Responses Of In Vitro Grapevine Plantlets To Grapevine Leafroll-associated Virus Infection And Peg-induced Drought Stress,and Preliminary Evaluations Of Tolerance Of Grapevine Rootstocks To Grapevine Leafroll Disease

Grapevine?Vitis?is one of the most economically important fruit crops worldwide.Viral diseases,a major biotic stress,have for long time threatened sustainable development of grapevine production.Grapevine leafroll disease?GLD?is among the most damaging viral diseasesand widely spread in the grapevine-growing regions of the world.Drought is a major abiotic stress that has limited high yield and quality of grapevine production.Virus infection and drought stress frequently occur simultaneously in many of grapevine-growing regions,particularly in the northwest of China,a major region for production of both table and wine grapevines.The objectives of the present study were:?1?to establish a fast and highly efficient method for detection of Grapevine leafroll-associated virus-3?GLRaV-3?using in vitro indicators grown under PEG-induced drought stress;?2?to reveal the mechanisminvolved in enhancing symptom development of GLRaV-3 in grapevine in vitro plantlets grown under PEG-induced drought stress;?3?to analyze effects of GLRaV-3infection and PEG-induced drought stress,singly or in combinations,on growth,physiological metabolism,hormone levels and expression of the genes related;?4?to evaluate the tolerance of grapevine rootstocks to grapevine leafroll diseases using three different grafting methods in different scion/rootstockcombinations.Results obtainedare summarized as follows:?1?.An in vitro graft-free protocol for indexing of Grapevine leafroll-associated virus-3?GLRaV-3?was developed.In vitro GLRaV-3-infected shoots of Vitis vinifera‘Cabernet Sauvignon'were cultured on half-strength Murashige and Skoog?1962?medium?MS?supplemented with 0%,2%and 4%polyethylene glycol?PEG?8000 to induce development of the virus symptoms.Symptom expression started after 14,14 and 6 days cultured on medium containing 0%,2%and 4%PEG,respectively.After 4 weeks of culture,81%of‘Cabernet Sauvignon'showed symptoms when cultured on MS medium containing 4%PEG.Under stress of 4%PEG,‘6-12-2'?V.pseudoreticulata×V.vinifera?,‘Red Globe'?V.vinifera?and‘Kyoho'?V.vinifera×V.labrusca?started to show symptoms after 5,6 and 12days culture,respectively,and 100%,72%and 60%of plants expressed symptoms after 4weeks culture,respectively.PEG-induced drought stress improvedin vitro biological indexing of GLRaV-3 in red grapevine cultivars.The techniques developed in the present study would have potential application to GLRaV-3 indexing of red grapevine cultivars.?2?.In vitro‘Cabernet Sauvignon'plantlets infected with GLRaV-3 were grown on MS supplemented with 4%PEG to induce drought stress.Eleven anthocyanin-related peaks were detected by HPLC in the infected plantlets with or without PEG-induced drought stress,but the peaks were significantly higher in infected plantlets grown underPEG-induced drought stress.Increased accumulation of total anthocyanin compounds was related to the development of GLD symptoms in the infected plantlets under PEG stress.The highest level of up-regulated gene expression was found in GLRaV-3-infected leaves with PEG-induced drought stress.Analyses of variance and correlation of anthocyanin accumulation with related gene expression levels found that GLRaV-3-infection was the key factor in increased anthocyanin accumulation.This accumulation was related with up-regulation of the two key genes,MYBA1 and UFGT,and their expression levels were further enhanced by drought stress.?3?Responses of in vitro-grown plantlets?V.vinifera‘Cabernet Sauvignon'?to GLRaV-3and PEG-induced drought stress were studied.Results showed that stress induced by either virus infection or PEG had negative effects on vegetative growth,caused significant decreases in total soluble protein and increases in free proline,induced obvious cell membrane damage and cell death,and markedly increased accumulations of O₂^·-and H₂O₂.GLRaV-3-infection dramatically reduced the chlorophyll of leaves and lowered its photosynthetic capacity.Co-stress by virus and drought had much severer effects than single stress on the said parameters.Virus infection alone did not cause significant alternations in activities of POD,ROS and SOD,and contents of MDA,which,however,markedly increased in the plantlets when grown under single PEG-induced drought stress and co-stress by the virus and drought.Levels of ABA increased,while those of IAA decreased in the plantlets stressed by virus infection or drought.Simultaneous co-stress by the virus and PEG-induced drought had co-effects on the levels of ABA and IAA.Up-regulation of expressions of ABA biosynthesis genes and down-regulation of expressions of IAA biosynthesis genes were responsible for the alternations of ABA and IAA levels induced by either the virus infection or drought stress and co-stress by them.Experimental strategies established in the present study using in vitro system facilitate investigations on‘pure'biotic and abiotic stress on plants.?4?.Evaluation of tolerance of grapevine rootstocks to GLD was conducted in grafted plants using 3 grafting methods and 3 virus infected scions?Franc free of virus,LR131infected with GLRaV-1,and LR132 infected with GLRaV-1 and GVA?and 4 healthy rootstocks.In in vitro grafting,micrografts failed when LR132?V.vinifera‘Canbernet Franc'?was used in combinations with all rootstocks.Similarly high survival rates were obtained when LR131?V.vinifera‘Canbernet Franc'?and Franc?V.vinifera‘Canbernet Franc'?were used as scions,and however LR131 delayed scion bud break and root formation on rootstock and lowered the vegetative growth of the grafts in LR131 scion/Freedom?V.champinii×1613C?and 101-14?V.riparia×V.ruprestris?rootstock combinations.Histological observations showed that LR132 delayed the callus formation at graft conjunctions and no vascular bundle was established between scion and rootstock.Obvious callus formation and subsequent vascular bundle development were observed in both LR131 and Franc micrografts.A higher GLRaV-1 concentration was detected in LR131/AXR?V.vinifera×V.rupestris?micrografts than in LR131/Freedom ones.In green grafting,survival rate was lower on AXR than onother rootstocks.Markedly reduced survival rate and biomass were obtained in LR132grafted on Freedom and 101-14.LR131 and LR132 showed much severer leafroll symptoms when grafted onFreedom and 101-14 than grafted on St.George?V.rupestris?and AXR.In LR132 grafts,St.George had the highest level of both GLRaV-1 and GVA,and 101-14 had the lowest level of both GLRaV-1 and GVA;In bench grafting,Franc had a high survival rate of90%-100%on all rootstocks and more than 95%of themsurvived after transferred to field.LR131 had 78%survival rate,regardless of the rootstocks and more than 95%of them survived after transferred to field.LR132 only survived by 18%,58%,47%and 47%when grafted on Freedom,St.George,101-14 and AXR,respectively.All the LR132/Freedom died and only 27%of LR132/101-14 survived after transferred to field;LR132 on St.George and AXR kept a high survival rate at 93%and 87%,respectively,after transferred to field.Overall,grafting methods,virus status of scion,rootstock genotypes and their interactions all affected the survival rate of grafts.St.George had the highest tolerance to leafroll virus in grafts,followed by AXR with also good leafroll virus tolerance.Both Freedom and 101-14 were sensitive to leafroll virus in grafts.The present results provide important information in choosing rootstocks,especially in grapevine-growing regions with severe leafroll virus damage.