| Verticillium wilt is caused by "Verticillium dahliae Kleb" a cosmopolitan fungus,this pathogen is divided into defoliation(D)and non-defoliation(ND)pathotypes on some hosts,including tomato,cotton,and olive.In China,Verticillium wilt of cotton has a direct economic impact of ca.250-310 million US dollars.Xinjiang province accounts for more than 60%of cotton output in China,and Verticillium wilt is challenging this productivity as the most virulent D-strain appeared to be expanding its niche in Xinjiang annually.Therefore,investigation of etiology of Verticillium wilt,the prevalence,and the adaptation of D-pathotypes on cotton is now a new challenge for better control of Verticillium wilt of cotton in Xinjiang.A survey was conducted during the 2017 cropping season to assess the situation of Verticillium wilt of cotton in Xinjiang province.About 7400 samples of cotton stems were treated for the isolation of V.dahliae out of the total 7500 samples collected from 44 fields during the 2017 cropping season.A total of 1345 isolates were separated from the samples,all of which were genotyped using pathotype markers.We detected a total of 776 isolates as defoliating(D);517 isolates as non-defoliating(ND),while 52 isolates have an absence or both of D or ND markers;while all the 1345 isolates are race type 2(R-2),so also all of them are mating type 2(MAT-1-2-1).In vitro screening for growth responses of ten isolates(5 D&5ND)at six temperature(15?,20?,25?,28?,30?,&32?,)conditions were tested and temperature was found to have significant effect on mycelial growth at all temperature tested,with overall decreasing growth responses in both directions from 25?.There are four main results:1)The D-strains growth faster than the ND-strains under the higher temperature(30?,D strains:HB19=40.33 mm;ND strains:XJ02=26.93 mm;Std Error=0.858;P=0.05)and at(32?,D strains:XJ28=28.40 mm;ND strains:XJ02=18.27 mm;Std Error=0.858;P=0.05);2)D-pathotypes showed significantly more melanization compared to ND-pathotype under growth condition at 15?,30? and 32?.3)The conidia length displays significant different between D and ND-pathotypes under 25? growth condition[D(HB10)=8.35?m Vs ND(XJ63)=8.1 ?m,SE=0.12;P=0.05]and 30?[D(HB19)=9.21?m Vs ND(XJ02)9.04?m SE=0.12;P=0.05].4)D-pathotypes displayed higher virulence compared to ND-pathotypes at both temperature tested(DI;D=38.25 Vs.ND=31.25 at 25?)and(DI;D=39.25 Vs.ND=29.75 at 28?).The D-strains also present higher pathogenicity at 28?(DI=39.25)than at 25?(DI=38.25).To further understand the molecular bases of variation in their temperature tolerance and RNASeq study using D-strain(XJ28)and one ND-strain(D08047)was conducted to globally identify the response genes of V.dahliae pathotypes under temperature stress.A total of 3469 DEGs were detected as V.dahliae response to temperature stress,and the DEGs were obtained using p<0.0005,Padj<0.05,and the absolute value of Log2Ratio>1.0 as a threshold.The ND-strain recorded only a total of 98 DEGs at average growth temperature,while at 32?,it expressed a total of 690 DEGs,whereas,at 28?,a total of 851 DEGs were detected.While D-strain at average temperature has only 45 DEGs,but at 32?,a total of 572 DEGs was detected,while at 28?,a total of 468 DEGs were detected.The above information indicated that both D and ND-strains expressed few DEGs at 25?,while D-strain has the highest number of DEGs at 32?(572);in contrast,the ND-strain recorded the largest number of DEGs at 28?(851).Comparing D-strains and ND-strain at normal growth temperature recorded a total of 1868 DEGs,while at 28?,a total of 2278 DEGs was detected,but at 32?,only a total of 793 DEGs was recorded.We constructed Ben diagram to look at DEGs at special conditions,and in relationships.We consider specific DEGs of both pathotypes at the higher temperature,where the D-strain recorded 140 specific DEGs at 32?,while the ND-strain recorded a total of 347 DEGs specifics at 32?.The common DEGs of D-strain at 32? are only 72,while common DEGs of the ND-strain at 32? are 111.Whereas common DEGs of both D and ND-strains at 32? are 420,while specific DEGs of D and ND-strains at 32? are 179.KEGG was used to comprehensively illustrate and give an overview of the pathways DEGs involved in V.dahliae response to temperature stress.V.dahliae used various pathways to respond to heat stress depending on pathotypes and amount of stress.For example,at XJ28(25? vs 32?)are metabolic pathway,and biosynthesis of secondary metabolites are most enriched,while at D08407(25? vs 32?)its metabolic pathway,and endocytosis.We observed that the majority of the genes were involved in metabolic pathways,and as D-strain tends to repressed genes,the ND-strain seems to induce genes in response to heat stress.We screened those candidate DEGs into 48 target genes of V.dahliae response to high temperature.Expression pattern clustering was used to illustrate the expression pattern of these DEGs for both pathotypes and temperature stress.One genes cluster was conspicuously repressed at XJ28(25?);at XJ28(28?)and D08047(32?),but induced in XJ28 at 32?,and these are unique thermal response genes of the D-strain at a higher temperature(32?).Another genes cluster was conspicuously repressed at D08047(25?);D08047(28?),also XJ28(32?),but induced in D08047 at 32?,and these are unique temperature response genes of ND-strain at 32?.We finally summarised and separated these 48 target genes in to fold change and Functional annotation tables.Common to XJ28(32?)compared to its self at 25?,at 28?,also compared with ND at 32? are 20,and include some Secretomes,Cysteine-rich,and CAZYmes,with various ortholog functions,encoding protein or protein families,e.g.,Alcohol dehydrogenase superfamily,zinc-type,ATP-dependent,DEAH-box type,conserved site,Amino acid/polyamine transporter I,etc.While the ND-strains,have 28 target genes,including some Secretomes,Cysteine-rich,CAZYmes,and Transcription factors with various ortholog functions,encoding protein or protein families,e.g.,Protein kinase-like domain,Short-chain dehydrogenase/reductase SDR,Zn(2)-C6 fungal-type DNA-binding domain,etc. |
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