Study On The Pathogenic Mechanism And Resistance Effect Of Ginger Stem Rot Pathogenic Microorganisms

Ginger（Zingiber officinale Rosc）is a valuable spice with both culinary and medicinal uses.However,its quality and yield are severely impacted by pathogenic bacteria that accumulate over time due to asexual reproduction and intensive cropping.One of the most serious diseases affecting ginger is stem rot,and current control measures pose environmental and food safety concerns.In this study,we isolated,screened,and purified pathogenic bacteria from diseased ginger plants to identify their species and pathogenicity,and we analyzed their potential pathogenic mechanisms through whole-genome sequencing.Additionally,we isolated and screened bacteria with high abundance from both inter-and non-inter-root soils of healthy ginger plants for biocontrol measures.We conducted antagonistic experiments and selected the best strains for whole-genome sequencing to uncover their potential biocontrol mechanisms.Finally,we investigated the inhibitory effects of plant endogenous substances,such as anisidine and flavonoids,on pathogenic bacteria,and we determined the expression of ginger defense enzymes and defense genes.This study will lay the theoretical foundation for more in-depth research on stem-based rot of ginger in the future and thus has both theoretical and applied implications.A total of seven strains of pathogenic bacteria were identified in this study,including Fusarium verticillioides,Fusarium proliferatum,Penicillium oxalicum,Trichoderma longibrachiatum,Nigrospora oryzae,Fusarium solani,and Globisporangium spinosum.Among these strains,Fusarium proliferatum was the most pathogenic,with a disease severity index（DSI）of 80 and 100% incidence in all ginger blocks.Whole-genome sequencing of Fusarium proliferatum revealed higher quantities of secreted proteins in the major promoting superfamily and smaller quantities of the ABC superfamily of multidrug transporter proteins.These proteins protect pathogenic bacteria from plant defense systems and secrete pathogenic factors such as toxins.The analysis of carbohydrate-active enzymes showed dominance in the cellulolytic enzyme family,the hemicellulolytic enzyme family,and the lignocellulolytic enzyme family,which degrade plant cell walls to facilitate pathogenic bacteria invasion and colonization.The most abundant secondary metabolites synthesized were analyzed through polyketide synthase（PKS）and nonribosomal polypeptide synthase（NRPS）,with NRPS genes acting as an important class of pathogenic factors.The virulence genes gene02921,gene12011,gene12873,gene02680,and gene07704 affect pathogenicity through MAPK,HIF-1 signaling,and ABC transporter protein transport across membranes.gene01380,gene05790,and gene10497 affect the pathogenicity of pathogenic bacteria through the metabolic pathways involved in amino acid and nucleotide metabolism.Furthermore,by comparing the pathogen-host interactions database,several genes including Lae A（PHI:482）,AKT7（PHI:4194）,ACC（PHI:8670）,EAMY₃222（PHI:7438）,and EAMY₃005（PHI:7393）were found to be involved in multiple pathogen-host interaction mechanisms that influence the pathogen virulence of Fusarium proliferatum.In addition,pathogenic genes that have not been validated in relevant studies were annotated in the pathogen-host database: FGSG,Gz Ara001,Gz C2H052,Gz HMG023,Gz HOMEL013,Gz Myb010,Gz Not001,Gz OB,Gz Rad001 Gz ZC,Movps1 These genes may affect the pathogenic ability of the pathogen to cause disease in plants and are specific to ginger,and deserve further investigation.The pathogen Fusarium proliferatum may regulate pathogenicity through different pathways together to make the plant diseased,and not in a single way.In this study,biocontrol bacteria were isolated and screened,resulting in the identification of Bacillus aryabhattai,Bacillus megaterium,Bacillus simplex,Bacillus proteolyticus,and Bacillus marisflavi.The different bacterial strains showed varied antagonistic abilities against different pathogens,with Bacillus megaterium showing the strongest ability to inhibit the growth of Globisporangium spinosum,Nigrospora oryzae,and Fusarium proliferatum,with inhibition rates of 92.9%,82.8%,and 35%,respectively.The genome of Bacillus megaterium was sequenced to further investigate its mechanism of antagonism against pathogenic bacteria.The analysis of carbohydrate-active enzymes revealed the presence of five butyrase gene families（gene0021,gene0760,gene1528,gene1775,p B-gene0010）,three glucanase gene families（gene1647,gene3419,gene3432）,and one CBM50 family（gene4149）.These enzymes can degrade the cell walls of pathogenic fungi,thereby inhibiting their growth.Furthermore,the secondary metabolite synthesis gene cluster analysis identified the presence of Terpene,T3 PKS,Siderophore,Phosphonate,and Lanthipeptide gene clusters,suggesting the potential secretion of other active substances that contribute to the antimicrobial effect.It is hypothesized that Bacillus megaterium inhibits the growth of pathogenic bacteria by secreting polysaccharide-degrading enzymes and producing nucleoside antibiotics.However,since the secondary metabolites have not been fully characterized,there may be new active substances that have not yet been studied or reported that contribute to fungus inhibition.Punicalagin and flavonoids are plant endogenous substances that have been shown to have inhibitory effects on the growth of pathogenic bacteria.Flavonoids showed a stronger inhibitory effect than Punicalagin,with an inhibition rate of up to 100% against some pathogenic bacteria at a concentration of 20 g/L.The inhibitory effect of Punicalagin ranged from 23% to 91% against different pathogenic fungi at the same concentration.Furthermore,the application of flavonoids was found to have a significant effect on the defense enzymes（peroxidase,polyphenol oxidase）and related defense genes（PAL,GLU,WRKY8）in plants,which can enhance the plants’ disease resistance potential.