Primary Pathogenicity Analysis Based On The Gene Expression,Diversity And Transmission Of Candidatus Liberibacter Asiaticus

Citrus Huanglongbing(HLB)is one of the most devastating diseases,which affects the development of the citrus industry worldwide.The bacterium has not been able to obtain pure culture in vitro,which hinders the research on its biological characteristics,genetic variation,and molecular mechanism of pathogenicity.The available of the whole genome sequence of HLB bacteria provides a theoretical reference for the research of the genetic diversity of HLB bacteria.In this study,to investigate the possible molecular mechanism of pathogenicity of Candidatus Liberibacter asiaticus(CLas),we firstly checked the expression level of putative pathogenesis-related effectors genes in different symptomatic samples,and some of the putative defence related genes in citrus.Secondly,genetic diversity was carried out with Pakistan isolates.CLas transmission from citrus to tobacco and periwinkle via dodder(Cuscuta indecora)was also performed.The main results are as follows1.Pathogenesis related effector gene expression analysis: we collected the samples showing different symptoms from main citrus-producing areas in China,including(Jiangxi,Yunnan and Hainan)provinces.Symptoms of Yellowing(Y),Blotchy mottling(BM),Vein corking(VK)and Zinc deficiency(ZD)were analyzed.The expression level of nine effector genes analysed by q RT-PCR were followed: CLas＿03230,CLas＿04025 and CLas＿04560showed higher expression level in VK symptom.The CLas＿02075,CLas＿02145,CLas＿00530 and CLas＿05320 showed less expression level.CLas＿03230,CLas＿4560,and RS03120 showed higher expression levels in ZD.CLas＿02075 and CLas＿05320 showed fewer expression levels.In BM symptom.CLas＿04560 showed higher expression level than CLas＿02145,CLas＿00530,CLas＿05320,CLas＿03230,CLas＿5315,and CLas＿04025.CLas＿05315 showed higher expression levels in yellowing samples than CLas＿03230,CLas04560 and RS03120.For the gene expression analysis of 3 defence-related genes in citrus,HSP and Drle1 showed high expression in VK,ZD,BM and Y samples.The CSSP gene showed high expression in VK,BM and Y symptoms but showed less expression in ZD symptom.2.Diversity analysis of CLas from Pakistan based on the Cthr gene: 19 samples were collected from Khanewal,Pakistan.The 13 samples we got positive bands of the Cthr gene.Then the Cthr gene was amplified,cloned and sequenced.The results revealed that single and multiple bands were got from different isolates.Among 13 samples,we got a single band from 5 isolates,double bands from 7 isolates and three bands from 1 isolate.The sequences ranged from 950-1800 bp.From Kinnow-1 isolate three types of sequences 1517 bp,1770 bp,and 1518 bp were obtained.In Kinnow-5 two types of sequences of 1614 bp and 1419 bp were got.Whereas,in sweet orange-4.2 and sweet orange-4.3,1566 bp and 1374 bp sequences were got respectively.Based on the phylogenetic tree analysis,Kinnow-23 isolate is showed closer similarly with the representative Chinsese isolate CLas str.Gxpsy and CLas str.UF0506 from Florida.Whereas the rest of all sequences indicated resemblance to CLas str.A4(Chinsese isolate).While no sequence showed connection to the CLas str.SC2.3.CLas transmission via dodder from citrus to periwinkle(Catharanthus roseus)and tobacco(Nicotiana tabacum cv.Xanthi and N.tabacum cv.Hongda): periwinkle and tobacco seeds were pot-cultured in growth chamber separately.Dodder(Cuscuta indecora)seeds were grown in periwinkle and tobacco seedling pots one week later.C.indecora tendrils were put on CLas infected citrus lemon for 20 d.After establishing a firm parasite on citrus,the tip of tendrils was connected to periwinkle and tobacco seedlings to transmit CLas.The dodder started haustorium formation on periwinkle and tobacco after 6 and 15 d respectively.The results revealed that the haustorium formation was faster in periwinkle compared to tobacco species.The periwinkle plants showed yellowing symptoms at 35 dpi whereas there was no symptom on tobacco(N.tabacum cv.Xanthi and N.tabacum cv.Hongda)until 6 months after infection when light blotchy mottling symptom started to appear.To verify the presence of CLas,g DNA was extracted from N.tabacum cv.Xanthi and N.tabacum cv.Hongda,periwinkle and dodder at different time points.PCR analysis with the 16 S primers showed strong positive bands in periwinkle 40 d after connection with dodders.Lighter bands in N.tabacum cv.Hongda compared with N.tabacum cv.Xanthi which proves the higher infection rate in periwinkle than in tobacco spp.Our research laid a firm foundation for future studies on the pathogenicity of CLas.