Sequencing Analysis And Detection Of Grapevine Leafroll Associated Virus-3 And Salt Tolerance Study In In Vitro Grapevine

Grapevine(Vitis)is one of the most economically important fruit trees in the world and China.Viral diseases cause huge losses to the grape industry.Grapevine leafroll disease(GLRD)is among the most damaging viral diseases,and grapevine leafroll-aassociated virus 3(GLRa V-3)is one of major causal agents of GLRD,and occurs widely in all grapevine-growing regions in the world and China.Salinity is one of the major abiotic stress to the grapevine production.GLRD and salt stress frequently occur simultaneously in the grapevine-growing regions.The objective of the present study was,therefore,(1)to investigate occurrence of GLRa V-3 in major grapevine-growing regions in Shaanxi and sequenced the full length of of GLRa V-3.Virus localization was conducted in red-and white-berried cultivars.Virus tolerance was evaluated in two cultivars and a wild(Vitis pseudoreticulata)accession Hunan-1;(2)to establish in vitro micrografting-based biological method for diagnosing GLRa V-3 in both red-and white-berried grapevine cultivars;(3)to investigate cross-talk between GLRa V-3 and salt stress in GLRa V-3-infected in vitro grapevine plantlets.The main results obtained were as followings:(1)A survey of occurrence of GLRa V-3 was conducted in 3 wine and 4 table grapevine cultivars in the major grapevine-growing regions of Shaanxi province.Results showed that for wine grapevines,occurrence frequencies were 92%,88% and 70% for 'Cabernet Sauvignon','Chardonnay' and 'Riesling' respectively.For table grapevines,occurrence frequencies were 73%,72%,86% and 85%,for 'Hutai-8','Red Globe','Shine-Muscat' and'Summer Black',respectively.Thus,the mean occurrence frequency of GLRa-V-3 was 81%in Shaanxi province.Sequencing analysis found that the full-length sequence of GLRa V-3was 18026 bp with 14 open reading frames,and shared 85.9% identities to GRLa V-3 LN,a isolate reported in China,and and 99.06% identities to 'ISAB-BR',a isolate from Brazil.GLRa V-3 isolate reported in Shaanxi was identified as a new GLRa V-3-Sau isolate.Immunohistochemistry for virus localization found that distribution pattern was similar in red-berried cultivar ‘Cabernet Sauvignon' and white-berried cultivar ‘Chardonnay',and GLRa V-3 is restricted in phloem tissue of vascular bundles.Virus transmission by micrografting of GLRa V-3-infected ‘Cabernet Sauvignon' scions upon healthy‘Chardonnay',‘Thompson Seedless' and ‘Hunan-1'(V.pseudoreticulata)rootstocks found virus transmission efficiency was similarly higher in ‘Chardonnay' and ‘Thompson Seedless' than in ‘Hunan-1',indicating that ‘Hunan-1' was relatively tolerant to GLRa V-3.(2)A micrografting-based method was developed for efficient diagnosing GLRa V-3.Healthy red-berried 'Cabernet Sauvignon' [Ca Sa(VF)] was used as rootstocks,and virus-infected 'Cabernet Sauvignon' [Ca Sa(VI)] and white-berried 'Chardonnay' [Ch(VI)]were used as scions.Micrografts of Ca Sa(VI)/Ca Sa(VF)and Ch(VI)/Ca Sa(VI)were cultured on 1/2 MS for different durations to induce virus symptoms in Ca Sa(VF)rootstocks.About 80% and 20% of Ca Sa(VI)rootstocks displayed typical GRLa V-3-induced symptoms in micrografats of Ca Sa(VI)/ Ca Sa(VF)and Ch(VI)/Ca Sa(VF)after 12 weeks of micrografting development.In order to improve symptom induction,Ca Sa(VF)rootstocks were removed from micrografts after 5 weeks of micrografting and cultured om 1/2 MS supplemented with 75 m M Na Cl.About 88% and 85% of Sa Ca(VF)rootstocks expressed GLRa V-3-induced symptoms after 4 weeks of culture under 75 m M Na Ca-induced stress.Interestingly,symptoms induced in Ca Sa(VF)micrografted with Ca Sa(VI)were leafrolling and leaf reddish coloration,while those in Ca Sa(VF)micrografted with Ch(VI)were leafrolling and leaf yellowing.On In order to explain why the different symptoms were induced in the same rootstock micrografted with red-and white grapevines,studies were conducted on vascular bundle development,relative expression levels of GLRa V-3 RNA,virus sequence,and anthocyanin biosynthesis genes.No significant differences were found in vascular bundle developments,relative expression levels of the virus RNA and gene sequencies.However,expression levels of anthocyanin biosynthesis genes LDOX,UFGT,My BA1 and anthocyanin levels were much lower in Ca Sa(VF)rootstocks micrografted with Ch(VI)than those in Ca Sa(VF)with Ca Sa(VI),thus failing to formation of reddish coloration in leaves of Ca Sa(VF)rootstocks micrografted with Ch(VI).(3)Healthy and GLRa V-3-infected in vitro plantlets of red-berried grapevine 'Cabernet Sauvignon' were cultured on 1/2 MS medium supplemented with 0,25,50 and 100 m M Na Cl,respectively.After 3 weeks of culture,100% and 90% of the healthy and the virus-infected shoots developed roots when cultured without Na Cl.Rooting percentages of the healthy shoots were 50%,30% and 10% when culture under 25 m M,50 m M and 100 m M Na Cl,while corresponding figures were 80%,60% and 60% in the virus-infected shoots.Vegetative growth of the healthy shoots was better than those of the virus-infected ones when cultured without Na Cl stress.However,vegetative growth of the virus-infected shoots was better than that of the healthy ones when cultured under 25-50 m M Na Cl stress.For the healthy plantlets cultured under Na Cl stress,levels of total soluble sugar and free proline increased,while levels of total soluble protein decreased,as Na Cl concentrations increased from 0 m M to 100 m M.Activities of superoxide dismutase(SOD)and peroxidase(POD),and malondialdehyde(MDA)contents increased as Na Cl concentrations increased from 0 m M to 100 m M.Activity of catalase(CAT)maintained relative stable regardless of Na Cl concentrations.Levels of indole-3-acetic acid(IAA)decreased as Na Cl concentrations increased from 0 m M to 100 M.Level of abscisic acid(ABA)was highest at 25 m M Na CL and decreased when cultured at higher than 25 m M Na Cl concentrations.Expression levels of IAA biosynthesis genes TAR2,TAR3,TAR4 and YUC1 in the healthy plantlets showed increased trends as Na Cl concentrations increased from 0 m M to 100 m M.Expression levels of ABA biosynthesis gens NCED1 and NCED2 in the healthy plantlets reduced as culture duration increased from 96 h to 2 weeks,while that of ZEP reached the highest after 1 week of culture.For the virus-infected plantlets cultured under Na Cl stress,levels of total soluble sugar and free proline increases,while that of total soluble protein drecerased,as Na Cl concentrations increased from 0 m M to 100 m M.Activities of SOD and CAT increased as Na Cl concentrations increased from 0 m M to 100 m M.Activity of POD reached the highest at 50 m M Na Cl,and then decreased.Contents of MDA increased as Na Cl increased from 0 m M to 25 m M,and maintained relatively stable as 50-100 m M Na Cl.Levels of IAA increased as Na Cl concentrations increased from 25 m M to 100 M.Level of ABA was no significant difference from 25 m M Na Cl to 100 m M Na Cl.Expression levels of IAA biosynthesis genes TAR2,TAR3,TAR4 and YUC1 in the GLRa V-3infected plantlets showed increased trends as Na Cl concentrations increased from 0 m M to100 m M.Expression levels of ABA biosynthesis gens NCED1 and NCED2 in the GLRa V-3infected plantlets reduced as culture duration increased from 96 h to 2 weeks,while that of ZEP increased as culture duration increased from 96 h to 2 weeks.The above results demonstrated that the healthy grapevine plantlets rooted and grew better than infected ones when cultured without Na Cl stress.However,when cultured under Na Cl stress,GLRa V-3-infected grapevine plantlets rooted and grew better than the healthy ones.These results indicate that GLRa V-3 infection improves tolerance of grapevines to moderate salt stress.Analyses of physiological metabolism,enzyme activities,levels of ABA and IAA,as well as expression levels of biosynthesis genes for ABA and IAA provided experimental data explaining why GLRa V-3 infection improves tolerance of grapevines to moderate salt stress.This study provided data of occurrence of GLRa V-3 in major grapevine-growing regions in Shaanxi province.Analysis of the full length of GRLa V-3 and its comparison with GLRa V-3 previously reported from other countries and China identified a new isolate GLRa V-3-Sau found in Shaanxi.Preliminary studies showed that ‘Hunan-1',the Chinese wild Vitis species(V.pseudoreticulata)was,to a certain content,tolerance to GLRa V-3.A micrografting-based method was developed for efficient diagnosing GLRa V-3 in both redand white berried grapevine.Our results also show that GLRa V-3 infection improves tolerance of grapevines to moderate salt tolerance.Therefore,the present study is of theoretical and applied importance in the grapevine industry.