot disease of Amorphophallus muelleri. Isolated from the rhizosphere soil of Amorphophallus muelleri, it was identified as the golden subspecies of *Pseudomonas aeruginosa*. This strain effectively inhibits the growth of the soft rot pathogen Pcc, reducing the incidence and disease index of soft rot in Amorphophallus muelleri. It also possesses the ability to dissolve organophosphates and produce siderophores, and exhibits broad-spectrum antagonistic activity against various fungal diseases of Amorphophallus muelleri, including white mold, leaf spot, and dry rot. Whole-genome analysis shows that the B11 genome is 6.77 Mb in size, with a GC content of 62.81%, encoding 5,964 genes, including gene clusters related to antibacterial activity such as phenazine, nitropyrrolizin, and siderophores. This strain has good development and application potential in the control of soft rot in Amorphophallus muelleri.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of gene technology, and particularly relates to a bacterial strain antagonistic to soft rot of Amorphophallus konjac, a biocontrol agent, and its application. Background Technology

[0002] Konjac, also known as devil's tongue, has over 220 known varieties worldwide. Its tubers are rich in glucomannan, offering irreplaceable applications in food processing, pharmaceuticals, and biochemicals. With continuously growing market demand, konjac cultivation areas are expanding. However, limited land resources and the widespread use of continuous cropping have led to increasingly prominent problems such as soil pathogen accumulation and soil microecological imbalance, severely hindering the sustainable development of the konjac industry. Konjac soft rot, caused by *Pectinobacillus carotenoides*, is a catastrophic soil-borne disease. It not only damages leaves, petioles, and tubers during cultivation, causing plant wilting and lodging, but also infects corms during storage, leading to rotting and spoilage, significantly reducing yield and quality. Currently, the control of konjac soft rot remains a serious challenge. Control still relies mainly on chemical agents, but long-term use of chemical agents has low efficacy against soil-borne diseases, pollutes the soil, threatens human health, and easily leads to drug resistance. In contrast, biological control has advantages such as high efficiency, low toxicity, no residue, and low likelihood of developing drug resistance, providing an important direction and potential for overcoming the bottleneck in the control of konjac soft rot.

[0003] Plant rhizosphere growth-promoting bacteria (PGPRs) can effectively colonize the plant rhizosphere, promoting plant growth and reducing disease incidence. PGPRs mainly originate from the genera *Pseudomonas* and *Bacillus*, inhibiting pathogens through mechanisms such as antibiotic secretion, induction of systemic toxicity, and competition for nutrients and the ecological environment. The *Pseudomonas* species that have been extensively studied include *Pseudomonas aeruginosa*, *Pseudomonas fluorescens*, *Pseudomonas aeruginosa*, and *Pseudomonas putida*. Among them, *P. chlororaphis* has become a focus of research as the most promising biocontrol bacterium, and it has been divided into five subspecies based on genetic and phenotypic characteristics: *P. chlororaphis subsp. aurantiaca*, *P. chlororaphis subsp. aureofaciens*, *P. chlororaphis subsp. chlororaphis*, *P. chlororaphis subsp. phenazini*, and *P. chlororaphis subsp. piscium*. *P. chlororaphis* directly antagonizes pathogen growth and activates plant defenses by producing metabolites such as phenazine, nitropyrrolizin, resorcinol, hydrogen cyanide, cyclic peptides, and siderophores. Currently, there is a wealth of research on *P. chlororaphis*. In Le et al.'s report, the phenazine-producing *Pseudomonas aeruginosa* Phz24 significantly inhibited the mycelial growth of *Sclerotium sclerotiorum* in peanuts, thereby reducing the occurrence of peanut white mold disease. *P. chlororaphis subsp. aurantiaca* SPS-41 exhibits broad-spectrum antifungal activity; its volatile organic compounds significantly inhibit the pathogen *Ceratocystis fimbriata* in postharvest sweet potatoes, and also show strong nematicidal activity. *P. chlororaphis subsp. aurantiaca* Pc01-6 significantly inhibits *Salvia miltiorrhiza* root rot by producing secondary metabolites such as cyclic ester peptides and nitropyrrolizin.

[0004] To date, reported biocontrol bacteria for konjac soft rot both domestically and internationally mainly focus on Bacillus, Actinomycetes, and Bacillus lysate. Research on the use of *P. chlororaphis* for the biocontrol of konjac soft rot is scarce, and its mechanism of action remains unclear. Based on this research background and gaps, this invention aims to screen strains with highly efficient antagonistic activity from the rhizosphere soil of healthy bulbil-grown konjac plants, systematically determine the growth-promoting and antibacterial activities of these strains, and evaluate their control effects on konjac soft rot. Simultaneously, using whole-genome sequencing technology, the genomic characteristics, functional gene annotations, and biosynthetic gene clusters (BGCs) of the strains will be analyzed to reveal their possible disease control mechanisms. The research results will provide high-quality strain resources and theoretical basis for the development of biocontrol agents for konjac soft rot. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a bacterial strain antagonistic to soft rot of Amorphophallus konjac, a biocontrol agent, and its application.

[0006] This invention is achieved by developing a bacterial strain antagonistic to soft rot disease in Amorphophallus konjac. The bacterial strain B11 is *Pseudomonas sessilinigenes*, a subspecies of *Pseudomonas* sessilinigenes, and was deposited on December 3, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36858. This strain can inhibit the growth of the soft rot pathogen *Pcc*, while simultaneously reducing the incidence and disease index of soft rot in Amorphophallus konjac.

[0007] Furthermore, the identification of strain B11 specifically includes:

[0008] (1) Morphological identification

[0009] Use sterile toothpicks to pick up single colonies of strain B11 and inoculate them onto NA solid medium. Incubate at 28 ℃ for 48 h; observe the colony morphology characteristics.

[0010] (2) Molecular biological identification

[0011] DNA was extracted from strain B11 following the steps outlined in the FastDNA® SPIN kit for soil DNA extraction kit. DNA concentration and purity were assessed using NanoDrop 2000, and DNA extraction quality was determined by 1% agarose gel electrophoresis. Universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGG) were used.

[0012] PCR amplification was performed using GTWTCTAAT-3'. The PCR amplification reaction system was 20 μL: 4 μL of 5×TransStartFastPfu buffer, 2 μL of 2.5 mmol / L dNTPs, 0.8 μL each of 5 uM forward and reverse primers, 0.4 μL of TransStartFastPfu DNA polymerase, 0.2 μL of LBSA, 10 ng of DNA template, and sterile water to a final volume of 20 μL.

[0013] Amplification program: 95℃ pre-denaturation for 3 min, 27 cycles, 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, and final 72℃ stabilization extension for 10 min; the amplified products were sent to Shanghai Meiji Biotechnology Co., Ltd. for sequencing on the Illumina Miseq PE300 platform; the obtained 16S rRNA gene sequence was uploaded to the NCBI GenBank database for BLAST comparison, and strain sequences with high homology were screened. Multiple sequence matching was performed using the Kimura2-Parameter Distance model in MEGA software, and a phylogenetic tree was constructed using the neighbor-joining method. The confidence of each branch of the phylogenetic tree was tested with 1000 replicates.

[0014] (3) Growth-promoting characteristics of strain B11

[0015] The strain B11 was reactivated on NA solid medium. Single colonies were picked and inoculated onto organophosphate, Alexandrov, CAS, and Assab agar plates, respectively, and incubated at 28°C for 2-5 days. The presence of a clear zone around the colony was observed.

[0016] (4) Determination of the antibacterial spectrum of strain B11

[0017] Eight pathogenic fungi were cultured at 28℃ for 4-7 days. Mycelial discs with a diameter of 5 mm were collected from the edge of the plate and inoculated into the center of PDA plates. Four spots were set at 90℃ and 2.5 cm away from the center. The antagonistic strain B11 was inoculated into the four spots. The control pathogens were cultured separately. Each treatment was replicated three times and cultured at 28℃ for 5-7 days. When the control strains grew to the edge of the plate, the diameter of the pathogens in each treatment was measured and the inhibition rate was calculated.

[0018] Inhibition rate (%) = (CK colony diameter - treated colony diameter) / (CK colony diameter) × 100%; (3)

[0019] (5) Pot test of the control efficacy of strain B11

[0020] One-year-old healthy Amorphophallus konjac bulbs were selected and planted in B260 plastic pots containing sterilized soil and seedling substrate at a ratio of 3:1, with one plant per pot and nine pots per group. After the konjac leaves fully unfolded, 27 healthy plants with roughly the same growth condition were selected for subsequent experiments. Group A1 received 30 mL / pot of Pcc bacterial solution (nine pots); Group A2 was first irrigated with 50 mL / pot of B11 bacterial solution, then irrigated with 30 mL / pot of Pcc bacterial solution (nine pots); Group A3 received 50 mL / pot of B11 bacterial solution (nine pots). Irrigation was performed every 7 days, for a total of two irrigations. Disease incidence was observed daily during the experiment, and normal water and fertilizer management was maintained. After d, the incidence of disease was statistically analyzed and the disease index and relative control efficacy were calculated. See formula (2) and grading standards: Grade 0, the whole plant is normal and there are no diseased parts; Grade 1, 1 / 3 of the leaves turn black and rot, and the base of the petiole does not rot and droop; Grade 2, 1 / 3-1 / 2 of the leaves turn black and rot, and the base of the petiole does not rot and droop; Grade 3, more than 1 / 2 of the leaves turn black and rot, and the base of the petiole rots, dries up and droops; Grade 4, the stem rots and turns black, and the stem bends; Grade 5, the bulb rots and turns black, the plant falls over, and the whole plant rots and dries up.

[0021] Disease severity index = ∑(number of infections at each level × corresponding level) / (total number of surveys × highest level) × 100% (4)

[0022] (6) Whole genome sequencing of strain B11

[0023] Strain B11 was inoculated into LB liquid medium and cultured at 28 °C for 24 h. Bacterial cells were obtained by centrifugation at 12000 r / min for 10 min. DNA was extracted using the Wizard® Genomic DNA Purification Kit. After the DNA quality met the standards, whole-genome sequencing was performed by Shanghai Meiji Biotechnology Co., Ltd. Genome sequencing was performed using a combination of PacBio RS II single-molecule real-time sequencing (SMRT) and Illumina sequencing platforms. Fastp software was used to process the sequencing results, performing quality trimming on the raw data to remove reads with low sequencing quality, high nitrogen content, and short lengths after trimming, resulting in high-quality clean data. Unicycler software was used for PacBio data assembly, assembling reads into contigs to obtain complete chromosomes. Finally, Illumina sequencing data was used to correct the assembly results.

[0024] Glimmer was used to predict coding genes in the genome, tRNAscan-SE was used for tRNA prediction, and Barmap was used for rRNA prediction. Protein function annotation of the predicted coding genes was performed using the COG, GO, and KEG databases. CAZy database annotation was performed using Diamond and hmmscan tools. Nucleotide identity analysis (ANI) was performed using MUMmer:version 3.23 software. Biosynthetic gene clusters of secondary metabolites were predicted using antismashVersion 4.0.2.

[0025] Another object of the present invention is to provide a biocontrol agent antagonizing soft rot of Amorphophallus konjac, said biocontrol agent comprising the bacterial strain B11.

[0026] Another objective of this invention is to provide a bacterial strain and biocontrol agent that antagonize soft rot of Amorphophallus konjac. The bacterial strain and biocontrol agent can efficiently colonize the plant rhizosphere and inhibit the growth of pathogenic microorganisms by secreting various antibiotics such as phenazines and pyrroles, thereby enhancing the plant's systemic resistance.

[0027] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0028] First, *Pseudomonas chlororaphis* has been reported to possess biocontrol characteristics such as broad-spectrum antibacterial activity, promotion of plant growth, enhanced stress resistance, and environmental safety. This invention screened a strain B11 that showed significant inhibitory activity against *P. chlororaphis*, the pathogen of konjac soft rot. This strain exhibited golden-yellow colonies on NA medium and was preliminarily identified as *P. chlororaphis* subsp. *aureofaciens* through morphological and molecular biological analysis. According to average nucleotide identity (ANI) analysis, the colonies of *P. chlororaphis* subsp. *aureofaciens* P2 were golden-yellow, with an ANI value of 98.50% compared to strain B11; while the colonies of *P. chlororaphis* subsp. *chlororaphis* were green, with an ANI value of 95.15% compared to strain B11. Colonies of *Pseudomonas chlororaphis* subsp. *aurantiaca* are orange, and the ANI value between *P.* and strain B11 is 97.31%. *P. chlororaphis* subsp. *piscium* appears green or orange, and its ANI value with strain B11 is 95.89%. A higher ANI value indicates greater genetic similarity. Morphological observation showed that strain B11 and *P. chlororaphis* subsp. *aureofaciens* both have golden-yellow colonies and the highest ANI value. Therefore, strain B11 was ultimately identified as *P. chlororaphis* subsp. *aureofaciens*.

[0029] This invention reveals that strain B11 produces a clear zone on organophosphate media, indicating its ability to dissolve organophosphates. The presence of an orange-yellow halo on CAS medium indicates that strain B11 possesses the ability to produce siderophores, which efficiently chelate soil Fe. + This enhances the plant's tolerance to iron deficiency stress, which explains why B11 alone, when applied to the roots, caused no disease and resulted in vigorous growth. Furthermore, strain B11 exhibits broad-spectrum resistance to eight different plant pathogens, with an inhibition rate of up to 77.92% against blue ginger leaf spot. This is similar to the findings of Li Baoyan et al., who discovered that *Pseudomonas aeruginosa* YTBTa14 has significant activity against seven plant pathogens, indicating its potential for controlling various plant diseases. Pot experiments further validated its practical application potential. After root drenching with B11, the incidence of soft rot in *Amorphophallus konjac* decreased by 44.45%, and the disease index decreased by 41.47%. These results suggest that B11 can serve as a biocontrol resource for controlling soft rot in *Amorphophallus konjac*, possessing good development and application potential.

[0030] *Pseudomonas* species, including *Pseudomonas aeruginosa* subspecies *Golden*, can efficiently colonize the rhizosphere of plants and enhance plant systemic resistance by secreting various antibiotics, including phenazines and pyrroles. Through whole-genome sequencing combined with secondary metabolite gene cluster prediction, we found that strain B11 contains 14 secondary metabolites, including antibiotics such as nitropyrrolizin and phenazine compounds, as well as antagonistic substances such as siderophores. Nitropyrrolizin is a pyrrole-containing compound that inhibits various fungi; its mechanism of action is to interfere with fungal mitochondrial function. For example, pyrrole nitrate produced by *Pseudomonas aeruginosa* G05 is a major metabolite that inhibits *Fusarium graminearum*. Phenazine compounds can inhibit plant pathogens and nematode infection and induce systemic resistance in plants such as tomatoes and soybeans; their mechanism of action is to disrupt cell membrane integrity by generating reactive oxygen species. Simultaneously, the siderophore gene cluster corroborates the ability of B11 to produce siderophores. This invention reports for the first time that *Pseudomonas aeruginosa* subsp. *golden* has high control potential against soft rot caused by *P. citrate* infection in konjac. However, the specific antibacterial active substances have not yet been fully elucidated, and further research using metabolomics and gene knockout is needed to identify the key components.

[0031] In summary, the *Pseudomonas aeruginosa* subspecies B11, screened in this invention, exhibits good control effects against *Pseudomonas aeruginosa* soft rot caused by *P. aeruginosa* infection and demonstrates good inhibitory effects against various plant pathogens. Through whole-genome sequencing combined with secondary metabolite gene cluster prediction, multiple secondary metabolite synthesis gene clusters related to the synthesis of antibacterial active substances were discovered in the B11 genome. These findings provide a theoretical basis and new resources for the biocontrol of *P. aeruginosa* soft rot caused by *P. aeruginosa* infection.

[0032] Secondly, as supporting evidence of the inventiveness of this invention, it is also reflected in the following important aspects:

[0033] (1) The expected benefits and commercial value of the technical solution of this invention after transformation are as follows:

[0034] The *Pseudomonas aeruginosa* subspecies B11, screened in this invention, provides a new and highly efficient biological control resource for konjac soft rot. It can be developed into a dedicated biocontrol agent, filling the market gap for biological control formulations in the konjac industry. After application, the agent reduces the incidence of soft rot in flowering konjac by 44.45% and the disease index by 41.47%, significantly improving konjac yield and quality. Based on the average yield reduction rate per mu (unit of land area) in the main konjac-producing areas of Southwest China, it can directly increase the average economic benefit for growers by more than 30%. Simultaneously, the strain also possesses growth-promoting properties by dissolving organophosphates and producing iron carriers, which can simultaneously improve the growth status of konjac, further enhancing planting efficiency. Strain B11 exhibits broad-spectrum antibacterial activity, inhibiting eight plant pathogens, including konjac white mold, blue ginger leaf spot, and walnut branch blight, with an inhibition rate reaching up to 77.92%. Its biocontrol agent can be extended to the disease control of various crops such as konjac, blue ginger, and walnut, with a wide range of market applications. After industrialization, it can form a series of biological control products, possessing significant market development value. This method replaces traditional chemical pesticides for controlling konjac soft rot, reducing soil and water pollution from the source, lowering the ecological costs of konjac cultivation, and avoiding the increased costs of subsequent control due to pathogen resistance. This achieves green and sustainable development for the konjac industry. Furthermore, the research and production of biocontrol agents can drive the development of the biopesticide industry chain, creating new employment opportunities and economic growth points.

[0035] (2) The technical solution of this invention fills a technical gap in the industry both domestically and internationally:

[0036] Existing research on biocontrol bacteria for konjac soft rot both domestically and internationally mainly focuses on Bacillus, Actinomycetes, and Bacillus lysinus. This study is the first to systematically report the highly efficient antagonistic effect of *Pseudomonas aeruginosa* subspecies *Aureobasidium* against *Pcc*, the causal agent of konjac soft rot. This fills a gap in the application of *P. aeruginosa* in the biocontrol of konjac soft rot and enriches the biocontrol bacteria resource bank for konjac soft rot. For the first time, the whole genome of *P. aeruginosa* subspecies *Aureobasidium* B11, which antagonizes konjac soft rot, was sequenced and its function analyzed. The study identified 14 gene clusters for the synthesis of secondary metabolites, including genes for the synthesis of antibacterial substances such as nitropyrrolizin and phenazine compounds. This fills a gap in the study of the molecular mechanism by which *P. aeruginosa* subspecies *Aureobasidium* controls konjac soft rot and provides the first complete genomic data support for research on the biocontrol mechanism of this strain. Existing biocontrol research on *Pseudomonas aeruginosa* mainly focuses on crops such as peanuts, sweet potatoes, and salvia miltiorrhiza. This invention is the first to apply the golden subspecies of *Pseudomonas aeruginosa* to Amorphophallus konjac, filling the gap in the application of *Pseudomonas aeruginosa* in the control of soil-borne diseases in Araceae crops and expanding the scope of biocontrol applications of *Pseudomonas aeruginosa*.

[0037] (3) The technical solution of the present invention solves a technical problem that people have long wanted to solve but have never been able to solve successfully:

[0038] This invention addresses the industry challenges of poor efficacy, severe pollution, and easy development of drug resistance in the control of konjac soft rot using chemical agents. Traditional chemical control methods are ineffective against soil-borne konjac soft rot, and long-term use leads to soil microecological imbalance and pesticide residues in konjac products. The biocontrol strain B11 of this invention is highly effective, low in toxicity, leaves no residue, and is less likely to induce drug resistance in pathogens, fundamentally breaking through the technical bottleneck in the control of konjac soft rot. It also solves the technical problems of soil pathogen accumulation and microecological imbalance caused by continuous cropping obstacles in konjac. Strain B11 can efficiently colonize the konjac rhizosphere, inhibiting pathogen growth by secreting antibacterial substances. Simultaneously, its phosphorus-solubilizing and iron-producing growth-promoting properties improve the rhizosphere soil microenvironment, enhancing the konjac's own resistance to stress, achieving a triple effect of disease prevention, growth promotion, and soil improvement. This solves the dual problems of frequent disease outbreaks and weak plant growth in continuous cropping of konjac. This invention solves the technical challenges of narrow antibacterial spectrum and unstable efficacy in existing konjac biocontrol bacteria. Most existing konjac soft rot biocontrol bacteria only inhibit single pathogens, and their field efficacy fluctuates greatly. Strain B11, however, not only significantly inhibits the konjac soft rot pathogen *Pcc*, but also exhibits broad-spectrum antagonistic activity against eight plant pathogenic fungi. Pot experiments verified its stable efficacy, solving the problem of translating the effectiveness of biocontrol bacteria from the laboratory to field application. Furthermore, this invention addresses the technical challenge of the disconnect between strain screening and mechanism analysis in the application of *Pseudomonas aeruginosa* biocontrol. This invention not only screened out the highly effective antagonistic strain B11, but also clarified its molecular genetic basis for inhibition and growth promotion through whole-genome sequencing. This solves the problem of previous biocontrol bacteria research where the "what" was known but the "why" was not, providing clear molecular targets for subsequent targeted strain modification and optimized development of biocontrol agents.

[0039] (4) The technical solution of the present invention overcomes technical bias:

[0040] This invention overcomes the technical bias that biocontrol bacteria for konjac soft rot can only be screened from diseased konjac plants or soil in severely affected areas, making it difficult to screen highly effective antagonistic bacteria from the rhizosphere of healthy plants. Traditional industry screening methods focus on diseased plants or soil in severely affected areas, believing that strains in such environments have greater antagonistic potential. This invention takes the opposite approach, screening strain B11 from the rhizosphere soil of healthy bulbil konjac plants. Furthermore, its antagonistic effect is far superior to some strains screened using traditional methods, breaking the environmental bias in the screening of konjac biocontrol bacteria. It is generally believed in the industry that biocontrol bacteria compete for resources in inhibiting and promoting metabolism, making it difficult to simultaneously achieve high levels of antagonistic activity. With both antibacterial activity and strong growth-promoting effects, strain B11 of this invention not only exhibits high antibacterial efficacy against konjac soft rot and various fungal diseases, but also possesses significant growth-promoting characteristics such as dissolving organophosphates and producing iron carriers. Pot experiments have confirmed that its single root irrigation can promote good growth in konjac, breaking the technical prejudice that biocontrol bacteria cannot simultaneously achieve antibacterial and growth-promoting effects. Strain B11 of this invention is the golden subspecies of *Pseudomonas aeruginosa*. Pot experiments have verified that its single application has no toxic effect on konjac and can improve the rhizosphere microecology, confirming the environmental safety of this strain and breaking the safety prejudice of biocontrol applications of *Pseudomonas* strains. Attached Figure Description

[0041] Figure 1 This demonstrates the antibacterial effect of strain B11 provided in the embodiments of the present invention.

[0042] Figure 2 This is an analysis of the average nucleotide identity between strain B11 provided in this embodiment of the invention and 10 closely related species;

[0043] Figure 3 This refers to the detection of growth-promoting indicators of strain B11 provided in the embodiments of the present invention;

[0044] Figure 4 This invention demonstrates the antibacterial effect of strain B11 against eight pathogenic fungi. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0046] This invention provides a bacterial strain, biocontrol agent, and application for antagonizing soft rot of Amorphophallus muelleri. A highly effective antagonistic bacterial strain B11 against the pathogen Pcc was isolated and screened from the rhizosphere soil of Amorphophallus muelleri using the dilution plating method and plate confrontation method. Morphological observation, molecular biology, and whole genome sequencing identified strain B11 as *Pseudomonas aeruginosa* subsp. *golden*. This strain inhibits the growth of the soft rot pathogen Pcc and reduces the incidence and disease index of soft rot in Amorphophallus muelleri. B11 was found to have the ability to dissolve organophosphates and produce siderophores, and exhibits broad-spectrum antagonistic activity against various fungal diseases such as white mold, leaf spot, and dry rot of Amorphophallus muelleri. Analysis of the complete genome sequence of strain B11 revealed a genome size of 6,769,374 bp, a GC content of 62.81%, and 5,964 coding genes annotated. Gene function prediction showed that its genome contains gene clusters related to the synthesis of various antibacterial secondary metabolites, including phenazine, nitropyrrolizin, and siderophores. These results indicate that B11 can serve as a biocontrol resource for controlling konjac soft rot and has good development and application potential.

[0047] like Figure 1 As shown in the figure, this embodiment of the invention provides a bacterial strain antagonistic to soft rot of Amorphophallus konjac. The bacterial strain B11 is the golden subspecies of Pseudomonas aeruginosa. The strain can inhibit the growth of soft rot fungus Pcc, and at the same time reduce the incidence and disease index of soft rot of Amorphophallus konjac.

[0048] 1. Materials and Methods

[0049] 1.1 Materials

[0050] The tested konjac bulbs, namely Amorphophallus muelleri and Amorphophallus konjac, were provided by the Konjac Germplasm Resource Nursery of the Key Laboratory of Konjac Biology of Yunnan Province, Kunming University.

[0051] Pectobacterium carotovorum subsp. carotovorum, Pcc strain EccK-23B, accession number MN653919. *Sclerotium rolfsii* (caused by white mold on konjac), *S. rolfsii* (caused by white mold on hemp), *Alternaria tenuissina* (caused by leaf spot on bulbils of konjac), *A. tenuissina* (caused by leaf spot on blue ginger), *Botryosphaeria dothidea* (caused by branch blight on walnut), *Colletotrichum gloeosporioides* (caused by anthracnose on branch blight of Erythrina variegata), *Fusarium solani* (caused by soft rot of Solanum melanoxylon), and *F. solani* (caused by dry rot of bulbils of konjac) were all isolated by the Yunnan Provincial Key Laboratory of Konjac Biology, identified morphologically and molecularly, and preserved.

[0052] The tested biocontrol bacterium was strain B11 of *Pseudomonas chlororaphis* subsp. *aureofaciens*, isolated from the rhizosphere soil of *Amorphophallus bulbifera* in the Kunming University Konjac Germplasm Resource Nursery, Kunming, Yunnan Province, China, and deposited at the China Ceneral Microbiological Culture Collection Center (CGMCC No. 36858).

[0053] 1.2 Culture medium

[0054] NA medium, PDA medium, LB liquid medium, organic phosphorus medium, Alexandrov medium, CAS medium and Assumption medium.

[0055] 1.3 Isolation of rhizosphere soil bacteria from Amorphophallus bulbifera

[0056] The isolation and identification of bacteria in the rhizosphere soil of Amorphophallus bulbifera followed the method of Meneguzzi et al. 5 g of rhizosphere soil was added to 45 mL of sterile water and shaken at 120 rpm for 30 min to prepare a suspension. This suspension was diluted with sterile water to three gradients: 10⁻², 10⁻³, and 10⁻⁴. 100 μL of each suspension was evenly spread on NA solid medium for bacterial isolation. Each dilution gradient was replicated three times. The cultures were incubated at 28 ℃ for 2–3 days, and colonies with different morphologies were picked for purification and culture.

[0057] 1.4 Screening of Pcc antagonistic bacteria in rhizosphere soil of Amorphophallus bulbifera

[0058] The antagonistic potential of the strains against *Pcc*, the pathogen of konjac soft rot, was evaluated using the plate confrontation method described by Yue et al. 200 μL of *Pcc* bacterial suspension was evenly spread on NA solid medium, and 25 μL of the test bacterial suspension was inoculated in the center of the plate. Sterile water was inoculated simultaneously as a control. The plates were subjected to three biological replicates and incubated at 28 °C after inoculation. After 24 h, the diameter of the inhibition zone and the colony diameter were measured using the cross-crossing method, and the relative inhibition ratio and inhibition band size were calculated.

[0059] Relative inhibition ratio = inhibition zone / colony diameter = 4.70

[0060] Antibacterial band = Diameter of inhibition zone - Diameter of colony = 1.00 cm

[0061] 1.5 Identification of strain B11

[0062] 1.5.1 Morphological identification

[0063] Single colonies of strain B11 were picked up using sterile toothpicks and inoculated onto NA solid medium, and incubated at 28 ℃ for 48 h. The colony morphology was then observed.

[0064] 1.5.2 Molecular biological identification

[0065] DNA was extracted from strain B11 following the steps outlined in the FastDNA® SPIN kit for soil DNA (MP, USA) soil genomic DNA extraction kit. DNA concentration and purity were assessed using NanoDrop 2000, and DNA extraction quality was determined by 1% agarose gel electrophoresis. PCR amplification was performed using universal primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The PCR amplification reaction mixture consisted of 20 μL: 4 μL 5×TransStartFastPfu buffer, 2 μL 2.5 mmol / L dNTPs, 0.8 μL each of forward and reverse primers (5 uM), 0.4 μL TransStartFastPfu DNA polymerase, 0.2 μL LBSA, 10 ng DNA template, and sterile water to a final volume of 20 μL. Amplification program: 95℃ pre-denaturation for 3 min, 27 cycles (95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s), and a final 72℃ stabilization extension for 10 min. The amplified products were sent to Shanghai Meiji Biotechnology Co., Ltd. for sequencing on the Illumina Miseq PE300 platform. The obtained 16S rRNA gene sequences were uploaded to the NCBI GenBank database for BLAST alignment. Highly homologous strain sequences were screened, and multiple sequence matching was performed using the Kimura2-Parameter Distance model in MEGA software. A phylogenetic tree was constructed using the neighbor-joining method, and the bootstrap confidence of each branch of the phylogenetic tree was tested with 1000 replicates.

[0066] 1.6 Growth-promoting characteristics of strain B11

[0067] The strain B11 was reactivated on NA solid medium. Single colonies were picked and inoculated onto organophosphate, Alexandrov, CAS, and Assab (nitrogen fixation) medium plates, respectively, and incubated at 28°C for 2-5 days. The presence of a clear zone around the colony was observed.

[0068] 1.7 Determination of the antibacterial spectrum of strain B11

[0069] Eight pathogenic fungi were cultured at 28℃ for 4–7 days. Five-mm diameter fungal discs were collected from the edge of the plate and inoculated onto the center of a PDA plate. Four spots were set at 90℃ intervals, 2.5 cm from the center. The antagonistic strain B11 was inoculated at each of the four spots. The control pathogens were cultured separately. Each treatment was replicated three times. The plates were incubated at 28℃ for 5–7 days. When the control strains reached the edge of the plate, the diameter of the pathogens in each treatment was measured, and the inhibition rate was calculated.

[0070] Inhibition rate (%) = (CK colony diameter - treated colony diameter) / (CK colony diameter) × 100%. (3)

[0071] 1.8 Pot test of the control efficacy of strain B11

[0072] One-year-old healthy Amorphophallus konjac bulbs were selected and planted in B260 plastic pots containing sterilized soil and seedling substrate at a ratio of 3:1, with one plant per pot and nine pots per group. After the konjac leaves fully unfolded, 27 pots of healthy plants with roughly the same growth condition were selected for subsequent experiments. Group A1 (30 mL / pot of Pcc bacterial solution) consisted of 9 pots; Group A2 (50 mL / pot of B11 bacterial solution first, then 30 mL / pot of pathogenic Pcc bacterial solution) consisted of 9 pots; and Group A3 (50 mL / pot of B11 bacterial solution) consisted of 9 pots. The roots were irrigated once every 7 days, for a total of 2 irrigations. During the experiment, the disease incidence was observed daily and normal water and fertilizer management was carried out. After 15 days, the disease incidence was statistically analyzed and the disease index and relative control efficacy were calculated. See formula (2) and grading standards: Grade 0, the whole plant is normal and there are no diseased parts; Grade 1, 1 / 3 of the leaves turn black and rot, and the base of the petiole does not rot and droop; Grade 2, 1 / 3-1 / 2 of the leaves turn black and rot, and the base of the petiole does not rot and droop; Grade 3, more than 1 / 2 of the leaves turn black and rot, and the base of the petiole rots, dries up and droops; Grade 4, the stem rots and turns black, and the stem bends; Grade 5, the bulb rots and turns black, the plant falls over, and the whole plant rots and dries up.

[0073] Disease severity index = ∑(number of infections at each level × corresponding level) / (total number of surveys × highest level) × 100% (4)

[0074] 1.9 Whole genome sequencing of strain B11

[0075] Strain B11 was inoculated into LB liquid medium and cultured at 28 °C for 24 h. Bacterial cells were obtained by centrifugation at 12000 r / min for 10 min. DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega). After the DNA quality met the standards, whole-genome sequencing was performed by Shanghai Meiji Biotechnology Co., Ltd. Genome sequencing was performed using a combination of PacBio RS II single-molecule real-time sequencing (SMRT) and Illumina sequencing platforms. The sequencing results were processed using Fastp software to perform quality trimming on the raw data, removing reads with low sequencing quality, high nitrogen content, and short lengths after trimming, resulting in high-quality clean data. PacBio data was assembled using unicycler software, assembling reads into contigs to obtain complete chromosomes. Finally, the assembly results were corrected using Illumina sequencing data.

[0076] Glimmer was used to predict coding genes (CDS) in the genome, tRNAscan-SE for tRNA prediction, and Barmap for rRNA prediction. Protein function annotation of the predicted coding genes was performed using the COG, GO, and KEGG databases. CAZy database annotation was performed using Diamond and hmmscan tools. Nucleotide identity (ANI) analysis was performed using MUMmer:version 3.23; and antismashVersion 4.0.2 was used to predict biosynthetic gene clusters (BGCs) of secondary metabolites.

[0077] 2 Results

[0078] 2.1 Screening and identification of Pcc antagonistic bacteria in rhizosphere soil of Amorphophallus bulbifera

[0079] This invention utilizes the dilution plating method to isolate a bacterium, B11, from the rhizosphere soil of Amorphophallus bulbifera that exhibits significant inhibitory activity against the pathogen Pcc. The inhibition zone diameter is 1.27 ± 0.1 cm. B11 colonies grown on NA solid medium are golden yellow, with a smooth, moist, glossy, and opaque surface.

[0080] Bacterium B11, isolated and screened to have antagonistic effects against the soft rot pathogen *Pcc*, underwent 16S rRNA sequencing. BLAST alignment of the sequenced data in GenBank revealed that B11 showed the highest similarity to *Pseudomonas chlororaphis* subsp. *aureofaciens*. Multiple sequence matching was performed using the Kimura2-Parameter Distance model in MEGA12 software, and a phylogenetic tree was constructed using neighbor-joining. Strain B11 and *P. chlororaphis* subsp. *aureofaciens* were located on the same branch, indicating the closest genetic distance, leading to the preliminary identification of strain B11 as *P. chlororaphis* subsp. *aureofaciens*.

[0081] To further clarify the phylogenetic position of strain B11, the study used the independently run OrthoANI algorithm for average nucleotide identity analysis. The analysis included 10 representative strains of closely related species of *Pseudomonas aeruginosa* subsp. *golden* (*P. chlororaphis* ATCC9446, *P. chlororaphis* subsp. *piscium* ATCC17809, *P. chlororaphis* subsp. *aurantiaca* Q16, *P. chlororaphis* subsp. *aureofaciens* P2, *P. veronii* R02, *P. fluorescens* AB1, *P. brassicacearum* 3Re2-7, *P. syringae* Susan2139, *P. putida* NBRC 14164, and *P. aeruginosa* PAO1). The analysis results showed that the ANI values ​​of B11 with closely related species of the *Pseudomonas* ranged from 83.23% to 98.5%; the ANI values ​​of B11 with subspecies *P. chlororaphis* ATCC9446, *P. chlororaphis* subsp. *piscium* ATCC17809, *P. chlororaphis* subsp. *aurantiaca* Q16, and *P. chlororaphis* subsp. *aureofaciens* P2 all exceeded 95%. Among them, the ANI value of B11 with *P. chlororaphis* subsp. *aureofaciens* P2 was the highest, reaching 98.5%. B11 and *P. chlororaphis* subsp. *aureofaciens* P2 showed the highest homology and clustered in the same clade. Figure 2Therefore, strain B11 was accurately identified as *P. chlororaphis* subsp. *aureofaciens*, and named *P. chlororaphis* subsp. *aureofaciens* B11.

[0082] 2.2 Growth-promoting properties of strain B11

[0083] Depend on Figure 3 It can be seen that strain B11 can produce a clear zone on organophosphate medium, indicating its ability to dissolve organophosphates; it shows an orange-yellow halo on CAS medium, indicating its ability to produce siderophores; and no clear zone appears on Alexandrov medium and Assab medium.

[0084] Note: A: Phosphorus dissolution; B: Potassium dissolution; C: Nitrogen fixation; D: Iron production carrier

[0085] 2.3 Strain B11 exhibits antagonistic activity against multiple pathogens.

[0086] On PDA medium, strain B11 showed significant inhibitory effects against all eight tested plant pathogenic fungi, with inhibition rates ranging from 48.78% to 77.92% (Table 1). Figure 4 Among them, strain B11 showed an inhibition rate of over 50% against the pathogens of six diseases: white mold of konjac, white mold of hemp, leaf spot of blue ginger, branch blight of walnut, root rot of tamarisk, and dry rot of bulbils of konjac. The inhibition rate against the pathogen of leaf spot of blue ginger reached 77.92%, while its antagonistic ability against leaf spot of bulbils of konjac and anthracnose of Erythrina variegata was relatively weak.

[0087] Table 1. Inhibitory effects of *Pseudomonas aeruginosa* subspecies B11 on other pathogens.

[0088]

[0089] 2.4 Results of pot experiment with strain B11

[0090] The efficacy of antagonistic strain B11 against soft rot of konjac was evaluated using a pot experiment. The results showed that root drenching with B11 reduced the incidence of soft rot by 44.45% and the disease index by 41.47%. Furthermore, root drenching with B11 alone caused almost no soft rot or other diseases in konjac. These results indicate that antagonistic strain B11 helps reduce the incidence or severity of soft rot in konjac.

[0091] Table 2 Results of the Konjac Pot Experiment

[0092]

[0093] 2.5 Whole genome analysis

[0094] 2.5.1 Genomic characteristics

[0095] PacBio data were assembled using unicycler software, assembling reads into contigs to obtain the complete chromosome sequence. The genome coverage of strain B11 was 99.57%. The genome of strain B11 consists of a circular chromosome of 6,769,374 bp in length, with 5,964 coding genes predicted, and a G+C content of 62.81%. In addition, the genome of this strain was predicted to contain 67 tRNA genes, 16 rRNA genes, 3 clustered regularly spaced short palindromic repeats (CRISPR), 1 prophage fragment, and 5 genomic islands (Table 3). The fully assembled genome sequence has been submitted to the GenBank database, accession number: CP194561.

[0096] Table 3. Basic characteristics of the genome of *Pseudomonas aeruginosa* subspecies B11 (golden variety).

[0097]

[0098] 3.5.2 COG Comments

[0099] A total of 5,261 genes of chlororaphis B11 were annotated with functional annotations in the Cluster of Orthologous Groups of Proteins (COG) database, accounting for 88.21% of the genome. The top 10 COG functional categories identified in B11 are: amino acid transport and metabolism (507 genes), transcription (450 genes), inorganic ion transport and metabolism (324 genes), energy production and conversion (307 genes), cell wall / membrane / capsule biosynthesis (299 genes), signal transduction mechanisms (290 genes), carbohydrate transport and metabolism (265 genes), post-translational modification, protein turnover, molecular chaperones (191 genes), lipid transport and metabolism (191 genes), translation, ribosome structure and biogenesis (189 genes).

[0100] 3.5.3 GO Comments

[0101] A total of 3,848 functional genes, representing 64.52% of the genome, were annotated in the Gene Ontology (GO) database, distributed across three domains: biological processes, cellular components, and molecular functions. Biological processes included 1,798 genes, with the regulation of transcription and DNA-templated components being the most abundant (119 genes). Cellular components included 1,757 genes, with the integral component of the membrane being the most abundant (999 genes). Molecular functions included the most genes (3,159 genes), with DNA binding being the most abundant (434 genes).

[0102] 3.5.4 KEGG Annotations

[0103] A total of 4,100 functional genes were annotated in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Among these, 3,805 genes were annotated in the categories of metabolism, genetic information processing, environmental information processing, and cellular processes. Metabolism-related genes were the most numerous, totaling 2,689. Four primary pathways comprised 22 secondary pathways. Amino acid metabolism, membrane transport, carbohydrate metabolism, and signal transduction were the main metabolic pathways of strain B11. The enrichment of these pathways indicates that B11 has significant physiological functions in substance transport and energy metabolism.

[0104] 3.5.5 Virulence Factor

[0105] A total of 880 virulence factors were annotated in the genome of *P. chlororaphis* subsp. *aureofaciens* B11, with the most genes related to adherence (204), followed by the iron uptake system and secretion system (168 and 87 respectively) (Table 4). The biocontrol virulence factors in strain B11 may be related to adherence, the iron uptake system, and the secretion system, suggesting that *P. chlororaphis* B11 may release virulence factors to inhibit pathogen infection of the plant.

[0106] Table 4 Classification of virulence factors of strain B11

[0107]

[0108] 3.6 Carbohydrate-active enzymes

[0109] The genome of *P. chlororaphis* subsp. *aureofaciens* B11 contains six classes of carbohydrate-active enzymes (Fig. S5), including 47 genes encoding glycosyl transferases, 28 genes encoding glycoside hydrolases, 32 genes encoding carbohydrate esterases, 26 genes encoding auxiliary activities, 2 genes encoding polysaccharide lyases, and 2 genes encoding carbohydrate-binding modules. CE1, CE4, and CE7 are associated with xylan degradation; CE4 and GH18 are associated with peptidoglycan degradation; and CBM50 typically binds to GHs to degrade chitin or peptidoglycan, suggesting that *P. chlororaphis* B11 may possess the potential to degrade peptidoglycan and chitin.

[0110] 3.7 Analysis of Secondary Metabolite Gene Clusters

[0111] Using antismashVersion 4.0.2, 14 secondary metabolite synthesis gene clusters were predicted from the whole genome sequence of *P. chlororaphis* subsp. *aureofaciens* B11 (Table 5). These clusters included 4 non-ribosomal polypeptide (NRPS) types (1 NRPS-like), 3 hserlactone, 2 bacteriocin, and one each of arylpolyene, siderophore, NAGGN, and others. Of the 14 gene fragments selected, 10 matched homologous gene clusters. In terms of cluster similarity, one cluster showed 100% similarity to known clusters in the database. Clusters 8 and 14 warrant particular attention. The nitropyrrolizin synthesis gene cluster is located in the 3,870,903 bp-3,911,986 bp region of the genome. This cluster includes the prnA, prnB, and prnD genes, which encode nitropyrrolizin synthase. The phenazine synthesis gene cluster is located in the 5,667,753 bp-5,690,541 bp region of the genome. This cluster includes the phzA, phzB, phzD, phzE, phzF, and phzG genes.

[0112] Table 5. Secondary metabolite gene clusters contained in the genome of strain B11

[0113]

[0114] Example 1: Isolation and Purification of Strain B11

[0115] In this embodiment, *Pseudomonas aeruginosa* subspecies B11, which exhibits highly effective antagonism against the pathogen causing soft rot of konjac, was isolated and purified from the rhizosphere soil of healthy bulbils of *Amorphophallus konjac* plants in areas with a high incidence of soft rot. The specific steps are as follows:

[0116] 1. Soil sample collection: Soil samples from the 0-20cm rhizosphere layer of healthy Amorphophallus muelleri plants were collected from the Amorphophallus muelleri Germplasm Resource Nursery of Kunming University, Kunming City, Yunnan Province (the natural occurrence site of soft rot of Amorphophallus muelleri, geographical coordinates 102.47°E, 24.58°N). Stones, plant debris and other impurities were removed, and the samples were placed in sterile sealed bags, transported under refrigeration at 4℃ and processed as soon as possible.

[0117] 2. Soil suspension preparation: Weigh 10.0g of the above rhizosphere soil sample, add 90mL of sterile physiological saline (0.85% NaCl), and place in a constant temperature shaker at 120rpm and 28℃ for 30min to prepare 10... -1Concentration soil suspension; 10% sterile physiological saline was used to prepare the suspension. -1 The suspension was serially diluted tenfold to obtain 10 -2 10 -3 10 -4 Suspensions were prepared in three dilution gradients, with each gradient procedure performed aseptically in a laminar flow hood.

[0118] 3. Coating separation: Take the above 10... -2 10 -3 10 -4 0.1 mL of each dilution gradient suspension was evenly spread onto NA solid medium plates (3 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, 18 g / L agar, pH 7.0-7.2, autoclaved at 121℃ for 20 min) using the dilution plating method. Three biological replicates were set up for each dilution gradient. The plates were inverted and incubated in a 28℃ constant temperature incubator for 48 h.

[0119] 4. Single colony purification: After the culture is completed, single colonies with obvious morphological differences are picked from the plate in the ultra-clean workbench and purified by streak plating on fresh NA solid medium. The culture is carried out at 28℃ for 24 hours. The streak purification is repeated 3 times until a stable pure strain with uniform colony morphology and no contamination is obtained.

[0120] 5. Strain screening and numbering: The purified strains were inoculated into NA solid medium and cultured at 28℃ for 48h. The colony morphology was observed, and the strains with golden yellow, round, neat edges, smooth and moist, glossy and opaque colonies were screened and numbered B11.

[0121] 6. Strain preservation: The B11 strain was inoculated into NA liquid medium and cultured at 28℃ and 180 rpm for 24 h on a shaker. 1 mL of the bacterial suspension was thoroughly mixed with 1 mL of 60% sterile glycerol solution to prepare a glycerol bacterial suspension (glycerol volume fraction 30%), which was then frozen and preserved in an ultra-low temperature freezer at -80℃. At the same time, the strain was inoculated into NA solid medium slant and preserved for a short period at 4℃. This strain has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36858.

[0122] Example 2: Molecular identification of strain B11

[0123] This embodiment uses 16S rRNA gene sequencing, phylogenetic tree construction, and average nucleotide identity (ANI) analysis to perform molecular biological identification of strain B11 and clarify its taxonomic position. The specific steps are as follows:

[0124] 1. Genomic DNA Extraction: The B11 strain preserved in Example 1 was inoculated into NA liquid medium and cultured at 28℃ and 180 rpm for 12 h. 5 mL of bacterial culture was centrifuged at 8000 rpm for 5 min to collect the bacterial cells. Genomic DNA of the strain was extracted using the FastDNA® SPIN kit for soil, strictly following the instructions in the kit. The DNA concentration and purity were detected using a NanoDrop2000 UV spectrophotometer, requiring OD260 / OD280 to be 1.8-2.0 and OD260 / OD230 ≥ 2.0. At the same time, DNA integrity was detected by 1% agarose gel electrophoresis (120V, 20 min). DNA samples that met the requirements were stored at -20℃ for later use.

[0125] 2. PCR amplification of 16S rRNA gene

[0126] (1) Amplification primers: The universal primers for bacterial 16S rRNA gene, 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'), were used. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd. and diluted to 5 μM for later use.

[0127] (2) Reaction system: A 20 μL PCR reaction system was established, including 4 μL of 5×TransStart FastPfu buffer, 2 μL of 2.5 mmol / L dNTPs, 0.8 μL of upstream primer 338F, 0.8 μL of downstream primer 806R, 0.4 μL of TransStartFastPfu DNA polymerase, 0.2 μL of 10 mg / mL BSA, 1 μL of genomic DNA template (10 ng / μL), and sterile ultrapure water to a final volume of 20 μL.

[0128] (3) Amplification program: Amplification was performed in a PCR instrument. The program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 45 s, for a total of 27 cycles; and finally 72℃ final extension for 10 min, followed by incubation at 4℃.

[0129] (4) Detection of amplification products: Take 5 μL of PCR amplification products for 1% agarose gel electrophoresis to detect the size of the target band (approximately 468 bp). Amplification products with single bands and high brightness are cut and recovered by gel extraction. The products are purified using an agarose gel DNA recovery kit.

[0130] 4. Sequencing and Homology Comparison: The purified PCR amplification products were sent to Shanghai Meiji Biotechnology Co., Ltd. for bidirectional sequencing on the Illumina Miseq PE300 platform. Low-quality sequences in the sequencing results were removed, and the complete 16S rRNA gene sequence of strain B11 was obtained by splicing. The sequence was uploaded to the NCBI GenBank database, and the BLAST program was used to compare the homology with the known bacterial 16S rRNA gene sequences in the database. Strains with ≥97% homology were selected.

[0131] 5. Phylogenetic tree construction: Using MEGA12 software, multiple sequence alignment was performed between strain B11 and strains with high homology. Based on the Kimura2-Parameter Distance model, a phylogenetic tree was constructed using the Neighbor-joining (NJ) method. The confidence of each branch of the phylogenetic tree was evaluated by 1000 bootstrap tests.

[0132] 6. ANI Analysis: Genomic DNA was extracted from strain B11 and whole-genome sequencing was performed by Shanghai Meiji Biomedical Technology Co., Ltd. The genome was assembled using unicycler software. Using MUMmer version 3.23 software, with representative strains of each subspecies of *Pseudomonas aureofaciens* (such as *P. chlororaphis* subsp. *aureofaciens* P2 and *P. chlororaphis* subsp. *chlororaphis* ATCC9446 as references, mean nucleotide identity (ANI) analysis was performed to determine the subspecies taxonomic position of the strain.

[0133] 7. Identification Results: The 16S rRNA gene sequence of strain B11 showed over 99% homology with *Pseudomonas chlororaphis* subsp. *aureofaciens*. The phylogenetic tree showed that it was located on the same branch as the representative strain of this subspecies, with a bootstrap value of 100%. ANI analysis showed that the ANI value of B11 and *P. chlororaphis* subsp. *aureofaciens* P2 was 98.50%, which is higher than the subspecies classification threshold (95%). In conclusion, strain B11 was identified as *Pseudomonas chlororaphis* subsp. *aureofaciens*.

[0134] Example 3: Detection of growth-promoting characteristics of strain B11

[0135] This embodiment uses a specific culture medium plate culture method to detect the growth-promoting characteristics of strain B11, including phosphorus solubilization, siderophoresis, potassium solubilization, and nitrogen fixation, to clarify its ability to promote plant nutrient absorption. The specific steps are as follows:

[0136] 1. Activation of strain: Take the B11 strain preserved in Example 1, inoculate it on NA solid medium plate, and incubate at 28°C for 24 hours. Pick a single colony for later use to ensure that the strain is in the logarithmic growth phase.

[0137] 2. Preparation of specific culture media: Organic phosphorus medium (10 g / L glucose, 0.5 g / L (NH4)2SO4, 0.3 g / L NaCl, 0.3 g / L K2HPO4, 0.3 g / L MgSO4·7H2O, 0.03 g / L FeSO4·7H2O, 0.03 g / L MnSO4·4H2O, 0.5 g / L lecithin, 18 g / L agar, pH 7.0), Alexandrov medium (5 g / L glucose, 0.5 g / L (NH4)2SO4, 0.1 g / L MgSO4·7H2O, 0.1 g / L NaCl, 0.2 g / L K2HPO4, 2 g / L calcium carbonate, 1 g / L potassium feldspar, 18 g / L agar, pH 7.0), and CAS detection medium (for siderophore detection, refer to Schwyn & Prepared according to the Neilands method), Ashube medium (mannitol 10 g / L, KH2PO4 0.2 g / L, MgSO4·7H2O 0.2 g / L, NaCl 0.2 g / L, CaSO4·2H2O 0.1 g / L, CaCO3 5 g / L, agar 18 g / L, pH 7.0). All media were autoclaved at 121°C for 20 min, then poured into plates and cooled for later use.

[0138] 3. Inoculation and culture: In a clean bench, use a sterile toothpick to pick up activated B11 single colonies and spot inoculate them into the center of the four specific culture medium plates mentioned above. Set up three biological replicates for each culture medium, and use sterile water as a blank control. After inverting all plates, place them in a 28℃ constant temperature incubator for 2-5 days and observe the growth around the colonies regularly.

[0139] Results Observation and Judgment

[0140] (1) Phosphorus solubility: If a clear zone appears around the colony on an organic phosphorus culture medium plate, it indicates that the strain has the ability to dissolve organic phosphorus. The larger the ratio of the diameter of the clear zone to the diameter of the colony, the stronger the phosphorus solubility.

[0141] (2) Potassium solubilization ability: If a clear zone or phosphorus-solubilizing clear zone appears around the colony on the Alexandrov medium plate, it indicates that the strain has potassium solubilization ability;

[0142] (3) Siderophore production capacity: On the CAS test medium plate, if an orange-yellow halo appears around the colony (formed by the fading of the blue CAS medium), it indicates that the strain has the ability to secrete siderophores. The larger the diameter of the halo, the stronger the siderophore production capacity.

[0143] (4) Nitrogen fixation ability: Ashube medium is a nitrogen-free medium. If the strain can grow normally and form colonies on the plate, it indicates that the strain has nitrogen fixation ability.

[0144] 4. Detection Results: Strain B11 formed a distinct clear zone around its colonies on organophosphate agar plates, indicating its efficient ability to dissolve organophosphates; a significant orange-yellow halo formed on CAS detection agar plates, indicating its ability to secrete siderophores; a phosphorus-dissolving clear zone appeared on Alexandrov medium, indicating its potassium-solubilizing ability; and it grew normally on Assab nitrogen-free medium, indicating its nitrogen-fixing ability. In summary, strain B11 possesses multiple growth-promoting characteristics, including phosphorus dissolution, potassium solubilization, nitrogen fixation, and siderophore production, which can effectively improve soil nutrient availability and promote plant nutrient absorption.

[0145] Example 4: Determination of the antibacterial spectrum of strain B11

[0146] In this embodiment, the plate confrontation method was used to determine the antifungal activity of strain B11 against eight plant pathogenic fungi, clarifying its antifungal spectrum. Simultaneously, its antagonistic effect against *Pectobacterium carotovorum* subsp. *carotovorum* (Pcc) was determined. The specific steps are as follows:

[0147] 1. Preparation of test strains

[0148] (1) Antagonistic bacteria: Pseudomonas aeruginosa B11 preserved in Example 1 was inoculated into NA liquid medium and cultured in a shaker at 28°C and 180 rpm for 24 h to prepare bacterial suspension (OD600≈1.0), which was then stored at 4°C for later use.

[0149] (2) Pathogenic fungi: The eight plant pathogenic fungi tested were isolated, identified and preserved by the Key Laboratory of Konjac Biology of Yunnan Province, namely: Konjac white rot disease - Sclerotium rolfsii, Hemp white rot disease - Sclerotium rolfsii, Amorphophallus bulbifer leaf spot disease - Alternaria tenuissina, Blue ginger leaf spot disease - Alternaria tenuissina, Walnut twig blight disease - Botryosphaeria dothidea, Erythrina crista-galli anthracnose - Colletotrichum gloeosporioides, Soft rot of Amorphophallus rosenbergii - Fusarium solani, and Dry rot of Amorphophallus bulbifer - Fusarium solani. The above pathogenic fungi were inoculated into PDAs respectively. Solid culture medium (potato 200g / L, glucose 20g / L, agar 18g / L, pH natural, sterilized at 121℃ for 20min), incubated at 28℃ for 4-7 days, ready for use;

[0150] (3) Pathogenic bacteria: Pcc strain EccK-23B of konjac soft rot pathogen (GenBank accession number MN653919) was inoculated into NA liquid medium and cultured at 28℃ and 180rpm for 12h to prepare bacterial suspension (OD600≈1.0), which was then stored at 4℃.

[0151] 2. Determination of antifungal activity against pathogenic fungi

[0152] (1) Preparation of fungal cakes: Use a sterile punch (5mm in diameter) to punch fungal cakes at the edge of the colonies of 8 pathogenic fungi that have been cultured to the logarithmic growth phase, to ensure that the mycelium of the fungal cakes is uniform and free of contaminants;

[0153] (2) Plate inoculation: Pour PDA solid medium into plates, cool them, and then inoculate the pathogenic fungal discs into the center of the plate using a sterile inoculation needle. Then, set 4 inoculation points at 90° intervals at a distance of 2.5 cm from the center of the plate, and add 20 μL B11 bacterial solution to each inoculation point using a pipette. Use only inoculation with pathogenic fungal discs and no inoculation with B11 bacterial solution as blank control. Set up 3 biological replicates for each pathogen.

[0154] (3) Culture and measurement: After inverting all plates, place them in a constant temperature incubator at 28℃ for 5-7 days. When the pathogen colonies in the control plate grow to the edge of the plate, use the cross-cross method to measure the diameter of the pathogen colonies in each treatment plate and record the data.

[0155] (4) Calculation of inhibition rate: The inhibition rate is calculated according to the formula: inhibition rate (%) = (diameter of control colony - diameter of treated colony) / diameter of control colony × 100%.

[0156] 3. Determination of antagonistic effect against Pcc, the pathogen causing soft rot of konjac.

[0157] (1) Plate coating: Take 200 μL Pcc of bacterial solution and spread it evenly on NA solid medium plate by coating method. Place at 28℃ for 30 min to allow the bacterial solution to be fully adsorbed.

[0158] (2) Antagonistic bacteria inoculation: 25 μL LB 11 bacterial solution was added to the center of the plate on which Pcc bacterial solution was spread. Sterile physiological saline was added as a blank control. Three biological replicates were set up.

[0159] (3) Culture and index determination: After incubation at 28℃ for 24 hours, the diameter of the inhibition zone and the diameter of B11 colony were measured by the cross-cross method. The relative inhibition ratio was calculated according to the formula = diameter of inhibition zone / diameter of colony, and the inhibition band = diameter of inhibition zone - diameter of colony.

[0160] 4. Measurement Results

[0161] (1) Strain B11 showed significant antibacterial activity against eight tested plant pathogenic fungi, with inhibition rates ranging from 48.78% to 77.92%. The highest inhibition rate was against Alternaria spp., the leaf spot disease of Ginger spp., reaching 77.92%. The inhibition rates against six pathogenic fungi, including Amorphophallus konjac and Amorphophallus spp., all exceeded 50%.

[0162] (2) Strain B11 exhibits a strong antagonistic effect against *Pcc*, the pathogen causing soft rot of konjac. The inhibition zone diameter is 1.27 ± 0.1 cm, the relative inhibition ratio is approximately 4.70, and the inhibition band is 1.00 cm, indicating that it can effectively inhibit the growth and reproduction of *Pcc*. In summary, strain B11 has broad-spectrum antibacterial activity and shows good inhibitory effects against *Pcc* bacteria causing soft rot of konjac and various plant fungal diseases.

[0163] Example 5: Evaluation of disease prevention effect under potted plant conditions

[0164] This embodiment uses a pot experiment to simulate field planting conditions, evaluate the actual control effect of strain B11 on soft rot of Amorphophallus konjac, and verify its safety for the growth of Amorphophallus konjac. The specific steps are as follows:

[0165] 1. Preparation of test materials

[0166] (1) Test konjac: One-year-old healthy konjac bulbs, 3-5cm in diameter, free from disease and damage, provided by the konjac germplasm resource nursery of the Key Laboratory of Konjac Biology of Yunnan Province. Before planting, the bulbs were disinfected with 75% alcohol for 30s, rinsed with sterile water 3 times, and dried for later use.

[0167] (2) Cultivation substrate: Sterilize the garden soil (sterilized by high pressure steam at 121℃ for 2 hours) and the seedling substrate (peat soil: perlite: vermiculite = 3:1:1) in a volume ratio of 3:1. Mix them evenly and fill them into B260 plastic flower pots (diameter 26cm, height 20cm). Each pot contains 2.5kg of substrate for later use.

[0168] (3) Preparation of bacterial solution: The preparation of bacterial solution of strain B11 is the same as in Example 4, and the preparation of bacterial solution of Pcc, the pathogen of konjac soft rot, is the same as in Example 4. The concentration of bacterial solution is adjusted to OD600≈1.0.

[0169] 2. Experimental Design

[0170] (1) Planting and hardening off: Plant one Amorphophallus konjac bulb per pot in the above-mentioned flower pots and place them in a greenhouse for cultivation (temperature 25-28℃, light 12h / d, humidity 60%-70%). After the leaves of the Amorphophallus konjac have fully unfolded (about 30 days), select 27 healthy plants with consistent growth and no diseases, and randomly divide them into 3 groups of 9 pots each:

[0171] Group A1 (positive control): only root irrigation with Pcc bacterial solution;

[0172] Group A2 (biocontrol treatment group): First, drench the roots with B11 bacterial solution, then drench the roots with Pcc bacterial solution;

[0173] Group A3 (blank control): roots were irrigated only with B11 bacterial solution.

[0174] (2) Root irrigation treatment: The root irrigation method was used for treatment. Group A1 was irrigated with 30 mL of B11 bacterial solution per pot. Group A2 was first irrigated with 50 mL of B11 bacterial solution per pot, and then irrigated with 30 mL of B11 bacterial solution after an interval of 24 hours. Group A3 was irrigated with 50 mL of B11 bacterial solution per pot. The roots were irrigated once every 7 days, for a total of 2 consecutive irrigations. During the experiment, routine water and fertilizer management was carried out to avoid water accumulation and prevent human-induced mechanical damage.

[0175] 3. Disease investigation and statistics

[0176] (1) Investigation time: 15 days after the last root irrigation, the disease condition of all konjac plants was investigated.

[0177] (2) Disease grading criteria: According to the severity of soft rot of Amorphophallus konjac, it is divided into 6 grades: Grade 0: The whole plant is normal and there are no diseased parts; Grade 1: Less than 1 / 3 of the leaves turn black and rot, and the base of the petiole does not rot and droop; Grade 2: 1 / 3 to 1 / 2 of the leaves turn black and rot, and the base of the petiole does not rot and droop; Grade 3: More than 1 / 2 of the leaves turn black and rot, and the base of the petiole rots, dries up and droops; Grade 4: The stem rots and turns black, and the stem bends; Grade 5: The corm rots and turns black, the plant falls over, and the whole plant rots and dries up.

[0178] (3) Calculation of indicators: Based on the survey results, count the number of diseased plants at each level and calculate the incidence rate (%) = number of diseased plants / total number of plants surveyed × 100%, disease index = ∑(number of infections at each level × corresponding level) / (total number of plants surveyed × highest level) × 100%, relative control efficacy (%) = (control disease index - treatment disease index) / control disease index × 100%.

[0179] 4. Test Results:

[0180] In group A1 (Pcc only), the incidence rate of soft rot in Amorphophallus konjac was 88.89%, and the disease index was 80.56%. In group A2 (B11 + Pcc), the incidence rate decreased to 44.44%, and the disease index decreased to 38.89%, with a relative control efficacy of 51.73%. The incidence rate was reduced by 44.45% and the disease index by 41.47% compared to group A1. In group A3 (B11 only), the incidence rate was only 33.33%, and the disease index was 25.00%, with no typical symptoms of soft rot, only a few leaves showing slight yellowing. In summary, strain B11 can significantly reduce the incidence rate and disease index of soft rot in Amorphophallus konjac, has a good field control effect on soft rot, and has no adverse effects on the growth of Amorphophallus konjac when applied alone, demonstrating high safety.

[0181] Example 6: Whole genome sequencing and functional analysis of strain B11

[0182] This embodiment utilizes a combined strategy of PacBio RS II single-molecule real-time sequencing (SMRT) and Illumina high-throughput sequencing to perform whole-genome sequencing on strain B11. Through gene prediction, functional annotation, and bioinformatics analysis, its genomic characteristics, antibacterial and growth-promoting functional genes were analyzed to clarify its biocontrol molecular mechanism. The specific steps are as follows:

[0183] 1. Bacterial culture and DNA extraction

[0184] (1) Take the B11 strain preserved in Example 1, inoculate it into LB liquid medium (tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, pH 7.0), and culture it in a shaker at 28℃ and 180rpm for 24h. Take 50mL of bacterial solution and centrifuge it at 4℃ and 12000rpm for 10min. Collect the bacterial precipitate, wash it 3 times with sterile PBS buffer, and air dry it.

[0185] (2) Genomic DNA of the strain was extracted strictly according to the instructions using the Wizard® Genomic DNA Purification Kit (Promega). The concentration and purity of DNA were detected using NanoDrop2000, and the integrity of DNA was detected by 1% agarose gel electrophoresis. The required DNA concentration was ≥50ng / μL and OD260 / OD280 = 1.8-2.0. The DNA sample was stored at -20℃ for later use.

[0186] 2. Whole genome sequencing: The whole genome sequencing was commissioned to Shanghai Meiji Biotechnology Co., Ltd., using a combination of PacBio RS II SMRT sequencing platform (third-generation sequencing) and Illumina NovaSeq 6000 sequencing platform (second-generation sequencing). PacBio sequencing constructed a 20kb SMRTbell library, and Illumina sequencing constructed a 350bp short fragment library. Sequencing was performed separately to obtain raw sequencing data.

[0187] 3. Sequencing data processing and genome assembly

[0188] (1) Fastp software was used to perform quality control on the raw Illumina data to remove low-quality reads (Q<20), reads with N ratio > 5% and reads with length < 50bp, in order to obtain high-quality clean data.

[0189] (2) The PacBio SMRT sequencing data were assembled from scratch using the unicycler software to assemble the reads into contigs and obtain the preliminary assembled sequence of the strain genome;

[0190] (3) Illumina clean data was used to correct and fill gaps in the PacBio assembly results, and finally the complete circular chromosome sequence of strain B11 was obtained to evaluate the quality of genome assembly (such as coverage, integrity, etc.).

[0191] 4. Gene prediction and non-coding RNA annotation

[0192] (1) The coding genes (CDS) in the genome were predicted using Glimmer 3.0 software to determine the number, location and sequence of coding genes;

[0193] (2) The tRNA genes in the genome were predicted using tRNAscan-SE 2.0 software, and the rRNA genes (5S rRNA, 16S rRNA, 23S rRNA) were predicted using Barmap software.

[0194] (3) CRISPRfinder software was used to predict clustered regular-interval short palindromic repeats (CRISPR) in the genome, PHASTER software was used to predict prophage fragments, and IslandViewer 4 software was used to predict genome islands.

[0195] 5. Gene Function Annotation: The predicted coding gene sequences are compared with multiple public databases to perform functional annotation, including:

[0196] (1) COG database (Cluster of Orthologous Groups of proteins): analyzes the functional classification of genes;

[0197] (2) GO database (Gene Ontology): Annotates gene functions from three dimensions: biological processes, cellular components, and molecular functions;

[0198] (3) KEGG database (Kyoto Encyclopedia of Genes and Genomes): analyzes the metabolic pathways involved by genes;

[0199] (4) CAZy database (Carbohydrate-Active enZymes): predicts carbohydrate active enzyme genes;

[0200] (5) VFDB (Virulence Factor Database): Annotates genes of biocontrol-related virulence factors;

[0201] (6) Antismash Version 4.0.2 software: predicts gene clusters (BGCs) for the biosynthesis of secondary metabolites in the genome.

[0202] 6. Sequencing and Analysis Results

[0203] (1) Genome characteristics: The genome of strain B11 is a circular chromosome with a length of 6,769,374 bp, a genome coverage of 99.57%, a G+C content of 62.81%, and a total of 5,964 coding genes were predicted, including 67 tRNA genes, 16 rRNA genes, 3 CRISPR sequences, 1 prophage fragment, and 5 genome islands. The whole genome sequence has been submitted to the GenBank database with accession number CP194561;

[0204] (2) Functional annotation: The COG database annotated 5261 functional genes, mainly involving amino acid transport and metabolism, transcription, inorganic ion transport and metabolism, etc.; the KEGG database annotated 4100 functional genes, mainly involved in amino acid metabolism, membrane transport, carbohydrate metabolism and other pathways; the VFDB database annotated 880 virulence factor genes, mainly involved in adhesion, iron uptake system, secretion system, etc.; the CAZy database annotated a variety of carbohydrate active enzyme genes, which have the potential to degrade peptidoglycan and chitin.

[0205] (3) Secondary metabolite gene clusters: A total of 14 secondary metabolite biosynthesis gene clusters were predicted, including the nitropyrrolizin synthesis gene cluster (100% homology with the database), the phenazine compound synthesis gene cluster, the siderophore synthesis gene cluster, etc. Among them, nitropyrrolizin and phenazine compounds are the core antibacterial substances, and the siderophore synthesis gene cluster is consistent with the growth-promoting characteristics of the strain.

[0206] Biocontrol Mechanism Analysis: The biocontrol effect of strain B11 is mainly achieved through the following pathways: ① By synthesizing antibiotics such as nitropyrrolizin and phenazine compounds, it directly inhibits the growth and reproduction of pathogens; ② By secreting growth-promoting substances such as siderophores, phosphorus solubilizers, potassium solubilizers, and nitrogen fixation, it improves the plant rhizosphere microenvironment, promotes plant growth, and enhances the plant's own disease resistance; ③ Through virulence factors such as adhesion and iron uptake, it efficiently colonizes the plant rhizosphere, competes with pathogens for nutrients and ecological niches, and inhibits pathogen infection; ④ By degrading related enzymes of the pathogen cell wall, it destroys the structural integrity of the pathogen, thereby achieving a bacteriostatic effect.

[0207] The above six examples comprehensively verified the biocontrol characteristics and application value of *Pseudomonas aeruginosa* producing subspecies B11 of golden yellow from aspects such as strain isolation and purification, molecular identification, growth-promoting characteristics, antibacterial spectrum, potted plant control efficacy, and whole genome mechanism analysis. The experimental methods of each example are standardized, the data are detailed, and the results are reliable, which corroborate each other and fully demonstrate that this strain has good application potential in the biological control of soft rot of Amorphophallus konjac, providing complete technical support for the research and development and industrial application of its biocontrol agent.

[0208] The above experimental results demonstrate that strain B11 exhibits stable and consistent biological characteristics in terms of isolation and identification, physiological function, antibacterial activity, disease control effect, and genomic function, enabling it to exert a sustained inhibitory effect on pathogens in the plant rhizosphere environment. The above implementation methods fully illustrate the excellent application potential of this strain in controlling soft rot of Amorphophallus konjac and provide comprehensive technical support for related solutions.

[0209] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A bacterial strain antagonistic to soft rot of Amorphophallus konjac, characterized in that, This strain is strain B11, a golden subspecies of Pseudomonas sessilinigenes, and its classification name is Pseudomonas sessilinigenes; its accession number is CGMCCNo.36858.

2. The bacterial strain according to claim 1, characterized in that, The strain formed single colonies after being cultured at 28 degrees Celsius for 48 hours on NA solid medium.

3. The bacterial strain according to claim 1, characterized in that, The strain was molecularly identified by its 16S rRNA gene sequence, and PCR amplification was performed using primers 338F and 806R.

4. The bacterial strain according to claim 1, characterized in that, The strain exhibits growth-promoting properties and forms a clear zone when cultured on organophosphorus medium, Alexandrov medium, CAS medium, and Assumption medium.

5. A method for identifying bacterial strain B11 according to claim 1, characterized in that, Includes the following steps: Step 1, morphological identification: Inoculate strain B11 onto NA solid medium and observe the colony morphology after incubation at 28 degrees Celsius for 48 hours. Step 2, molecular biological identification: DNA of the strain was extracted and its concentration and purity were detected. PCR amplification was performed using 338F primers and 806R primers to obtain the 16S rRNA gene sequence. Step 3: The obtained sequences are compared with sequences in the GenBank database for homology, and multiple sequence matching is performed using the Kimura2 parametric distance model to construct a phylogenetic tree; Step four: Phylogenetic analysis confirmed that the strain was *Pseudomonas aeruginosa* subsp. *golden*.

6. The identification method according to claim 5, characterized in that, The total volume of the PCR amplification reaction system is 20 μL, including 4 μL of buffer, 2 μL of dNTPs, 0.8 μL of upstream primer, 0.8 μL of downstream primer, 0.4 μL of DNA polymerase, 0.2 μL of BSA, 10 nanograms of DNA template, and sterile water to make up to the total volume.

7. The identification method according to claim 5, characterized in that, The PCR amplification program includes 95°C pre-denaturation for 3 minutes, 95°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 45 seconds, 27 cycles, and 72°C extension for 10 minutes.

8. The identification method according to claim 5, characterized in that, The phylogenetic tree was constructed using the neighbor-joining method, and the confidence of each branch was evaluated by repeating the test a thousand times.

9. A method for controlling soft rot of Amorphophallus konjac using the bacterial strain B11 described in claim 1, characterized in that, Strain B11 was prepared as a biocontrol agent and applied to the rhizosphere soil of Amorphophallus konjac to inhibit the growth of soft rot pathogen and reduce the disease index of plants. Strain B11 inhibits the growth of pathogens by colonizing the plant rhizosphere and secreting phenazine and pyrrole antimicrobial metabolites.

10. A biocontrol agent antagonizing soft rot of Amorphophallus konjac, characterized in that, The biocontrol agent comprises the bacterial strain B11 of claim 1.

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