| Fusarium crown rot(FCR),caused by Fusarium spp.,has become a widely distributed soil-borne disease throughout the word.This disease has been reported in Australia,the USA,Mediterranean countries,south America,Africa and China,and caused serious economic losses.The FCR has been an important limiting factor due to causing yield reduction and toxins contamination which does harm to the health of humans and animals.FCR is a new reported disease in China,the largest producer of wheat in the world.In recent years,FCR has become more and more serious owing to accumulation of pathogen population caused by planting susceptible varieties and returning the straws year by year in China.In this study,the occurrence and distribution of the FCR were systematically investigated in Huanghuai wheat growing region(HHWGR)of China,and the distribution pattern of FCR,pathogens and population structure of Fusarium pseudograminearum were clarified.The results showed that F.pseudograminearum is the predominant pathogen species in HHWGR,meanwhile the abundant genetic diversity was detected in this species.The genome of strong pathogenic strain WZ-8A isolated by our group was further sequenced by using a new generation of sequencing technology.Furthermore,identification and functional analysis of nine genes coding salicylate hydroxylase were carried out by sequences searching from F.pseudograminearum genome and RNA-seq analysis.In total,our results will provide a theoretical basis for disease monitoring and control strategies being adopted,as well as being helpful for the study on the molecular mechanism in pathogenicity of F.pseudograminearum.The main results were as follows:FCR is serious damage in Henan,Shandong,Hebei and Shanxi province,where F.pseudograminearum is the frequently isolated in these regions,followed by F.graminearum.Whereas F.graminearum is most common in northwest of Shaanxi Province and north of Jiangsu and Anhui province.Throughout the HHWGR,the isolation frequency of F.pseudograminearum accounts for about 50%,followed by F.graminearum,the total frequency of the two pathogen species is above 80%.The pathogenicity results showed that F.pseudograminearum caused the most severe FCR symptom among the nine tested Fusarium spp.These results demonstrated F.pseudograminearum was the dominant pathogen species causing FCR in the HHWGR.In addition,both of F.pseudograminearum and F.culmorum caused the obvious more serious(P < 0.05)yield losses(20.2%~26.6%)than F.graminearum under the condition of artificial inoculation in the field assay.In HHWGR,genetic diversity is abundant in F.pseudograminearum population.Phylogenetic analysis of EF-1? gene sequence showed that 194 F.pseudograminearum isolates were differentiated into two distinct clades which were close to strains from Australia and China respectively,indicating that gene variation was detected in F.pseudograminearum population in HHWGR,however no geographical regularity was found in these variations;URP analysis results showed that a certain of gene flow was detected among different geographic populations(Nm = 2.2252),however the genetic diversity within a population accounts for a major proportion of the total level of diversity.Clusters were generated from these 194 isolates relating to their geographical distributions.Amongst these eight populations,Hebei population closes to the three Henan populations,while the Shaanxi and Shanxi population are closer to the two Shandong populations,and these eight populations were clustered into two distinct clades.These results suggested that these population differentiations might attribute to the nature of this species(such as sexual recombination being difficult in this species),their biotopes(climate,soil and farming system)and human factor(such as seed exchange and mechanical cross-zone operation,etc.).A high-resolution genetic map of WZ-8A has been obtained in this study.The quality of genome sequencing was evaluated by 140× sequencing depth and well assembling(contig N50 and scaffold N50 were 7.8Mb and 9.0Mb,respectively).Finally,a total size of 37.5Mb genome size containing 10 Contigs,6 scaffolds and 4 gaps were extracted and 12234 functional genes(account for 99.12%)were classified by genome annotations,including 1790 CAZyme genes,36 secondary metabolic gene clusters and 1266 putative secreted protein coding genes.By comparing the WZ-8A genome sequences with other Fusarium spp.,a high level of synteny relation was found between the strains WZ-8A and CS3270,however,a major of differential genes located in the end of the chromosomes and near the centromere in WZ-8A.A total of nine salicylate hydroxylase genes(Shys)were identified by sequences searching from F.pseudograminearum genome and RNA-seq analysis.The results of RNA-seq showed that Shy1,Shy2,Shy4,Shy5 and Shy8 were up-regulated expression during F.pseudograminearum infecting the wheat.Of these genes,the expression of Shy4 is induced by the process of infection and colonization.Additionally,shy9 is probable down-regulated,while the expression of Shy3,Shy 6 and Shy7 were not examined.Deletion mutants of these nine Shys genes(including two Nah G,six Nah W and one another type of genes)were obtained successfully by using the split-PCR method,while complement mutants were obtained by gene cloning and genetic transformation techniques.The results of basic phenotypes of these mutants showed that no significant change was detected in the single deletion mutants of the two Nah Gtype Shys genes in mycelia growth,sporulation and pathogenicity by comparing with the wild type.However,Fpg Shy1 plays an important role in the degradation process of salicylic acid.In the single deletion mutants of those Nah W-type genes,compared with the wild type,?Shy4,?Shy8 and ?Shy9 grew more slowly than the wild type and displayed different pigmentation on PDA,while sporulation of both of Shy4 and Shy8 mutants were significantly reduced(P<0.005).In addition,the results of pathogenicity test indicated that these three Nah W-type genes might be involved in pathogenic processes in F.pseeudograminearum. |
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