[Objective]Botrytis cinerea,the causal agent of gray mold in facility vegetables,is characterized by a wide host range and rapid post-infection spread,often leading to severe economic losses.This paper aims to establish an early and precise detection method for gray mold before its visible symptoms appear,and to provide technical support for the early monitoring and targeted control of gray mold in facility vegetable cultivation.[Method]A conserved gene sequence(BC1G_05726)from B.cinerea strain B05.10 was used as the target.Specific recombinase polymerase isothermal amplification(RPA)primers and CRISPR/Cas12a crRNAs were designed and screened.Key parameters including reaction time and the concentration ratio of Cas12a to crRNA were systematically optimized to establish a rapid visual detection method combining RPA and CRISPR/Cas12a.Fluorescence signals were visualized using a portable blue light transilluminator.The specificity,sensitivity,and applicability of the developed method were evaluated using complex environmental samples(infested soil,seeds,symptomatic plants,and air samples).[Result]The optimal crRNA(crRNA-1)and primer pair(RPA-bc-F/R)were selected.The optimized detection conditions were as follows:RPA reaction at 37℃for 20 min,followed by CRISPR/Cas12a reaction with final concentrations of 200 nmol·L-1 for Cas12a and 50 nmol·L-1 for crRNA for another 20 min.The entire detection process was completed within 40 min.The sensitivity assay revealed a detection limit of 10-4 ng·μL-1 of genomic DNA of B.cinerea,which was 100-fold more sensitive than conventional PCR.Specificity testing showed that only B.cinerea produced positive fluorescence signals,while none of the other 14 tested fungal and bacterial species or the negative control generated any signal,indicating excellent specificity of the assay.In application tests,the method successfully detected B.cinerea strains from different hosts.After artificial inoculation on cucumber leaves,the pathogen was stably detected as early as 1 d post-inoculation,preceding symptom appearance.Dynamic monitoring of air samples in the facility environment demonstrated that B.cinerea could be detected in the air at 60 h post-inoculation,at which time typical disease lesions had not yet developed on the plants,validating the early warning capability of this method.[Conclusion]The RPA-CRISPR/Cas12a-based visual detection method established in this study enables rapid,sensitive,and specific detection of B.cinerea under isothermal conditions without requiring complex instrumentation,and the results can be read by the naked eye.This method is suitable for on-site detection of the pathogen in soil,seed,plant,and air samples.
Wheat(Triticum aestivum)remains vital to global food security,yet diverse pathogens constantly threaten its stable production.To address these threats,the identification and utilization of resistant genetic resources is the most effective and eco-friendly approach to manage disease epidemics.Based on a systematic review of the molecular mechanisms of wheat immunity,this paper compares the infection strategies of biotrophic and necrotrophic pathogens and elucidates the evolutionary arms race between wheat and its pathogens.Particular emphasis is placed on key immune regulatory mechanisms,including resistosome assembly and allele-specific evolution,modular cooperation mediated by kinase-integrated immune receptors,and host physiological homeostasis reprogramming driven by non-canonical resistance genes.In addition,the molecular basis by which pathogen effectors promote susceptibility through hijacking host immune pathways or exploiting hypersensitive response-associated cell death is discussed.Building upon these mechanistic insights,we summarize current strategies for resistance resource discovery and precision improvement,including the construction of panoramic resistance-gene atlases,the exploitation of novel resistance resources from wild relatives,and receptor optimization through targeted editing of key genetic loci.In response to the continuing evolution of pathogen populations and the increasing prevalence of multiple concurrent diseases,we further propose several emerging directions for resistance improvement,including the decoupling of immune activation from cell death,the evolution-guided design of universal immune receptors,and the establishment of multi-kingdom immune ecological barriers.These concepts provide a theoretical framework and technical foundation for the rational design of broad-spectrum and durable disease resistance in wheat.
[Objective]Roots are the most critical underground organs of plants.This study investigates the dynamic effects of grazing disturbance on the root functional traits of meadow steppe communities,aiming to clarify the mechanisms by which grazing affects the structure and function of grassland community roots.It provides insights into the regulatory mechanisms and survival strategies of grassland plants under grazing disturbance.[Method]The root functional traits including total root length,number of root tips,number of root forks,root average diameter,root surface area,and total root volume were measured using minirhizotron technology under six grazing intensity treatments:no grazing(G0),light grazing(G1),light moderate grazing(G2),moderate grazing(G3),heavy grazing(G4),and extreme grazing(G5).Each grazing intensity treatment was replicated with nine minirhizotron tubes.[Result](1)Both below-ground biomass and root-soil ratio exhibited significant decreasing trends with increasing soil depth(P<0.05).The maximum below-ground biomass and root-soil ratio were observed in the 0-10 cm soil layer under G4 grazing treatment,but there were no significant differences in below-ground biomass among grazing treatments.(2)The root morphological characteristics of meadow steppe communities vary significantly with the progression of the growing season and are affected by grazing intensity,with total root length and number of root forks showing an increasing trend over the growing season,whereas both root average diameter and total root volume reached their maxima in July before subsequent declines.Under varying grazing intensity gradients,root traits exhibit distinct patterns:moderate grazing(G1-G3)promotes root growth in the early growing season,whereas heavy or extreme grazing increases total root length and number of root forks in the late growing season but still reduces root average diameter and total root volume.(3)Specific root length(SRL)and specific root area(SRA)showed an increasing trend as the growing season progressed,peaking in September,while root tissue density(RTD)fluctuated.From June to July,SRL and SRA were highest under G3 grazing intensity,while from August to September,SRL increased with grazing intensity,and SRA decreased.RTD was generally higher under G3-G5 intensities compared to G0-G2.(4)Over time,plant height and coverage increased seasonally and peaked in September,while root-shoot ratio(R:S)decreased.With increasing grazing intensity,both height and coverage significantly decreased significantly in each month,while R:S significantly increased.(5)SRL and SRA were significantly correlated with plant community characteristics,and total root length and RTD were strongly correlated with R:S.However,average root diameter and number of root tips showed no significant correlation with soil factors or plant community characteristics.[Conclusion]The response of root morphological traits to grazing intensity exhibited dynamic differences,reflecting trade-offs in resource acquisition and structural strength at different periods.The functional composition of plant communities was strongly correlated with root traits,warranting further research.This study provides valuable insights into the response mechanisms and adaptability of meadow steppe plant roots to grazing intensity and seasonal dynamics.
[Objective]Early maturity is an important target trait in cotton genetic improvement in China,and flowering time is a key indicator of earliness.H3K9 histone lysine methyltransferases(SUVs)are involved in plant development and flowering regulation in model plants such as Arabidopsis thaliana and rice.This study aimed to investigate the role of the SUV gene family in flowering regulation in upland cotton and to identify elite allelic variations of key members.The findings provide valuable genetic resources for breeding early-maturing cotton cultivars.[Method]Protein sequences of the SUV family from Arabidopsis were used as reference sequences.HMMER,MEME,and TBtools were employed to identify and analyze SUV family members in upland cotton at the whole-genome level.Candidate genes involved in flowering regulation were screened through integrated RNA-seq and RT-qPCR analyses.Virus-induced gene silencing(VIGS)was used to validate the functions of candidate genes in flowering regulation.Resequencing data from 419 upland cotton accessions were analyzed to identify SNPs within GhSUV1,which were further validated by Sanger sequencing.Allele frequencies and genetic differentiation were examined across different cotton ecological regions.[Result]A total of 200 SUV homologous proteins were identified in nine higher plant species,including rice.Phylogenetic analysis divided them into SUVH and SUVR clades,indicating a relatively conserved evolutionary pattern.In upland cotton,28 GhSUV genes were identified.Their expansion was mainly driven by polyploidization events.Most collinear gene pairs showed Ka/Ks<1,suggesting strong purifying selection.Gene structure and motif analyses revealed high similarity among members within the same clade.Promoter analysis showed that GhSUV genes are enriched in light-responsive and hormone-responsive cis-elements,indicating their involvement in multiple developmental regulatory pathways.RNA-seq and RT-qPCR analyses showed that GhSUV1 and GhSUV13 were expressed at higher levels in early-maturing materials.They were also highly expressed in leaves and certain floral organs.Functional validation using VIGS demonstrated that silencing GhSUV1 delayed squaring and flowering by 5.6 and 6.0 days,respectively.Silencing GhSUV13 delayed these stages by 5.8 and 6.8 days compared with the control.Moreover,suppression of GhSUV1 and GhSUV13 significantly reduced the expression of flowering-related genes GhSOC1,GhAP1,GhFT,and GhCAL,while significantly increasing GhSVP expression.These results indicate that both genes participate in flowering regulation.Sequence analysis identified a SNP in the exon region of GhSUV1 that was significantly associated with flowering time.The early-flowering GG allele increased in frequency with latitude.Genetic differentiation index and nucleotide diversity analyses revealed significant differences in the GhSUV1 genomic region between early-and late-maturing cultivars,suggesting that this locus has undergone breeding selection.[Conclusion]GhSUV1 and GhSUV13 are involved in flowering regulation in upland cotton.The elite allele of GhSUV1 is closely associated with early flowering and has been subject to breeding selection.These findings provide important genetic resources for the improvement of early-maturing cotton varieties.
[Objective]The GDSL esterase/lipase(GELP)family comprises a group of multifunctional hydrolases with broad substrate specificity and catalytic versatility,playing an essential role in plant growth and developmental processes.The GDSL lipase gene GhGELP23D from Gossypium hirsutum is highly expressed during cotton fiber elongation development.This study aims to investigate its function in plant cell elongation and provides references for the mechanism elucidation of GELP-mediated regulation of cell growth.[Method]The GhGELP23D was cloned from upland cotton fibers.Bioinformatic analyses were performed to characterize its physicochemical properties,structural features,and phylogenetic relationships.The promoter region was analyzed with PlantCARE to identify putative cis-elements.Expression patterns were detected based on public transcriptomic data and RT-qPCR validation.A GUS reporter construct driven by the GhGELP23D promoter was generated to examine the tissue-specific expression of GhGELP23D in stably transformed Arabidopsis.Subcellular localization of GhGELP23D was determined through transient expression in Nicotiana benthamiana leaves.Transgenic Arabidopsis lines heterologously expressing GhGELP23D(GhGELP23D-OE)were obtained using the floral-dip method,and phenotypic analyses were conducted.Additionally,virus-induced gene silencing(VIGS)was employed to suppress GhGELP23D expression in cotton.Silencing efficiency was verified by RT-qPCR,and the resulting changes in fiber length were assessed in GhGELP23D-VIGS plants.[Result]GhGELP23D encodes a 356-amino-acid protein that is stable,weakly basic,and hydrophilic,containing a typical signal peptide but no transmembrane domain.Transcriptomic analysis and RT-qPCR detection showed that GhGELP23D was highly expressed during cotton fiber elongation.Subcellular localization analysis revealed that GhGELP23D is predominantly localized to the extracellular space.Analysis of the GhGELP23D promoter indicated the presence of multiple cis-elements related to light responsiveness,hormone signaling,and stress response.Histochemical GUS staining showed that the GhGELP23D promoter can drive reporter gene expression in various Arabidopsis tissues,indicating broad tissue expression activity.Heterologous expression of GhGELP23D in Arabidopsis(GhGELP23D-OE)significantly enhanced plant growth and development,resulting in increased plant height and longer primary roots and root hairs.Furthermore,complementation of GhGELP23D in the mutant resulted in a pronounced recovery of primary root and root hair length similar to that of the wild type(WT).In cotton,the fiber length of GhGELP23D-VIGS lines was significantly reduced compared with that of empty-vector control plants,with an average decrease of approximately 13.14%.[Conclusion]GhGELP23D encodes an extracellular GDSL lipase that is highly expressed during the fiber elongation stage of upland cotton and plays an important role in plant cell elongation development.
