This study aims to establish a biocontrol bacteria screening system based on root exudates to screen biocontrol strains with strong rhizosphere colonization ability.Using this system,bacterial strains capable of synergistic control of cucumber Fusarium wilt in combination with spent mushroom substrate(SMS)were screened.The chemical composition of cucumber root exudates was modulated by pathogen stress and SMS induction.Chemotaxis,metabolic proliferation,and biofilm formation,which were colonization-related factors,were used as quantitative indicators for the tested strains.Efficient screening of rhizosphere-colonizing bacteria was achieved using capillary assays,96-well plate cultures,and biofilm formation tests.Among 200 bacterial strains isolated from the cucumber rhizosphere,significant differences were observed in chemotaxis,metabolic proliferation,and biofilm formation in response to root exudates.Using this screening system,Bacillus velezensis MTC-5 was screened as an outstanding strain across all screening indicators.Greenhouse experiments demonstrated that MTC-5 strain combined with SMS exhibited enhanced rhizosphere colonization ability and showed significant synergistic effects in disease suppression and plant growth promotion.Field trials revealed that the combined application of MTC-5 strain and SMS achieved a disease control efficacy of 74.5% against cucumber Fusarium wilt,which was 10.7,24.6 percent points higher than the efficacy of SMS or MTC-5 strain alone,respectively.It established a high-throughput biocontrol bacteria screening system based on root exudates,highlighting the critical role of root exudates in bacterial recruitment.The MTC-5 and SMS combination developed through this system demonstrated significant synergistic effects in controlling cucumber Fusarium wilt,providing new insights and technical support for biocontrol bacteria screening and application.

The aim was to explore the role of the small G protein StRab5b in potato resistance to Verticillium wilt.The relative expression levels of the StRab5b gene in the roots of potato tissue culture seedlings(Hutou and Xiapodi)after inoculation with Verticillium dahliae were determined by qRT-PCR.The biomass of V.dahliae in the roots,the lesion area on potato leaves,the content of hydrogen peroxide(H₂O₂),the content of malondialdehyde(MDA),and the activity of antioxidant enzymes were detected in the transgenic potato lines L7,L8,and L10 overexpressing StRab5b(StRab5b-L7,StRab5b-L8,StRab5b-L10)and the non-transgenic plants(CK)after inoculation with V.dahliae.The disease index was calculated using the pot culture method with the StRab5b-L7 transgenic line and the non-transgenic plants(CK).The results showed that the expression of the StRab5b gene was induced in potato after inoculation with V.dahliae.Compared with 0 h after inoculation,the relative expression levels of Hutou and Xiapodi increased significantly by 701.12% and 649.41% at 72 h after inoculation,respectively.After inoculation with V.dahliae,overexpression of StRab5b enhanced the resistance of potato to Verticillium wilt.Compared with CK,the lesion area on potato leaves in StRab5b-L7,StRab5b-L8,and StRab5b-L10 decreased significantly by 78.56%,66.56%,and 59.76% at 96 h after inoculation,respectively;the biomass of V.dahliae in the roots decreased significantly by 73.07%,51.69%,and 23.38% at 168 h after inoculation,respectively;the MDA content decreased significantly by 47.13%,33.08%,and 14.70% at 96 h after inoculation,respectively;the H₂O₂ content increased significantly by 83.67%,48.38%,and 20.15% at 72 h after inoculation,respectively,and the CAT activity increased by 53.54%,33.28%,and 12.62%,respectively;the SOD activity increased significantly by 53.01%,36.36%,and 16.54% at 48 h after inoculation,respectively;the POD activity increased by 55.58%,27.18%,and 1.73% at 96 h after inoculation,respectively;the APX activity increased significantly by 63.69%,44.27%,and 17.11% at 72 h after inoculation,respectively.Compared with CK,the disease rate and disease index of the StRab5b-L7 transgenic line decreased significantly by 43.33 percentage points and 86.07%,respectively.In conclusion,overexpression of the StRab5b gene enhanced the resistance of potato to Verticillium wilt.

Catalase(CAT)plays a critical role in plant resistance against biotic stresses.To investigate the functions of StCAT1 and StCAT3 in potato defense against Alternaria solani infection,this study cloned the cDNA sequences of StCAT1 and StCAT3 via RT-PCR.Sequencing results demonstrated that both genes were 1 479 bp in length,encoding 493 amino acids.Phylogenetic tree construction revealed that StCAT1 shared high homology with SlCAT1 from tomato,while StCAT3 exhibited high homology with both SlCAT3(tomato)and SpCAT3 from wild tomato.Following A.solani infection for 6 days,the expression of StCAT1 and StCAT3 was significantly upregulated by approximately 3.9-fold and 8.7-fold,respectively.A co-silencing vector targeting both StCAT1 and StCAT3 was constructed,and transient co-silencing was achieved using virus-induced gene silencing(VIGS).qRT-PCR screening identified an effective co-silenced potato line(VIGS-3)with suppressed StCAT1 and StCAT3 expression.Upon A.solani inoculation,the silenced line exhibited 42% larger lesion areas compared to the control,indicating that co-silencing StCAT1/StCAT3 compromised potato resistance to early blight.DAB(3,3'-diaminobenzidine)staining further demonstrated that H₂O₂ accumulation in co-silenced leaves was significantly elevated,with staining intensity being 3.8-fold higher than that in the control.These results demonstrate that StCAT1 and StCAT3 regulate H₂O₂ homeostasis to mediate potato resistance against A.solani,providing a theoretical foundation for elucidating the molecular mechanisms of CAT genes in potato-pathogen interactions.

To investigate the molecular mechanisms of the potato miR7997 family in response to Phytophthora infestans infection,this study analyzed the sequence characteristics,target gene prediction,expression patterns,and stress-responsive expression dynamics of Stu-miR7997 and its targets using the potato cultivar Desiree.The results revealed that the potato miR7997 family comprised three members(Stu-miR7997a/b/c)distributed across two chromosomes,with Stu-miR7997a/b sharing identical mature sequences.All precursor sequences formed canonical stem-loop secondary structures,with minimum folding free energies ranging from -37.00 to -49.50 kcal/mol,and mature sequences were located on the 5' arm.Stu-miR7997c exhibited distinct sequence length and functional element distribution compared to Stu-miR7997a/b.Promoter analysis identified light-responsive,hormone-responsive,transcription factor-binding,and stress defense-related cis-regulatory elements;target prediction identified 19 genes predominantly co-regulated by the miR7997 family.Tissue-specific expression profiling showed that Stu-miR7997a/b were highly expressed in stems,while Stu-miR7997c accumulated predominantly in roots.Upon P.infestans infection,all miR7997 members were significantly downregulated,whereas their target genes-including the transcription factor MYB92 gene,NAD(P)H-quinone oxidoreductase gene,and pectin lyase gene-were markedly upregulated.These findings suggest that the Stu-miR7997 family may indirectly modulate potato disease resistance by negatively regulating target genes.This study provides a theoretical foundation for further exploration of the miR7997 family in potato late blight resistance.

To investigate the function of the β-glucosidase(βGlu)gene PgβGlu4 from Pyrenophora graminea,which previous studies found to be highly expressed during the infection stage,we constructed a subcellular localization vector of PCE2-EGFP-PgβGlu4 and transformed rice protoplasts,observed the fluorescence distribution and analyzed its location of existence.Simultaneously,the PgβGlu4 gene RNAi vector was constructed,and QWC protoplasts were prepared by CaCl₂-PEG4000 mediated method for genetic transformation.The function of PgβGlu4 gene was studied by detecting the vegetative growth and pathogenicity of the RNAi mutants.Phylogenetic analysis of PgβGlu4 and other homologous proteins from different pathogens showed that PgβGlu4 had a closer evolutionary relationship with that from Pyrenophora tritici-repentis.The subcellular localization results showed that PgβGlu4 was mainly localized in the nucleus and cell membrane.Four PgβGlu4 gene RNAi mutants were verified by hygromycin.qRT-PCR analysis showed that the expression of PgβGlu4 gene in four RNAi mutants decreased by 66.31%,68.60%,54.37% and 69.89%,respectively,compared with the wild isolate.The colony diameter was smaller than that of the wild isolate,and their incidence rate was reduced by 56.69,52.76,47.43,and 53.30 percentage points.After infection with the mutant strain of RNAi-PgβGlu4,the relative chlorophyll content in barley leaves ranged from 30.3 to 35.0,which was significantly higher than that of the wild-type group.The effect of PgβGlu4 gene silencing on the height of barley plants before and after infection was significant compared with that of the wild-type.The results indicated that the PgβGlu4 gene was involved in the regulation of the growth,development,and pathogenicity of Pyronophora graminea.

Fusarium head blight(FHB)is a devastating fungal disease that seriously threatens the safety of wheat production.Marker-assisted selection(MAS)and pyramiding of resistance genes represent efficient strategies for FHB-resistant breeding.To establish a high-throughput screening system for FHB resistance genes and enhance wheat resistance in Sichuan Province,we performed genome-wide genotyping using a 100K SNP array on 14 Sichuan wheat varieties(lines)along with three FHB-resistant genetic materials.Based on the reported genetic linkage intervals of major FHB resistance genes(Fhb2,Fhb4,Fhb5),we identified SNPs co-segregating with Fhb5 or linked to Fhb2,Fhb4,and subsequently developed kompetitive allele-specific PCR(KASP)markers.Results showed that the genetic relationship of 17 wheat varieties(lines)could be clustered into two major groups:two northern wheat-derived resistant materials(NMAS070 and NMAS069)formed an independent cluster distinct from the Sichuan varieties(lines)while the remaining 15 varieties(lines)were clustered together and subdivided into two subgroups.Functional gene profiling revealed FHB-resistant parents carried superior resistance loci,whereas agronomic parents harbored favorable alleles for yield and quality traits.Through SNP screening,we identified 8 critical SNPs within the linkage intervals of Fhb2,Fhb4 and the co-segregation region of Fhb5.These SNPs enabled the successful development of 4,2,and 2 high-specificity KASP marker systems for Fhb2,Fhb4 and Fhb5,respectively.Validation experiments confirmed all KASP markers achieved precise genotyping and were effectively implemented in molecular breeding for FHB-resistance.This study established a high-efficiency KASP marker system for Fhb2,Fhb4 and Fhb5,providing a robust technical platform for improving FHB resistance breeding of wheat varieties in Southwest China.

The purport was to identify the black rot pathogen of bitter gourd (Momordica charantia L.) and screen highly effective biocontrol Bacillus spp.,so as to provide a theoretical basis for scientific prevention and control of black rot of bitter gourd.The pathogen of black rot was isolated and purified by tissue separation method from postharvest bitter gourd,and the pathogenicity of isolated pathogens was further verified according to Koch's rule.The pathogen was identified by observing morphological characteristics combined with molecular phylogenetic analysis.The representative strain NCKG8.2-4 was used as the test pathogen,and antagonistic experiments were conducted to compare the effects of Bacillus subtilis subsp. spizizenii TEB-1,Bacillus amyloliquefaciens SWB-2,Bacillus licheniformis SWB-1,Bacillus velezensis SB023 and Bacillus polymyxa SWP-1 on black rot pathogen of bitter gourd.The inhibition effect of the best antagonistic strains on test pathogen was further evaluated by the gradient inhibition experiment of the cell-free supernatant (CFS) from its fermentation broth.The results showed that NCKG8.2-4 strain was pathogen causing black rot of bitter gourd,and the ITS,β-Tubulin and GAPDH fragments sequences were 100% consistent with Stagonosporopsis cucurbitacearum.And it was respectively clustered with S.cucurbitacearum on a branch by phylogenetic analysis based on β-Tubulin or GAPDH sequences.The results of antagonistic experiment showed that the inhibitory area of SB023 against colony growth of black rot pathogen NCKG8.2-4 was the largest.At the same time,the inhibitory effect increased with the Mcrease of SB023 CFS content,and the inhibitory rate of 9% CFS for NCKG8.2-4 growth was up to 93.7% and the EC₅₀ of SB023 CFS was 3.48% to NCKG8.2-4 on PDA.The pathogen of black rot of bitter gourd was identified as S.cucurbitacearum,and Bacillus velezensis SB023 had a high inhibitory effect on the pathogen NCKG8.2-4.

Soybean mosaic virus(SMV)disease can cause significant yield losses and quality deterioration in soybeans,and breeding resistant cultivars remains the only effective strategy for SMV control.Identifying the functional genes associated with SMV resistance provides essential genetic resources for developing resistant varieties.Six MATE candidate genes involved in SMV resistance were identified using a near-isogenic line(NIL)of the qTsmv-3 locus and a transgenic Arabidopsis thaliana plant.A total of 128 MATE family genes were predicted in the soybean genome,which were classified into five subfamilies.Notably,all six MATE candidate genes located at the qTsmv-3 locus clustered within subfamily Ⅰ,exhibiting significant differences in expression levels and tissue specificity based on public data.Among them,Glyma.03G005600 showed the highest expression in aerial tissues(leaves and stems),while Glyma.03G005800 was predominantly expressed in underground tissues(roots and nodules).Following SMV inoculation,the resistant NIL(#NIL-NC)exhibited a 70% reduction in viral accumulation compared with the susceptible line(#NIL-SMC).Concurrently,the expression levels of GmICS1 and GmPR1,key genes in the salicylic acid(SA)-mediated defense pathway,were upregulated by 2.40,15.16 folds,respectively,in #NIL-NC,indicating that qTsmv-3 confers resistance through SA-dependent signaling.Among the 6 MATE candidate genes,only Glyma.03G005300 and Glyma.03G005600 displayed significant differential expression between NILs,which were down-regulated by 61.0% and 82.1%,respectively.Considering their expression patterns and responses to SMV infection,Glyma.03G005600 was identified as the most promising candidate gene for qTsmv-3.Further,the expression of GmICS1 and GmPR1 in transgenic Arabidopsis thaliana(OE_MATE),which carrying Glyma.03G005600,was significantly up-regulated by 4.22,9.12 folds compared with that of wild type(WT)after UV-B stress.These results strongly indicated that Glyma.03G005600 could significantly enhance or affect the expression of genes in salicylic acid signaling pathway,and preliminarily confirmed that Glyma.03G005600 was a key regulatory gene for qTsmv-3 locus.In all,the results laid a foundation for cloning the key genes regulating SMV resistance and provided gene resources for genetic improvement of SMV resistance in soybean.

This study explored the relationship between cotton GhERF14 gene and Fusarium oxysporum pathogenicity,analyzed the molecular mechanism of F.oxysporum pathogenicity,and tentatively explored the response of cotton GhERF14 gene to Fusarium wilt disease and its regulatory effect on related resistance genes,to provides some theoretical basis for breeding new cotton cultivars resistant to wilt.Gene cloning and virus-induced gene silencing(VIGS)were used to construct the non-conserved domain interference vector pTRV2-GhERF14.Using Real-time fluorescence quantification(qRT-PCR)technology and VIGS technology,the expression characteristics of GhERF14 and downstream genes related to lignin,ethylene(ET),jasmonic acid(JA),salicylic acid(SA),antioxidant enzymes and disease progression-related protein(PR)were analyzed after F.oxysporum stress and hormone treatment,and the role of GhERF14 in the process of cotton disease resistance was analyzed.The results indicated that inhibition of GhERF14 gene expression could significantly reduce the synthesis of jasmonic acid(JA),salicylic acid(SA)and ethylene(ET)and the expression of genes related to the signaling pathway.After GhERF14 gene silencing by VIGS technology,cotton plants were more susceptible to Fusarium wilt.These results suggested that GhERF14 may play an important role in the pathogenesis and host-pathogen interaction of F.oxysporum.

In order to deeply explore the diversity of mycoviruses in Ustilaginoidea virens,this study used an abnormal strain Uv263 of U.virens isolated from diseased rice samples collected from Hainan Province as experimental material to identify potential mycoviruses in this strain,and analyze the relationship between the genome organization and function of mycoviruses.The results showed that strain Uv263 was infected by a novel mycovirus named Ustilaginoidea virens RNA virus 7 (UvRV7).UvRV7 was a double stranded RNA virus with 5 082 bp in total length and 60.29% GC content.UvRV7 encoded two large open reading frames (ORF1 and ORF2),which encoded the coat protein (CP) and RNA-dependent RNA polymerase (RdRP),respectively.The BlastP comparison showed that the RdRP amino acid sequence of UvRV7 shared the highest similarity with that of Thelebolus microsporus totivirus 1,at 48.49%.The results of multiple alignment based on the amino acid sequence of UvRV7 RdRP showed that the RdRP sequence contained a total of eight conserved motifs,among which the most typical GDD motif in the RdRP conserved domain was identified in the Ⅵ motif.The phylogenetic analysis showed that UvRV7 was the most closely related to Thelebolus microsporus totivirus 1 and clustered with representative viruses of the genus Victorivirus in the family Totiviridae.The results of genome organization and evolutionary analyses both indicated that UvRV7 was a novel mycovirus in the genus Victorivirus.Transmission electron microscopy observations showed that UvRV7 formed a spherical viral particle of about 45 nm.Horizontal and vertical transmission experiments showed that UvRV7 could be efficiently transmitted vertically by conidia and efficiently transmitted horizontally between vegetatively compatible strains.Taken together,this study elucidated the genome organization and evolutionary relationships of the novel mycovirus UvRV7 in U.virens,and provided a potential biocontrol agent and theoretical basis for the biological control of rice false smut.

To understand the changes in endogenous hormones and related gene expression levels after konjac infection with southern blight disease and reveal the main hormone pathways involved in konjac's response to southern blight disease,the ultra performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) was employed to detect the changes in the contents of phytohormone (auxin,abscisic acid,trans-zeatin nucleoside,jasmonic acid,and salicylic acid) in the three-month-old Amorphophallus muelleri,which was infected with southern blight disease for 0,1,3 and 6 days.Moreover,the gene expression levels of abscisic acid,jasmonic acid,and salicylic acid pathways was analyzed by Real-time Quantitative PCR.The results showed that southern blight disease caused changes in the levels of various endogenous hormones in konjac.The content of indole-3-acetic acid tended to increased and then decreased with the southern blight disease infection,while the content of indole-3-butyric acid showed tended to decrease;the content of trans-zeatin nucleoside significant decreased with the infection of southern blight disease;the content of abscisic acid showed a significant increase followed by a decrease with the infection of southern blight disease;the content of jasmonic acid metabolites and salicylic acid metabolites were significantly increased than the control group with the infection of southern blight disease.The content of jasmonic acid of konjac infected with southern blight disease 1,3,and 6 d was 2.31,2.31,and 5.08 times that of the control group,and the content of salicylic acid was 5.53,4.60,and 7.38 times that of the control group,respectively.Eight genes related to abscisic acid,jasmonic acid,and salicylic acid pathways were activated and participated in the response process of konjac to southern blight disease.The above results indicated that the endogenous hormone homeostasis was disrupted after konjac infection with southern blight disease,activated the expression of plant hormone pathway related genes,and jasmonic acid and salicylic acid might play an important role in konjac's resistance to southern blight disease.

Virus-induced gene silencing(VIGS)is a post-transcriptional gene silencing technique widely used in plant gene function research.However,there are few reports on the establishment of a VIGS system for cabbage in China.The aim of this study was to establish a PCVA/PCVB-mediated gene silencing system using phytoene desaturase(PDS) as an effective visual indicator gene in cabbage.Cabbage,Chinese cabbage,and radish were used as plant materials.The PDS gene was silenced by constructing the PCVA-PDS vector.The PCVA/PCVB-PDS-GFP vector was then generated and transformed into Agrobacterium tumefaciens,which was used to infect cabbage and tobacco epidermal cells via injection and to perform vacuum infiltration in cabbage seedlings.Real-time Quantitative PCR was used to study the applicability of the VIGS system in cabbage,and the system was also applied to another representative Brassicaceae crops-Chinese cabbage and radish.The results showed that green fluorescence could be observed on the cell membranes of cabbage and tobacco leaf cells after infection with Agrobacterium tumefaciens transformed with PCVA/PCVB-PDS-GFP.After 14 d of vacuum infiltration in cabbage seedlings,photobleaching appeared on the new leaves,with the affected area gradually expanding.Real-time Quantitative PCR analysis indicated that the relative expression of PDS homologous genes in the experimental groups decreased 3.2-fold and 1.7-fold compared to the control group,respectively.After infecting Chinese cabbage and radish seedlings,whitening of the leaf veins and some leaves was observed,along with some leaf curling.In conclusion,the photobleaching observed in cabbage leaves after PDS gene silencing demonstrates that the VIGS system effectively replicates and spreads within cabbage plants.The whitening of leaves in Chinese cabbage and radish also indicates that the VIGS system can be applied to other Brassicaceae crops,thus expanding the application scope of this silencing system.The establishment of the cabbage VIGS system provides a theoretical foundation for functional gene studies in Brassicaceae crops.

In order to explore the molecular mechanism of the interaction between Bursaphelenchus doui and host plants,an FMRFamide-like neuropeptide gene,named Bd-FLP-12,was cloned from B.doui by using RACE-PCR technique in a specific cDNA library of the anterior end of B.doui.Further,the nucleotide sequence and amino acid sequence of Bd-FLP-12 were analyzed using bioinformatics methods,the gene copy number was identified by Southern Blot,and the developmental expression pattern was investigated by semi-quantitative RT-PCR.The Bd-FLP-12 gene encoded a protein of 90 amino acids with a mature peptide sequence of FLP-12 and a signal peptide sequence but no transmembrane structure,indicating that its encoded protein was a secretory protein.Southern Blot indicated that the Bd-FLP-12 gene was a single copy gene in the B.doui genome,and the RT-PCR result showed that the transcription level of Bd-FLP-12 was lower in the egg stage than in the other stages.In summary,the full-length sequence of the Bd-FLP-12 gene was cloned for the first time in B.doui,and the structure,nature and expression of the gene were characterized,which could serve as a foundation for further study of the gene function.

To investigate the transcriptome differences between resistant and susceptible varieties of cauliflower after inoculation with Xanthomonas campestris pv.campestris (Xcc),and to identify genes associated with cauliflower resistance to black rot disease,the susceptible variety Y1-2 and the resistant variety EC-247 of cauliflower were selected as the research subjects.Total RNA was extracted from cauliflower leaves at 0,1,3,and 5 days post-inoculation with Xcc,respectively.High-throughput parametric transcriptome sequencing was then conducted utilizing the Illumina RNA-Seq platform,followed by Real-time Quantitative PCR for validation of selected differentially expressed genes(DEGs).DEGs associated with disease resistance were screened and analyzed.The findings revealed that 6 355 genes exhibited significant differential expression between resistant and susceptible cultivars across the four time points.KEGG enrichment analysis focused on plant disease resistance pathways,identifying 47 genes involved in plant-pathogen interactions and 61 genes related to plant hormone signaling.Cluster analysis of these gene expression levels disclosed specific genes,including one CDPK,four CMLs,one PTK,one CaM,one RLK,and one SGT1 in the plant-pathogen interaction pathway,and three auxin-responsive protein genes,a TIFY gene,an indole-3-acetic acid-amido synthetase gene,two brassinazole-resistant protein genes,and a Shaggy-associated protein kinase zeta gene in the plant hormone signaling pathway.Notably,the expression of these genes was significantly higher in resistant varieties compared to susceptible ones,indicating their active response to pathogen infection at various time points.The results indicated that these differential genes might be related to disease resistance in cauliflower,which provided important genetic resources and scientific basis for molecular breeding of disease resistance in cauliflower.

This study evaluates the efficacy of bio-organic fertilizer in managing Verticillium wilt in sunflowers,examining its effects on plant growth and disease resistance,and exploring its potential as a sustainable disease control strategy in sustainable agriculture.Pot experiment was conducted to investigate the effects of bio-organic fertilizer at different concentrations and under various treatment conditions on the sunflower Verticillium wilt pathogen and its impact on sunflower growth and disease resistance.The results indicated that the treatment significantly reduced the disease index of Verticillium wilt by 14.57% compared to the control group,with a relative control efficacy of 28.54% under indoor conditions.The application of bio-organic fertilizer significantly promoted sunflower growth and development.Specifically,when 1 g of bio-organic fertilizer was mixed with every 50 g of field soil,the sunflower emergence rate increased by 8.67 percentage point,and key physiological parameters,including seedling height,stem diameter,and fresh weight,were significantly enhanced.Further investigation revealed that bio-organic fertilizer fermentation filtrates at various concentrations effectively inhibited the growth of Verticillium dahliae colonies and spore germination,with inhibitory effects diminishing as dilution increased.Additionally,volatile compounds from the fertilizer significantly suppressed the formation of microsclerotia.In terms of disease resistance mechanisms,the bio-organic fertilizer fermentation filtrates enhanced plant resistance by inducing induced resistance.Physiological analysis showed that the filtrates triggered reactive oxygen species (H₂O₂)bursts,elevated the activities of superoxide dismutase (SOD) and peroxidase (POD),reduced malondialdehyde (MDA) accumulation,and increased the activity of phenylalanine ammonia-lyase (PAL),thereby significantly enhancing disease resistance in sunflowers.This study highlights the control potential of bio-organic fertilizer for managing sunflower Verticillium wilt and promoting plant health,providing theoretical and experimental support for optimizing fertilizer formulations and developing effective disease control strategies to enhance agricultural production.

Cucumber powdery mildew is one of the main diseases that adversely impacts cucumber production,posing a significant challenge to its sustainable cultivation.Identifying genes related to cucumber resistance to powdery mildew can help understand the genetic principles and molecular mechanisms of cucumber resistance to powdery mildew,and provide diverse gene resources for disease resistant breeding.This study constructed F₁ and F₂ populations of QK×QG using cucumber resistant inbred line QK and susceptible inbred line QG as parents.Using the extreme trait mixed pool resequencing (BSA-seq) method,the genomic regions harboring the resistance genes of cucumber powdery mildew were preliminarily located.By integrating transcriptome data with gene annotation information,the association interval of the disease phenotype was narrowed,sequence variations were identified,and key genes were screened.The results showed that the resistance of powdery mildew may be controlled by recessive genes,and the population of F₂ showed a continuous normal distribution from resistant to susceptible.The BSA-seq analysis,combining the SNP-Index method and QTG(quantitative trait genomics)-seq method analysis highlighted the 19—21 Mb region of chromosome 5,where there were 77 annotated genes with SNP differences between samples,including 33 non-synonymous mutations.The transcriptome sequencing (RNA-seq) results showed that there were 309 upregulated genes and 697 downregulated genes in the susceptible material.The expression levels of 13 genes within the candidate segment of chromosome 5 showed significant differences after infection.Through a comprehensive analysis of differentially expressed genes and BSA,the candidate genes in this segment were narrowed to 3,and only SNP mutations were detected in the LOC101207011 gene.The candidate gene LOC101207011 was characterized by a mutation resulting in an amino acid change from Valine at position 656 to Leucine.This gene emerges as the primary focus of our investigation due to its potential role in conferring resistance to powdery mildew.

To explore candidate genes for resistance of Aphis gossypii to the insecticide imidacloprid,the transcriptome data of the two strains were obtained and compared with imidacloprid indoor resistant and relatively sensitive lines of Aphis gossypii by using Illumina HiSeq 2500 high-throughput sequencing technology.The gene annotation was performed using the NCBI database,and bioinformatics analysis of the differential genes at the transcriptional level included GO function,KEGG metabolic pathway,and other analyses.The relative expression of eight candidate differentially expressed genes(CYP6a2,CYP6a13,CYP6k1,CYP6j1,CYP4c1,AChE2,CarE and ALP3)was detected using qRT-PCR technology,and the evolutionary relationships of resistance related genes were analyzed.After sequencing and sequence splicing,a total of 70 101 Unigenes were obtained,with an average length of 654.37 bp.29 131,27 861 and 2 993 Unigenes were annotated in NR,GO and KEGG databases,respectively.According to the NR annotation analysis of the differential genes of insecticide resistance and sensitivity strains,a total of 22 differential genes that may be related to insecticide resistance were found,including 9 detoxification enzyme genes(CYP6a13,CYP6k1,CYP6j1,CYP4c1 and ALP3 each,two CYP6a2,two CarE),8 cuticle protein genes(CP)and their precursors(CPP),target enzyme genes(AChE2),2 transcription factors(WRKY1,leucine zipper transcription factor-like protein 1 gene,LZTFL1),one pancreatic lipase-related protein 2 gene(PLRP2)and one multidrug resistance-associated protein gene(MRP).The results of qRT-PCR indicated that the expression levels of CYP6a2,CYP6a13,CYP6k1,CYP6j1,CarE and ALP3 genes in insecticide resistance strains were significantly higher than those in sensitive strains.The phylogenetic tree analysis of CYP,ALP,GST and CP genes obtained by NR annotation indicated that the genetic relationship between the Aphis gossypii and Aphis glycines and Acyrthosiphon pisum was relatively close.We found that 87.50% of the candidate differentially expressed genes in the two strains showed consistent changes in expression levels at the transcriptional and mRNA levels.

The aim was to study the biological function of FolSid1 gene in Fusarium oxysporum f.sp.lini and its protein localization in Fusarium by cloning the gene.The gene sequence of FolSid1 was cloned by homologous comparison with F.oxysporum, and based on the principle of homologous recombination, a gene deletion box containing hydromycin resistance gene(hph)was constructed by Split Marker strategy,and the gene deletion mutant(ΔFolSid1)was obtained by PEG-mediated transfer into protoplasts of the wild type.pZESH1,a green fluorescent expression vector containing FolSid1 gene was constructed,and the subcellular localization of FolSid1-EGFP fusion protein was performed.The results showed that the sequence of FolSid1 gene consisted of 5 392 bp,which contained 3 introns.Compared with the wild type and the external insertion mutant, the knockout mutant ΔFolSid1 conidia showed a significant decrease in yield, although they did not differ in morphology and size; morphological observations revealed that the growth rate of colonies from the knockout mutant was significantly slower. The experiments of subcellular localization showed that FolSid1 protein was located in the cell membrane of mycelia cell.FolSid1 gene regulated the vegetative growth of mycelium,conidiogenesis and pathogenicity of Fusarium oxysporum f.sp.lini.

To understand the member characteristics of the WRKY transcription factor family in hydrangea and their roles in response to leaf spot disease,this study utilized the bioinformatics method to characterize WRKY family members in Hydrangea macrophylla Endless Summer,and systematically analyzed the protein physicochemical features,gene structure,systematic evolution,collinearity and expression patterns under the infection with Corynespora cassiicola of WRKY family members.The results showed that there were 84 non-redundant HmWRKY members in H.macrophylla genome.All of the HmWRKYs were hydrophilic proteins and unevenly distributed on 18 chromosomes of H.macrophylla,encoding 112—1 046 amino acids.In addition,HmWRKY members were divided into 3 subgroups(Group Ⅰ—Group Ⅲ),which contained a conserved DNA binding domain composed of WRKYGQK and C2H2.The sequence length of HmWRKY varies greatly from 512 bp to 40 338 bp,and 8 collinear gene pairs with Ka/Ks ratios less than 1 were detected,indicating that the HmWRKY family experienced purification selection in evolution.18 HmWRKY members showed significantly differential expression after infection with C.cassiicola,of which 9 up-regulated and 9 down-regulated.The results indicated that these HmWRKY genes might play important roles in response to leaf spot disease of hydrangea.

The purpose of this study is to develop a loop-mediated isothermal amplification(LAMP)technique for rapid detection of Panax notoginseng root rot pathogen Fusarium oxysporum, timely block the spread of pathogen,and specifically prevent and control root rot.Firstly,F.oxysporum transmembrane protein-like(TP)gene was screened from GenBank,and a set of LAMP specific primers was designed based on its conserved region.In addition,the reaction temperature,and concentrations of inner primer,outer primer,loop primer,Mg²⁺,and dNTPs in the F.oxysporum LAMP detection system were optimized,and then the specificity and sensitivity of this system were evaluated.Finally,the LAMP detection system was used to analyze the F.oxysporum in diseased P.notoginseng plants and soil.The results showed that the optimal reaction temperature for LAMP was 64 ℃,the concentrations of inner primer,outer primer,loop primer,Mg²⁺,and dNTPs were 1.6,0.2,0.1 μmol/L,6 mmol/L,and 0.4 mmol/L,respectively.The LAMP system can specifically detect the F.oxysporum from seven pathogens isolated from P.notoginseng root rot samples with a sensitivity of 0.2 pg/μL.Finally,an on-the-spot detection method for root rot pathogen F.oxysporum in P.notoginseng was developed by combining DNA extraction-free and calcein-green indicator,and this study provided a technical support for convenient detection of F.oxysporum in P.notoginseng planting process,cultivating soil,and seed/seedling trading.

In order to obtain Nipah virus nucleocapsid protein(N protein)with similar function and activity to natural proteins,the NiV N protein open reading frame gene was amplified by PCR,cloned into pFastBac^TMHTB vector,transformed into DH10Bac receptive cells,and transfected into sf9 insect cells.The protein was identified by Western Blotting and bioinformatics analysis was used to study the characteristics of N protein.The results showed that the recombinant plasmid pFastBac^TMHTB-NiV-N was obtained through PCR and double enzyme digestion identification.Recombinant protein Bacmid-NiV-N with immune activity was identified by SDS-PAGE and Western Blotting.According to molecular bioinformatics software analysis and prediction,the recombinant protein contained 532 amino acids with a molecular size of 58.34 ku.It was an unstable and hydrophilic protein without a transmembrane region.The secondary structure of the protein was mainly irregular curls,with 5 T cell epitopes and 18 B cell epitopes.The above research provides a material basis for further understanding the function of NiV N protein and establishing corresponding antigen detection methods.

Bioinformatics and expression patterns of Dicer-like(DCL),Argonaute(AGO)and RNA-dependent RNA polymerase(RDR)gene families in the whole genome of Solanum habrochaites were analyzed,so as to provide references for further study on the functions of DCL,AGO and RDR gene families in the response of S.habrochaites to abiotic and viral infection.Using Arabidopsis thaliana DCL,AGO and RDR genes as reference sequences,the genome of S.habrochaites LA1777 was searched by local perl language and software such as Pfam and SMART,and the members of ShDCL,ShAGO and ShRDR gene families were determined.Bioinformatics analysis of DCL,AGO and RDR family genes in S.habrochaites was carried out by means of ExPASy,GSDS 2.0,MEGA,Tbtools and SWISS-MODEL.According to abiotic stress treatment,Tomato chlorosis virus(ToCV)treatment and Real-time Fluorescence Quantitative PCR technology,the expression patterns of these genes were analyzed.Seven ShDCL,15 ShAGO and 6 ShRDR genes were identified from S.habrochaites,which were distributed on chromosome 5,7 and 6 respectively.The encoded proteins were similar in structure to DCL,AGO and RDR in other plants,and all of them contained conserved domains unique to this family.Phylogenetic analysis showed that these genes were divided into 4 subgroups,and there were high structural and functional similarities between S.habrochaites and S.lycopersicum.ShDCL2a,ShDCL2c,ShDCL3,ShDCL4,ShAGO1b,ShAGO3,ShAGO4b,ShAGO5,ShAGO7,ShAGO10a,ShAGO10b,ShRDR1,ShRDR2,ShRDR3a,ShRDR6a and ShRDR6b were significantly up-regulated after various abiotic stresses and ToCV infection.It is speculated that these genes play important roles in abiotic stress and virus infection.

The adult plant resistance gene Lr12 exhibits excellent resistance in production systems.To fine map and develop reliable molecular markers for Lr12,a cross was made between the susceptible variety Thatcher and the resistant near-isogenic line RL6011 containing the Lr12 gene.The F₁ generation resulting from this cross was self-pollinated to generate F₂ individual plants and F_2∶3 families.Field evaluations were conducted using a mixture of five highly virulent leaf rust pathotypes (PHTT, THKS, THTT, PHTS, and PHKS) to inoculate F₂ individual plants and F_2∶3 families for adult plant resistance assessment and genetic analysis of resistance.Subsequently,genotyping was performed using a 16K liquid chip on 10 resistant and 10 susceptible individuals from the F₂ generation to identify SNP markers closely linked to Lr12.This enabled the determination of the chromosomal physical interval containing the resistance gene,the development of SSR molecular markers,and the construction of a genetic linkage map.The results indicate that the segregation ratio of resistance to leaf rust in 3 494 F₂ individuals derived from the RL6011(Lr12)/Thatcher cross was consistent with a 3∶1 ratio ( {\mathrm{\chi }}_{3:1}^2=0.14;P=0.71). In the assessment of 685 F_2∶3 families, the segregation ratio among resistant individuals, resistant heterozygous individuals, and susceptible individuals conformed to a 1∶2∶1 ratio ( {\mathrm{\chi }}_{1:2:1}^2=2.01;P=0.37), suggesting that Lr12 is a dominant gene and the population segregation follows Mendelian single-gene inheritance patterns. Genetic linkage map analysis localized the adult plant leaf rust resistance gene Lr12 between SSR molecular markers YK12817 and YK12928,within a genetic interval of 0.38 cM.This corresponds to a physical interval of 2.09 Mb within the physical range of 579.44 Mb to 581.53 Mb on chromosome 4BL of the Chinese Spring reference genome(IWGSC.Ref.V1.0).These findings provide a solid basis for predicting candidate genes.

Mitogen-activated protein kinase kinase (MAPKK or MKK) plays an important role in plant growth,development and stress responses.In order to identify MAPKK genes related to rust resistance in foxtail millet and provide candidate genes for the study of rust resistance mechanism and disease-resistant molecular breeding of foxtail millet,the members of MAPKK gene family (SiMKKs) in foxtail millet were identified and analyzed at the whole genome level by bioinformatics methods.Real-time PCR was used to detect the expression level of SiMKKs gene in different tissues,under the stress of rust fungus and exogenous hormone treatment.Excel,MEGA and DnaSP were used to analyze the variation sites and haplotypes of the SiMKKs gene related to rust resistance in 70 re-sequenced foxtail millet varieties,and the excellent rust-resistant haplotypes were identified based on phenotype analysis.The results showed that a total of 10 SiMKKs were identified in foxtail millet,which were distributed on 5 chromosomes.The number of exons ranged from 1 to 11,and the encoded protein contained 331-523 amino acids.The SiMKKs were divided into 4 groups.Groups A and B contained S/T-X₅-S/T motif,while SiMKKs in groups C and D did not have this motif.Conserved Motif 1-Motif 6 existed in all SiMKK proteins.The promoter region of each SiMKK gene contained 1 to 3 biotic stress-related cis-acting elements,such as defense and stress response,methyl jasmonate(MeJA) response,salicylic acid(SA) response and elicitor activation.Except SiMKK10-1 and SiMKK10-3,the other 8 SiMKK genes were expressed with different degrees in different tissues,and under rust infection,SA and MeJA treatments.The highest expression of SiMKK4,SiMKK5 and SiMKK10-2 were in roots at booting stage,and the highest expression of SiMKK6-1 and SiMKK6-2 were in stems at booting stage.The expression of SiMKK4 was up-regulated in the resistant response and down-regulated in the susceptible response within 24 h after inoculation,and its expression was related to disease resistance.The expression of SiMKK4 was up-regulated within 16 h and then down-regulatedafter SA and MeJA treatments,and showed continuous changes during SA treatment.In addition,the expression patterns of the remaining 7 SiMKK genes in SA and MeJA treatments were also consistent.The coding region of SiMKK4 gene contained 7 haplotypes and Hap_1 was the dominant haplotype,and no key variation sites related to disease resistance were found.In summary,the expression of SiMKK4 is identified to be associated with resistance to rust disease in foxtail millet,and SiMKK4 may participate in the early disease resistance response of foxtail millet through SA and MeJA signaling pathways.

Abstract: Tetraploid wheat is the ancestor specie of common wheat and an important food crop.Aiming to provide new resistance sources for wheat variety breeding,the resistance tetraploid wheat germplasm was explored and their resistance genes were identified.TDI-1 is a cultivated emmer wheat that has been immune to powdery mildew in the field for many years.To determine the resistance genes carried by TDI-1,and provide a theoretical basis for genetic improvement of wheat resistance,a durum wheat TDU-1 that was susceptible to powdery mildew was used to hybridize with TDI-1,and their F₁ plants,F₂ population,and F_2:3 lines were obtained.Genetic analysis of resistance was conducted on parents TDI-1,TDU-1,and their hybrid offspring that were inoculated with powdery mildew isolate E09.Then,bulked segregant analysis method combined with molecular markers was used to map the resistance gene.The results showed that TDI-1 was susceptible to E09 during the seedling stage but immune during the adult stage.F₁ plants derived from the cross of TDI-1 and TDU-1 were immune to E09 during the adult stage.The resistance of adult F₂ individuals was separated,and the ratio of resistant and susceptible plants was 3:1($χ_{3:1}^{2}$=0.11,P=0.74);the ratio of the number of homozygous resistant,separated resistant,and homozygous susceptible F_2:3 lines was 1:2:1($χ_{1:2:1}^{2}$=0.47,P=0.79),indicating that the resistance to powdery mildew in the adult stage of TDI-1 was controlled by one dominant gene,temporarily named PmTDI-1.Subsequently,a set of molecular markers was used to amplify the parents and their F₂ population,and then four markers on chromosome 2A,including Xwmc407,NRM-2AS29,NRM-2AS45 and NRM-2AS84, confirmed to be linked to PmTDI-1. PmTDI-1 was between the flanking markers NRM-2AS45 and NRM-2AS84,with genetics distances of 1.8 cM and 4.6 cM,respectively.Therefore,the adult stage powdery mildew resistance gene PmTDI-1 was preliminarily localized on chromosome 2A.This study identified a novel dominant adult-plant-resistance powdery mildew gene PmTDI-1 from tetraploid wheat TDI-1.

A lysozyme gene,MseLYS,was cloned from oriental armyworm,and then,its prokaryotic expression vector was constructed by the prokaryotic expression system,and finally,the expression pattern of this gene was detected in different stages and tissues of oriental armyworm,to provide theoretical reference for exploring the function and structure of the MseLYS and lay a foundation for further study of the antibacterial function and physiological mechanism.We cloned the sequence of the lysozyme gene MseLYS from the midgut of Mythimna separata by reverse transcription PCR(RT-PCR)and rapid amplification of cDNA ends(RACE).The open reading frame(ORF)sequence with the signal peptide removed was ligated to the expression vector pET-30a(+),and the inducible expression was carried out by IPTG.Quantitative PCR was used to detect the spatiotemporal expression pattern of this gene.Sequence analysis showed that the full length cDNA of MseLYS was 729 bp,and its ORF was 426 bp,encoding a total of 141 amino acid residues.Meanwhile,the 5'non-coding region and 3'non-coding region included 75,228 bp,respectively.The isoelectric point and molecular weight of the protein encoded by MseLYS were 7.72 and 16.13 ku,respectively.Phylogenetic tree demonstrated that MseLYS was clustered with other species' C-type lysozyme,and was highly consistent with other insect amino acid sequences,suggesting that MseLYS was a C-type lysozyme.SDS-PAGE showed that the expressed protein was consistent with the expected size,which was about 20 ku,indicating that MseLYS can be expressed efficiently in BL21(DE3).Instar expression profiling analysis illustrated that there were significant differences in the expression levels of MseLYS gene among different developmental stages of larvae,males,and females.The expression levels of MseLYS gene were higher in the late larval and pupal stages of oriental armyworm,while the expression levels were lower in other developmental stages.The tissue expression analysis results indicated that there were significant differences in the expression levels of this gene among different tissues of male adults,with higher expression levels in the fat body and thorax;this gene expression also showed significant differences among different tissues of female adults,with higher expression levels in antennae,wings,and cuticle.To sum up,the full-length sequence of MseLYS is cloned,its prokaryotic expression vector is successfully constructed,which could efficiently express the target protein,and its expression pattern is clarified in different tissues and ages.

To study the resistance mechanism of TaHis gene to Fusarium head blight(FHB)in wheat,the full-length coding sequence of TaHis was cloned,and the bait vector pGBKT7-TaHis was constructed,which was then used as bait for screening a yeast two-hybrid library of wheat ear induced by FHB.After obtaining the interacting proteins,yeast two-hybridization and bimolecular fluorescence complementation were further used to verify the interaction between these proteins,and RT-qPCR was used to analyze the expression pattern of TaHis interacting protein induced by FHB in resistant and susceptible cultivars respectively.The results showed that the bait vector pGBKT7-TaHis was successfully constructed,and 18 yeast monoclones were obtained on the four deficient selection medium(SD/-Leu/-Trp/-His/-Ade)after yeast two-hybrid library screening.Blast analysis showed that a total of 5 proteins were obtained,and the coding sequence of serine/arginine-rich mRNA splicing factor SR45a-like(TaSR)was identified in 6 colonies.We cloned the full-length coding region of TaSR gene from Bainong 4299 and constructed pGADT7-TaSR vector.The experiment of yeast two-hybrid showed that the yeast cells co-transformed with pGADT7-TaSR and pGBKT7-TaHis grew well and appeared blue on SD/-Leu/-Trp/-His/-Ade/ X-α-Gal/AbA,indicating that TaSR and TaHis directly interacted in yeast cells.The vectors of YC-TaHis and YN-TaSR were constructed,and the bimolecular fluorescence complementary experiments were performed.The results showed that strong fluorescence signals were generated in tobacco cells co-transferred with YC-TaHis and YN-TaSR,which further verified the interaction between TaSR and TaHis.RT-qPCR analysis of TaSR gene expression showed that TaSR expression was up-regulated in resistant cultivar Bainong 4299,while down-regulated in susceptible cultivar Bainong 607 upon FHB infection,indicating a positive correlation between TaSR expression level and FHB resistance in wheat.To sum up,the interaction between wheat TaSR and TaHis was proved,and TaSR expression level was positively correlated with FHB resistance in wheat.

GhENODL6 has been discovered to be involved in the resistance of Gossypium hirsutum to Verticillium dahliae.To further reveal its role in cotton resistance against Verticillium wilt,GhENODL6 open reading frame was inserted into the yeast expression vector pNC-GBKT7 using Nimble Cloning technology to construct the recombinant plasmid pNC-GBKT7-ENODL6.The resulting plasmid was successfully transferred into yeast Y2HGold and exhibited normal growth on DDO medium,while failing to grow on QDO/X/A medium,indicating that the GhENODL6 protein did not exhibit toxicity towards the yeast host and lacked autoactivating activity.Subsequently,the Y2HGold yeast carrying the pNC-GBKT7-ENODL6 decoy vector was subjected to hybridization with a cDNA library and screened for colonies displaying blue coloration.Through PCR amplification,a 526 bp non-vector insert was obtained in these blue yeasts,which showed high sequence consistency with the WRKY47 gene within G.hirsutum genome.By employing homologous amplification techniques,we successfully cloned the WRKY47 open reading frame from upland cotton,which spanned 1 587 bp in length and encoded 528 amino acid residues.Furthermore,a yeast two-hybrid assay confirmed an interaction between WRKY47 and GhENODL6.To summarize our findings,the study constructed the recombinant vector pNC-GBKT7-ENODL6 and identified WRKY47 as an interacting protein of GhENODL6.

In order to determine whether Tomato brown rugose fruit virus(ToBRFV)occurs in Zhangjiakou and Yinchuan tomato producing areas,and to explore the genetic information and evolution of ToBRFV,so as to provide an important scientific basis for the diagnosis and control of ToBRFV and the genetic engineering of tomato resistance to viral diseases.Molecular detection was carried out in Zhangjiakou and Yinchuan suspected diseased fruits,and sequence analysis and genome-wide phylogenetic analysis of the virus gene were carried out by relevant molecular biology software.The results showed that the genome structure similarity between the virus isolates from Zhangjiakou and Yinchuan fruit was more than 99% similar to most of the ToBRFV isolates in GenBank,and the tomato fruit virus in the two places was determined to be ToBRFV.ToBRFV isolates were highly regional,and ToBRFV viruses from different regions of China were associated with many countries and regions from Europe and Asia,and the Zhangjiakou isolate was most closely related to the Chinese isolate(MT018320.1),and the Yinchuan isolate was clustered with the Peruvian isolate,indicating that the Yinchuan isolate may have originated from South America.The similarity of the four ORF amino acids of the Zhangjiakou isolate from China(MT018320.1)was the highest,while the similarity between the Yinchuan isolate and the Zhangjiakou isolate was low.In addition,this study found for the first time that the 444th base of the Yinchuan isolate CP protein changed from A to G,resulting in a meaningful mutation of the 131st amino acid of the CP protein from V(valine)to A(alanine).In summary,ToBRFV disease occurred in Zhangjiakou and Yinchuan tomato producing areas,and the virus came from different places in the two regions.

In order to quickly obtain homozygous and stable DH lines of Chinese cabbage resistant to clubroot disease,and to provide basic materials for breeding Chinese cabbage resistant varieties to clubroot disease,5 resistant genotypes of Chinese cabbage were used as materials for isolated microspore culture in this study,and phenotype and resistance of the obtained microspore regenerated plants were identified by combining molecular markers,morphological observation,and artificial inoculation of P.brassicae.The results showed that all 5 genotypes were induced to produce embryos,with a variation of 0.02 to 1.72 embryos per bud.Three genotypes,20aCR12,21aCR6,and 21aCR12,obtained regenerated plants,with regeneration rates of 27.09%,1.45%,and 26.07%,respectively.Resistance marker identification showed that molecular markers linked to CRa and CRb^kato all amplified the resistant bands in 50 obtained microspore plants.Phenotypic investigation showed that there were significant differences in the shape and color of basal leaves,bolting timing,and fertility among 50 microspore plants at reproductive growth stage,and 29 DH lines obtained through self-pollination also showed exhibited diversity in plant type,leaf traits,and heading traits at nutritional growth stage.Resistance inoculation showed that the disease index of 11 DH lines to races 4 and 1 of P.brassicae was less than 33.33,indicating resistance or tolerance to clubroot disease.Among them,there were 3 highly resistant DH lines with a disease index less than 5.0 for two races of P.brassicae,namely 20aCR12-23,20aCR12-29,and 21aCR12-39.The research results have shown that the homozygous DH lines resistant to clubroot can be obtained quickly by microspore culture combined with molecular marker assisted identification.

In order to study the important role of zinc finger protein C₂H₂ in the stress response to soybean cyst nematode disease(SCN),lay an important foundation for further research on anti-soybean cyst nematode.The transcriptome data of SCN3 roots inoculated with Dongnong L-10(SCN-resistant)and Heinong 37(SCN-susceptible)were utilized in the present study,and the differentially expressed gene,GmC₂H₂-2like,was screened out.The localization of this gene was observed under the inverted fluorescence microscope.The gene was bioinformatically analyzed,and its overexpression vector was cloned and constructed to transform soybean hairy roots to study its resistance function against cyst nematode disease.The results showed that GmC₂H₂-2like encoded 410 amino acid residues,with a molecular weight of 45.77 ku,an isoelectric point of 8.73,and 47 phosphorylation sites,and the protein tertiary structure contained four structures,namely,α-helix(25.61%),random coil(57.32%),extended strand(13.66%),and β-turn(3.14%);the results of the subcellular localization revealed that the encoded protein was located in the nucleus;analysis of the overexpressed trans-GmC₂H₂-2like soybean hairy roots revealed that the number of cyst nematodes per unit area of the root tissue was significantly lower than that of the empty vector control,suggesting that GmC₂H₂-2like has the function of inhibiting cyst nematodes.

To investigate the nature and function of PlSPL1 (SPL)gene of Paeonia lactiflora,further elucidate the commonalities and characteristic differences of the PlSPL1 gene in different species,and explore the role of PlSPL1 in the degree of stem straightening in Paeonia lactiflora. The full-length sequence of the PlSPL1 gene was obtained by RACE technology using the stalk of herbaceous peony Hongfeng as the research material,and the structural,physicochemical properties,and phylogenetic relationships of PlSPL1 were analyzed and predicted using bioinformatics software,then,the expression level of PlSPL1 in different developmental stages of herbaceous peony stems was analyzed using qRT-PCR technology,and subcellular localization analysis of the protein was conducted using laser confocal microscopy technology.The results showed that the open reading frame of the PlSPL1 gene was 3 000 bp,encoding 999 amino acids.The protein had a molecular formula of C₄₈₆₉H₇₆₈₂N₁₄₀₆O₁₄₉₇S₄₃,a molecular weight of 111.25 ku,a theoretical isoelectric point of 6.26,encoding a hydrophilic unstable acidic protein,phosphorylation modification predominantly on serine,no signal peptide,with transmembrane structure,and had a secondary structure consisting mainly of random coil.Phylogenetic tree analysis revealed that the PlSPL1 protein was most closely related to peony,followed by a closer relationship with grape;protein sequence comparison analysis revealed that the PlSPL1 protein had a conserved SBP domain which was unique to SPL transcription factor family.The relative expression analysis found that PlSPL1 gradually showed a decreasing trend with stem development,indicating that PlSPL1 was negatively regulating the stem development of herbaceous peony and it was hypothesised that PlSPL1 played important roles in degree of stem straightness;and the subcellular localisation showed that the PlSPL1 protein was localised in the nucleus.The above results indicate that PlSPL1 is participated in the stalk development of herbaceous peony.

In order to explore the role of RPM1 in sorghum disease resistance,a sorghum SbRPM1 gene was obtained from sorghum smut resistant variety SX44B by homologous cloning method.The bioinformatics analysis results showed that the total length of the cDNA of SbRPM1 gene was 2 802 bp,encoding 933 amino acids,and its protein had a theoretical molecular weight of 106.1 ku and an isoelectric point of 7.11,which was a hydrophilic protein.The SbRPM1 protein had no transmembrane structure,and its subcellular localization was in the cytoplasm.Conservative domain analysis showed that SbRPM1 protein contained RX-CC-like,NB-ARC and LRR domains,and belonged to CNL proteins in the NLRs family.Phylogenetic analysis showed that SbRPM1 protein was most closely related to the RPM1 protein of Miscanthus lutarioriparius.The expression pattern of SbRPM1 gene was detected by Real-time quantitative PCR,and the results showed that the expression of SbRPM1 gene was higher in leaves and inflorescence,followed by roots,and the lowest in stem.The expression of SbRPM1 gene was significantly up-regulated at 24—72 h in disease-resistant varieties after inoculation with Sporisorium reilianum pathogen,suggesting that this gene could be induced by S.reilianum and played an important role in sorghum disease resistance.In this study, the CDS sequence of the SbRPM1 gene was cloned for the first time in sorghum, and the structure, nature and expression of the gene were characterized.

In order to investigate the effect of iron ions on the biological characteristics and pathogenicity of Verticillium dahliae in sunflower,the growth rate,conidial production,number of microsclerotia,crude toxin secretion,cell wall degrading enzymes activity and pathogenicity of V.dahliae were measured after the exogenous addition of iron ions to the culture medium.The results showed that the growth rate and conidial production of mycelium showed an increasing trend after adding different concentrations of iron ions in an exogenous,compared with the control,the growth rate of V.dahliae was the fastest after adding 80 μmol/L of iron ions,and the colony diameter was 68.81 mm with a growth rate of 21.40%;the conidial production was 2.58×10⁷ conidia /mL with a growth rate of 21.13%;the number of microsclerotia also increased with the increase of exogenously added iron ion concentration,and after 5 days of culture,its increase was 51.53% compared with the control.The crude toxin secretion was increased after adding 80 μmol/L of iron ions,and the increase rate was nearly doubled.The activity of cell wall lyase increased with increasing concentration of exogenous iron ions and increased at 80 μmol/L reached the strongest.In addition,the pathogenicity of V.dahliae increased accordingly with the increase in the concentration of iron ions in the medium,as shown by the increase in the disease index from 35.00 to 62.20 when the concentration of iron ions added exogenously from 0 to 80 μmol/L,with a growth rate of 77.71%.In conclusion,the exogenous addition of iron ions not only accelerates the growth of V.dahliae,and promotes conidial production and microsclerotia formation,but also enhances the pathogenicity of V.dahliae.

To investigate the genetic mechanism behind Annong 1687's ability to resist stripe rust,expedite the dissemination of Annong 1687 wheat variety,offer guidance for enhancing the genetics of novel wheat varieties,and mitigate the damage inflicted by wheat stripe rust on Chinese wheat production.This study employed Annong 1687(Annong 1687 is a semi-winter wheat type with resistance to the less significant physiological species CYR32.It was developed through the crossing of Annong 1106 and Xinong 822)with its sister lines,and parents.The wheat 55K SNP microarray was used to scan the entire genome of Annong 1687 as well as parents and sister lines.Concurrently,Annong 1687 and sister lines were inoculated and characterized employing the physiological race CYR32.The resistance to stripe rust grades was noted at the adult stage of the plant,and the resulting combined inoculation and field phenotypes were evaluated for stripe rust resistance of the wheat lines.The study revealed that Annong 1687 and five sister lines (lines 24,26,28,30 and 140) exhibited moderate resistance to stripe rust,while two sister lines (lines 89 and 105) displayed moderate susceptibility to this disease.Based on genomic differential data comparing Annong 1687 to its sister strains,there was evidence of enriched differential SNP loci in the 31—37 Mb segment of chromosome 2A's short arm,indicating possible presence of a gene or genes responsible for the result.To eliminate any potential interference from other stripe rust resistance genes,we verified the known stripe rust resistance genes present on the parental and chromosome 2A.Our findings indicated the possibility of a new stripe rust resistance gene in the 31—37 Mb interval of the short arm of chromosome 2A in Annong 1687.Experimental and bioinformatic analyses demonstrated that the TraesCS2A01G070700 gene exhibited disparity in the NBS structural domain of resistant and susceptible lines within this interval.Additionally,susceptible lines possessed three missense mutations within the NBS structural domain.It was hypothesised that TraesCS2A01G070700 represented a candidate gene for resistance to stripe rust in Annong 1687.

In order to explore the mechanism of plant in response to Verticillium wilt pathogen infection,Dali wild eggplant (M239) was took as the material.By the artificial inoculation of Verticillium dahliae,four key physiological indicators of M239 root were determined at 0,6,12,24,and 48 h after inoculation with V.dahliae, which namely the peroxidase (POD) activity,catalase (CAT) activity,phenylalanine ammonilyase (PAL) activity and the soluble protein (SP) content.The result showed that 12 and 24 h after inoculation with V.dahliae were determined as the proper sampling time points for proteome analysis.On this basis,iTRAQ technique was used to analyze the protein changes of M239 root after inoculation with V.dahliae.Compared with CK,463 differentially expressed proteins (DEPs) were found in M239 root at 12 h after inoculation with V.dahliae,among which 305 DEPs were down-regulated and 158 were up-regulated.At 24 h after inoculation with V.dahliae in M239,456 DEPs were induced to express by pathogens,including 296 down-regulated and 160 up-regulated proteins.These proteins were mainly enriched in carbon metabolism,glyoxylate and dicarboxylic metabolism,biosynthesis of secondary metabolites,pyruvate metabolism,and metabolic pathways.Compared with the reported Verticillium wilt resistant materials,it was found that the number of proteins and metabolic pathways involved in the defense response of M239 in the process of responding to the infection of V.dahliae was less.Therefore,it failed to form an effective defense network,and finally manifested as susceptible to the disease.

To reveal the diversity characteristics of endophytic bacteria in different cultivars and organs,and clarify the correlation between endophytic bacteria community structure and host varieties,organ types and disease resistance and susceptible characteristics.Six different varieties of foxtail millet,Jigu 22,Honggu,Longgu No.11 and Xiaoqinggu,Shiliuzi and Nenxuan 16,which were resistant (susceptible) to Fusarium head blight,were selected as materials.The leaves,roots,stems,leaf sheaths and mature spikes of foxtail millet,were taken respectively.DNA was extracted for PCR amplification of 16S rDNA V3—V4 region,and Miseq library was constructed for high-throughput sequencing.Microbial diversity was analyzed by Major biological cloud platform.There were certain differences in endophyte species composition between susceptible and resistant varieties,the dominant populations of endophytes were Proteobacteria and Actinobacteriota,followed by Bacteroidota and Firmicutes,Myxococota and Chloroflexi were lower relative abundance followed by Gemmatimondota and Fusobacteriota.Alpha diversity analysis showed that the resistant cultivars had higher panicle community richness at leaf and maturity spikes and higher panicle diversity at leaf,root and maturity spikes.The susceptible varieties had higher richness of root and stem community and higher diversity of stem and leaf sheath;PCoA analysis showed that organ type had more effect on endophytic community structure than variety.Species composition analysis showed that the diversity of endophytic flora in foxtail millet was affected by different varieties and different parts,and the species of endophytic flora in different parts were quite different.The diversity of endophytic flora between susceptible cultivars Xiaoqinggu,Shiliuzi and Nenxuan 16 and resistant cultivars Jigu 22,Honggu and Longgu No.11 was quite different.The resistant cultivars had NB1-j in leaf sheath stage,and the ears had Acidobacteriota in mature stage.The study showed that the diversity and community structure of endophytic bacteria were affected by different organs and varieties of the foxtail millet,and the organ types had more influence on the community structure of endophytic bacteria than varieties.

To explore the effect of planting density on disease and quality of tobacco and the response characteristics of soil microorganisms in rainy regions,a field experiment was conducted in the Rainy Regions.The random block design was adopted to study the effects of different planting densities(CK was 16 650 and T₁ was 18 525 plants/ha) on yield of tobacco and soil microbial community structure during the experimental periods. Results suggested that:compared with the CK treatment,T1 treatment increased by 6.02% for the tobacco plant height,and 8.65% for yield,respectively. Compared with CK treatment,T1 treatment decreased by 15.38% for tobacco black shank,11.76% for tobacco black root rot,and 18.18% for tobacco anthracnose at the mature stage,respectively. T1 treatment increased by 1.62%for the species diversity and decreased by 0.99% for the richness of the rhizosphere soil bacterial community,increased by 3.76% for the species richness and decreased by 4.71% for the diversity of the fungal community when compared with CK treatment. The bacterial and fungal community structure of the T1 treatment was similar to that of the CK treatment,but the microbial dominant taxa in the soil samples was inconsistent. In the horizontal structure of phylum,T1 treatment relative abundance of bacteria in the soil Proteobacter,Acidobacteria and Actinobacteria,and fungal in the soil Ascomycota was higher. In the horizontal structure of genera,T1 treatment relative abundance of bacteria Sphingomonas and fungi Penicillium was significantly improved. Redundancy analysis (RDA)showed that environmental factors (sunshine duration,rainfall,and temperature)significantly affected the bacterial and fungal community structure,and the influence of soil bacterial community was rainfall>temperature>sunshine duration,the influence of soil fungal community was sunshine duration>rainfall>temperature,respectively. This study indicated that properly increasing the planting density of flue-cured tobacco in rainy regions could improve the soil microenvironment,and reduce tobacco diseases,which could improve the quality of flue-cured tobacco and increase the income of tobacco farmers.

To establish a competitive ELISA method for the rapid detection of antibodies to Akabane disease virus. Balb/C mice were immunized with AKV virus,and their splenocytes were immunofused with SP2/0 cells to obtain monoclonal antibodies against AKV. Using the SBV N protein expressed by Bac-to-Bac baculovirus as a diagnostic specific antigen,and goat anti-murine HRP-IgG as a secondary antibody,a competitive ELISA method for AKV antibody detection was established and optimized. A hybridoma cell line that could secrete monoclonal antibody AKV nucleoprotein antibody was obtained with continuous and stable reproduction,and the monoclonal antibody subtype was identified as:heavy chain IgG1 and light chain kappa,which could only react positively with AKV virus,and did not react specifically with viral antigens such as BTV,FAMD,and EHDV virus.The established detection method for AKV antibody ELISA showed that the optimal coating concentration of diagnostic antigen was 0.5 μg/mL,1∶1 000 antibody dilution ratio,1∶50 serum dilution ratio,1∶2 000 secondary antibody dilution ratio,5% BSA,37 ℃ blocking for 2 h,and it was determined that the serum suppression rate was positive when it was greater than or equal to 44%,and it was negative when it was less than or equal to 44%. The established ELISA method sensitivity and specificity identification results are consistent with the ID Screen AKV Competition assay.A hybridoma cell line secreting AKV N protein was successfully prepared,and the established ELISA detection method can be used to detect AKV antibodies in animals,which lays a foundation for further research and industrialization of AKV antibody diagnostic reagents.

Genomic-wide identification of systematic work on the SKS gene family in cotton has not been reported.Identifying SKS (Skewed5 similar) gene family members and analyzing evolutionary relationship in Gossypium hirsutum,and analyzing the mechanism of GhSKS13 gene regulating cotton disease resistance are expected to provide a new direction for the cultivation of resistant plants.Here,the members of upland cotton SKS family were identified by bioinformatics at the genome-wide based on published genomic data of upland cotton genetic standard line TM-1,and G.hirsutum variety CRI-14 was used as the material.And chromosome distribution,evolutionary relationships,gene structure,and collinearity of SKS family members were predicted.Then,the expression pattern of GhSKS13 was analyzed by Real-time Quantitative PCR and virus-induced gene silencing was employed for preliminary investigation of the function of GhSKS13 in cotton resistance to Verticillium dahliae.A total of 48 upland cotton SKS genes were identified,unevenly distributed on 19 chromosomes,clustered into 5 subgroups with highly conserved gene sequences.Collinearity analysis revealed that the upland cotton SKS gene family was subject to purifying selection.Expression pattern analysis showed that GhSKS13 was predominantly expressed in upland cotton root tissues and significantly up-regulated by V.dahliae infection. GhSKS13-silenced reduced plants resistance to V.dahliae and suppressed the expression of the pathogen-related genes including GhPR1, GhPR2,GhPR3 and GhPR5 compared to control plants. GhSKS13-silenced plants invaded by V.dahliae showed significantly lower hydrogen peroxide (H₂O₂) deposition compared to the control,suggesting that GhSKS13 promotes the formation of reactive oxygen species (ROS).In conclusion,this study clarified the phylogenetic relationships,chromosomal distribution characteristics and gene structure characteristics of upland cotton SKS family members,and elucidated the involvement of GhSKS13 in upland cotton plant resistance to V.dahliae.

In order to excavate the mycovirus resources in Ustilaginoidea virens and deeply analyze the relationship between the genome organization and function of a novel mycovirus,it took a U.virens strain Uv321 with abnormal phenotype,isolated from Hainan Province,as the research object,and identified the species of the novel mycovirus in the strain Uv321 on the basis of the previous meta-transcriptome data,a series of studies have been carried out around the novel mycovirus.The results showed that a novel mycovirus was identified in strain Uv321,named Ustilaginoidea viruses botourmiavirus 7 (UvBV7).The genome of UvBV7 is positive single-stranded RNA(+ssRNA),with a total length of 2 406 nt and a GC-content of 53.78%,containing an open reading frame(ORF)encoding RNA-dependent RNA polymerase(RdRP),which encodes 643 amino acids with a molecular weight of about 72.727 ku.The prediction of the protein secondary structure of the viral terminal showed that the 5' and 3' terminal bases of UvBV7 were complementary and paired,forming a hairpin structure.The BlastP alignment showed that UvBV7 had the highest similarity with the virus Erysiphe necator associated ourmia-like virus 72,which belonged to the genus Botoulivirus in the family Botourmiaviridae,but only 44.52%.The multiple alignment results based on the RdRP sequences of UvBV7 and other similar viruses showed that there were 8 conserved domains in the RdRP amino acid sequences of UvBV7 and the members of the family Botourmiaviridae.The GDD motif was found in the Ⅵ conserved domain,which is the typical highly conserved core motif of viral RdRP proteins.The phylogenetic tree constructed based on the amino acid sequence of the viral RdRP also indicated that UvBV7 clustered with the members belonging to the genus Botoulivirus.Therefore,UvBV7 is a novel mycovirus belonging to the genus Botoulivirus in the family Botourmiaviridae.The results of dual cultures of different strains of U.virens showed that UvBV7 could be transmitted horizontally between vegetative compatibility strains,but the mycelial tip-ribavirin,heat-ribavirin and protoplast regeneration-ribavirin treatments were unable to eliminate the mycovirus UvBV7 in strain Uv321.In conclusion,this study not only enriched the diversity of mycoviruses in U.virens,but also provided potential biocontrol agents with hypovirulence for the biocontrol of rice false smut.

The full-length sequence of Diapausin,a novel antimicrobial peptide gene,was obtained from the third instar of oriental armyworm(Mythimna separata)by RT-PCR and RACE.And then,the amino acid sequence of Diapausin gene was analyzed by bioinformatics software.Finally,the prokaryotic expression system was used to induce the protein expression.This study would lay a theoretical foundation for further parsing of the function and structure of Diapausin gene in M.separata. Results showed that the novel antimicrobial peptide gene Diapausin was successfully cloned from M.separata and named Mysdiap (GenBank No.AZJ51075).The total length of this gene was 537 bp,with the 5' and 3' non-coding ends of 63,279 bp,respectively.The full length of the open reading frame was 195 bp,encoding 64 amino acid residues.The molecular weight and the isoelectric point of the protein were 7.13 ku and 5.59,respectively.The first 24 N-terminal amino acid residues of Mysdiap were signal peptide sequences.Multiple alignment of amino acid sequences showed that Mysdiap had a highly conserved region ECCRAHG.The recombinant expression plasmid pET-Mysdiap was successfully constructed and induced to express proteins by IPTG in Escherichia coli.The molecular weight of the expressed recombinant protein was about 20 ku,which could exist in both inclusion bodies and soluble proteins.In summary,the full-length sequence of the novel antimicrobial peptide gene Mysdiap was cloned from M.separata and successfully induced to express proteins in E.coli.

Basic leucine zipper(bZIP)transcription factor protein is a kind of transcription factor with conservative structure and function in animals,plants and microorganisms.In order to clarify the function and mechanism of bZIP transcription factor in plant pathogenic fungi,and further determine its relationship with the growth,development and pathogenicity of the pathogen,the StbZIP9 gene was cloned from Setosphaeria turcica 01-23(GenBank No.XM _ 008032179.1).StbZIP9 is a member of the bZIP transcription factor family.The analysis of the gene structure and protein characteristics showed that the DNA sequence was 788 bp in length,with an open reading frame of 726 bp,encoding 241 amino acids.The encoded protein contained a highly conserved homologous domain BRLZ in fungi.The RNA-seq data of the gene during the growth and development of the pathogen and the process of infecting the host were analyzed.It was found that the expression level of StbZIP9 was 2 to 4 times higher than that in the appressorium and germ tube period compared with the mycelium period.After 24,72 h of infection of maize leaves,the gene expression increased from scratch and continued to increase,indicating that StbZIP9 was associated with appressorium development and germ tube formation and played an important role in the process of pathogen infecting host cells.Further,bioinformatics techniques were used to predict its binding conserved motifs and regulatory target genes.The binding motif was NNTWACGTNN,including the bZIP transcription factor recognition core sequence ACGT,and the downstream target genes of StbZIP9 were predicted according to the sequence.Combined with the expression pattern analysis using the RNA-seq data,four downstream target genes(protein IDs in the JGI database were :132893,163024,162798,40466)were obtained,and the functional annotation table was obtained.The functional annotation revealed its involvement in many biological processes, such as polymerization and transport of cell wall components, host infection, and spore dormancy. It will provide the basis for further elucidation of the molecular mechanism involved in the regulation of pathogen infection.

GRAS transcription factors exist extensively in plant genomes and play an important role in both biological and abiotic stress in plants.To study the significant role of the response to stress in soybean cyst nematode (SCN).The susceptible and disease-resistant varieties Dongnong L-10 and Heinong 37 were inoculated with SCN 3 physiological subspecies respectively.After magenta staining was used to confirm the success of insect grafting,transcriptome sequencing was performed on plant roots.A total of 20 candidate genes related to SCN were obtained.The secondary and tertiary structures,phosphorylation sites,hydrophilicity and hydrophobicity,promoter information and gene evolution relationship of encoded proteins were analyzed and studied in bioinformatics.mRNA was extracted from different tissue parts of the plant,including roots,stems and leaves,and reverse transcription cDNA was performed for key candidate genes by RT-PCR.The results showed that the genes in this family were closely related to MYB and MYC transcription factors.The protein encoded by the gene was mainly α-helix and random coil,and the secondary structure and tertiary structure were highly consistent.Protein was a soluble protein,the phosphorylation site was mainly serine.RT-PCR showed that there were certain expression levels in different parts of plant tissues,mainly in roots.The GRAS expression level of resistant cultivars was significantly higher than that of susceptible cultivars under the same soybean cyst nematode stress.GRAS transcription factors were identified as SCN-related resistance genes,providing an important source of genes for resistance to stress response.

Understanding the variation and population structure of the foxtail millet blast provides a theoretical basis for future cultivation of resistant varieties and control of the disease.Twenty pairs of SRAP primers were used for PCR amplification of strains of P.oryzae collected from 9 regions.NTSYSpc-2.11F software was used for data analysis,UPGMA method was used for cluster analysis,and Popgene 32 software was used to calculate the genetic diversity index among populations.The results showed that 8 pairs of primers were selected with good polymorphism.A total of 1 728 discernible bands were amplified by 8 pairs of primers,1 492 of which showed polymorphism,and polymorphism rate was 86.34%.The cluster analysis of 90 strains of P.oryzae showed that the similarity coefficient was between 0.77-0.85.All strains were divided into 27 genetic lineages (L1-L27) when the genetic similarity coefficient was 0.802.Among them,L1 was the absolute dominant group,containing 29 strains of P.oryzae,which from Shandong,Hebei,Shanxi,Henan and Liaoning Province,accounting for 32.22% of the total strains.According to the calculation and analysis of Popgene 32 software,Nei's genetic diversity index (H) and Shannon's information index (I) of foxtail millet blast in 9 regions ranged from 0.141 4 to 0.288 1,and 0.196 0 to 0.441 6,respectively.Nei's genetic diversity index and Shannon's information index in the summer sowing region of Hebei Province were the highest,with the richest genetic diversity,while Hainan population had the lowest genetic diversity.Compared with different regions of the populations,the genetic relationship between the Jilin population and the Hainan population was the farthest,while the genetic relationship between the Hebei summer sowing millet region population and the Hebei spring sowing millet region population was the closest.It can be seen that the genetic diversity of P.oryzae in different regions was rich,and there was genetic differentiation among strains,but there was no significant correlation between genetic differentiation and geographical origin.

In order to further definite the genetic basis of black rot resistance in cabbage,and breed high-quality disease resistant cultivars,F₁ progenies were obtained by crossing the inbred line 4674(high resistance to race 1)as male parent,with the inbred line 4673(high susceptibility to race 1)as female parent.The F₂ population containing 152 individuals were obtained by F₁ self-crossing. The F₂ population was inoculated by spraying at seedling stage. After 12-14 days, the phenotype of F₂ population was identified according to the identification method of cabbage seedling stage.A total of 175 markers with good polymorphism and clear bands were selected from 404 pairs.Subsequently, the 175 molecular markers were used for genotyping of F₂ population and constructing a genetic linkage map. Finally, the QTL for black rot resistance of cabbage was mapped with phenotypic data and genetic map.The results showed that there were 154 molecular markers linked to nine chromosomes,including 110 pairs of InDel markers and 44 pairs of SSR markers and covering a length of 714.29 cM,and the average distance between markers was 4.64 cM.Seven QTLs were located,of which three major QTLs were qBR-7-2,qBR-7-3 and qBR-4-3,mapped on the genomic markers(CG842110-CG842482 and M29-M39)of chromosome 7,and on the genomic markers between CD838151 and BOE417 of chromosome 4,respectively.The explainable phenotypic variation of QTLs were 16.0%,9.2% and 10.0%,and their LOD values were 5.75,3.20 and 3.47,respectively.

In order to explore virus species in aphids,Aphis craccivora natural populations from Jiangsu were collected and identified,and then the fragmented mRNA from aphid total RNA was used as template to construct the sequencing library.The virus species in A.craccivora were analyzed by virome sequencing technology,and the two novel viruses were further identified.The results showed that after splicing,alignment and classification annotation of sequencing data,177 viral contigs were finally obtained,which were consistent or highly homologous with the sequences of 24 viruses,including 5 plant viruses(involving 5 families)and 19 insect viruses(involving 9 families and 6 unclassified viruses).The virus with the highest sequencing abundance among plant viruses was detected,and after Blast,these viruses with higher homology were all from Cytorhabdovirus,and they had the same conserved domain,so it was considered as a novel Cytorhabdovirus,temporarily named Aphis craccivora associated rhabdovirus(AcARV).The phylogenetic analysis based on viral L proteins revealed that AcARV and Rice stripe mosaic virus(RSMV)clustered into the closest clade,indicating that the two viruses were the closest in evolution.Most identified insect virus species were rarely reported in aphids.Capsid protein VP1 of insect virus Himetobi P virus(HiPV)was detected in A.craccivora via RT-PCR and Western Blot,which confirmed the infection of HiPV in A.craccivora,and it was the first report that HiPV was found in aphids.HiPV VP1 gene of A.craccivora isolate shared 95.5%,99.0% nucleotide identities with Japan and Jiangsu Laodelphax striatellus isolates.These results clarify A.craccivora bears a variety of plant viruses,including a new rhabdovirus AcARV.Meanwhile,insect virus species are abundant in A.craccivora,and HiPV can infect aphids.

Yuanjiang common wild rice(YP)is a germplasm material known as a living fossil in plants,which also has a large number of resistance(R)genes.Therefore,knowing the R genes in YP will help to apply different R genes to cultivated rice.Here,we identified the bacterial blight(BB)R genes in Yuanjiang common wild rice(YP),progeny of introgression lines(L214 and G252)and its susceptible parent,cultivated rice line 35(HX35).Meanwhile,the resistance of YP and its introgression lines to several Xanthomonas oryzae pv.oryzae (Xoo)strains was determined at the late tillering stage of rice.Under indoor control conditions,YP and its introgression lines showed resistance to 9 different Xoo strains(C1,C2,C3,C4,C5,C6,C7,C9 and PXO99^A),indicating that they had broad-spectrum resistance to BB.The results of bacterial blight resistance gene detection showed that YP contained homologous genes of Xa10,while HX35,L214 and G252 contained susceptible homologous genes of xa23.The sequence alignment results showed that the sequence of the effector binding element (EBE_AvrXa10) region of the disease resistance gene Xa10 was significantly different from that of the Xa10 promoter in YP. Similarly, the recessive susceptible gene xa23 was consistent with the effector binding element (EBE_AvrXa23) region of the xa23 promoter in HX35, L214 and G252. The qRT-PCR results showed that the immune responses in YP,HX35,L214 and G252 were activated after inoculation with strain PXO99^A,but Xa10 in YP and xa23 in HX35,L214 and G252 were not induced to be expressed.It is thus hypothesized that YP,L214 and G252 may contain new genes for resistance to bacterial blight.

In order to improve blast resistance of the maintainer line Ruanhua B,to carry rice blast resistance genes Pi46 and Pi2 high-quality Indica H281 as the donor parent,Ruanhua B as recurrent parent,using marker-assisted selection(MAS)technology combined with pedigree breeding method,polymerization of two foreign genes with improved maintenance line Ruanhua B resistance,Ruanhua B was carried out on the characteristics of stable strain identification of resistance to rice blast,rice quality analysis,etc.Two BC₁F₆ populations,two BC₂F₅ populations and two BC₃F₄ populations with two homozygous target genes were obtained by backcrossing,multi-generation self-crossing and molecular marker detection.Field naturally induced identification showed that the improved lines of different backcrossing generations were resistant to rice blast.The sterility of backcross generation to sterile lines ranged from 52.7% to 100.0%.Agronomic traits and rice quality analysis showed that the improved lines basically conserved the main agronomic characters and rice quality characteristics of Ruanhua B.The results of SNP gene chip analysis showed that the background response rate of BC₁F₆ was 74.42%—77.77%,that of BC₂F₅ was 86.42%—87.75%,and that of BC₃F₄ was 92.27%—92.59%.Multiple resistance genes can be effectively polymerized by continuous backcross,self-cross and marker-assisted selection techniques to obtain a new maintainer line resistant to rice blast,and achieve rapid molecular improvement of maintainer line Ruanhua B.

Bipolaris sorokiniana is one of the important pathogens of maize root rot.The purpose of the present work was to establish a rapid detection method for B.sorokiniana based on the loop-mediated isothermal amplification(LAMP).Firstly,a set of primers for LAMP assay was designed according to the partial sequence of Brn1 involved in the melanin biosynthetic pathway.Then,the optimal reaction temperature was screened for this primer set,the specificity and sensitivity of LAMP reaction were detected,and the LAMP detection was evaluated by using the maize root rot samples artificially inoculated with B.sorokiniana.The results showed that the primer set designed could amplify the target gene Brn1 at 61—68 ℃,with 66 ℃ was the optimal temperature.In the specific detection,the primer set could specifically detect B.sorokiniana from the genomic DNA of 10 main pathogenic fungi isolated from maize root rot plants.In the sensitivity detection,the minimum detection limit for plasmid DNA carrying Brn1 was 10 copies/μL,and amplification could be achieved in about 25 min at the minimum concentration.And,for the detection of maize root rot samples,B.sorokiniana can be detected in 1 pg/μL of maize root tissue DNA.These results indicated that the LAMP detection method for B.sorokiniana established in this study has robust specificity and high sensitivity.