| Sweetpotato(Ipomoea batatas(L.)Lam)was one of the most important crops to provide staple food,feedstuff,bioenergy and industrial raw materials in the world.China was considered to have the largest production and consumption of sweetpotota in the world,occupying 71%of the world total yield reported from FAO at 2013.However,because of uncontrolling introduction of seeding,the occurrence of new diseases or those previously occurred in limited areas have brought new threats to sweetpotato production in China.A new stem rot of sweetpotato was observed recently in the fields in Guangdong,GuangXi,FuJian,HeNan,and ChongQing provinces,China,which was different from those of stem rot of sweetpotato in previous reports.Initial,the plant leaves grew yellow and black water-soaked rot occurred on bottom of the stems which could extent to the top of the stems gradually.Finally,the entire plant was collapsed and death.In the present study,the newly found disease was recorded and its pathogens were identified,genetic structure of pathogen populations in six regions have been analyzed in details,and the profiles of differential gene expression before and after pathogen infection were established using RNA-seq analysis.The main results are as follows:(1)Many strains of sweetpotato stem rot were isolated from the samples collected from Guangdong,Guangxi,Fujian,Henan Provinces,Chongqing City and etc.Based on examination using Koch's Rule in combination with morphological observations,physiological and biochemical tests,and 16S rDNA sequence analysis,the pathogenic bacteria were confirmed to be Dickeya dadantii(Erwinia chrysanthemi).This is the first report of D.dadantii(E.chrysanthemi)causing bacterial stem and root rot of sweetpotato in China.(2)The genetic diversity was investigated for the D.dadantii population of sweetpotato from six different regions using REP-PCR fingerprinting techniques.Forty-one obvious bands were amplified,in which there were 36 polymorphism bands.At each pair of primers,4-10 alleles were detected with an average of 7.2.On the level of species,effective number of alleles(Ne),diversity index(H)of Nei's genes,and Shannon's information index(I)were 1.7805,1.4768,and 0.2801,respectively.The results showed that D.dadantii populations were considered to have a rich genetic diversity,while there were significant differences between pathogen populations in diffeent regions.Genetic diversity in Zhangjiang pathogen population was the highest,the lowest genetic diversity was found in Nanning pathogen population.(3)When the genetic similarity coefficient(GSC)was 0.80,the 59 strains of bacterial stem and root rot of sweetpotato collected from six different regions were divided into five major groups using UPGMA method according to genetic distance and GSC.The group I contained 13 strains,in which 10 strains were collected from GuangZhou,and 3 strains from ZhanJiang.Group ? contained 6 strains collected from ZhanJiang.Group III included NanNing and HePu populations.Group IV contained 10 strains from DongXin population,and Group V contained 10 strains from WanZhou population.The results suggested that the groups divided according to GSC was associated with geographic origins in some degree.(4)Nine sweetpotato cultivars were inoculated with 62 strains from 7 regions using tuber inoculation method.The results showed that pathogens population collected from different regions were different in infection ability,In which HePu and ZhanJiang populations had the highest capacities to cause stem rot disease,while wanzhou population had the relatively weakest pathogenicity.Pathogen stains,collected from the same region,causing bacterial stem and root rot to sweetpotato had significant differences in infection ability,such as strain E59 from NanNing had the strongest infection capacity,while strain E173 from WanZhou had the least infection capacity.Therefore,it seems to indicate that D.dadantii has wide pathogenesis division in different regions.(5)The tolerant abilities of 1355 accessions of sweetpotato against D.dadantii were tested using stem injected inoculation method.None of them was immune,but there were considerable differences in resistance.The results showed that there were 75 accessions with high resistance,83 accessions with resistance,229 accessions with moderately resistance,284 accessions with susceptible,and 684 accessions of high susceptible.The result revealed that sweetpotato germplasm hadrich resources of resistance genes against D.dadantii.Besides,no correlation was found between the response of tuber and stem inoculation by D.dadantii,such as Guangshu 87 was resistance of stem while was susceptible of tuber.(6)Digital gene expression analysis was used to identify the transcriptional changes at the timepoints of Oh,3h,9h,18h and 24h incubated with D.dadantii in the resistant sweetpotato cultivar,Guangshu 87.After removing the lower quality sequences and adaptor sequences,the 7623245,8380087,7616466,7915727,and 6954337 clean reads,using to annotate 2865375,3469073,3164368,3311301,and 2571990 genes,respectively,were obtained from the samples incubated with D.dadantii at the timepoints of Oh,3h,9h,18h,and 24 h.We found that around 60%of total clean reads were mapped onto the reference gene with total mapped reads,and generated 54193,57583?57451?56098,and 53222 ? annotated genes from the five libraries(R0,R3,R9,R18 and R24).2891 differentially expressed genes were detected in R3 compared with R0,including 2167 and 724 of genes were up-and down-regulated,respectively.The comparison of R9 with R0 revealed that 4825 differentially expressed genes were significantly up-regulated and 2229 genes were down-regulated.By contrast,Up to 4944 genes were up-regulated and 4792 genes were down-regulated in R18 compared with R0;Besides 6328 differentially expressed genes were detected in R24 compared with R0,including 3193 and 3135 of genes were up-and down-regulated,respectively.There are 581 differentially expressed genes that were common genes detected in all five libraries.(7)Digital gene expression analysis was used to identify the transcriptional changes in susceptible weetpotato cultivars guangzishu number 2.After removing the lower quality sequences and adapter sequences,8624410,8618610,and 8615650 clean reads were obtained with 49 nt in length in the samples at 0,9,and 18 h post inoculation.The numbers of clean reads that could be mapped onto genes with perfect match in 0,9,and 18 h were 4053056,4126736,and 3710426,respectively.We found that around 60%of total clean reads were mapped onto the reference gene with total mapped reads,and generated 55813?56093,and 54549 annotated genes from the three libraries(S0,S9,and S18).488 differentially expressed genes were detected in S9 compared with S0,including 24 and 464 of genes were up-and down-regulated,respectively.By contrast,up to 1013 genes were up-regulated and 1409 genes were down-regulated in S18 compared with SO.There are 259 differentially expressed genes that were common genes detected in all three libraries.(8)To further characterized these differentially expressed genes,we used Gene Ontology for their annotation.These differentially expressed genes belonged to three main categories(biological process,cellular component,and molecular function),distributed in 47 categories in resistant sweetpotato cultivar guangshu 87,and distributed in 44 categories in susceptible sweetpotato cultivar guangzishu number 2,including the most dominant pathways such as cell,membrane,antioxidant activity,biological regulation,metabolic process regulation,and response to stimulus.Biological processes were 50%in three main categories,showing that it plays a major role in the infected host process.Kyoto Encyclopedia of Genes and Genomes(KEGG)annotation showed that,differentially expressed genes were identified involved in resistance related metabolic pathways of plant-pathogen interaction,phenylpropanoid biosynthesis,plant hormone signal traisduction,flavonoid biosynthesis,and peroxisome,etc.Visibility,various of disease resistance pathways and the differential expression genes which referred to defense reaction and signal transduction were inspired in sweetpotato after inoculation with D.dadantii.Differentially expressed genes involved in resistance related metabolic pathways in guangshu 87 were more than the number of guangzishu number 2 after inoculation.These shows that the molecular mechanism of response to pathogen infection was controlled by polygenic network system,and provide a reference to identification and functional analysis of candidate genes. |
PDF Full Text Request