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Elucidating the genetic makeup of Grapevine leafroll-associated virus 3 for managing leafroll disease in Washington State vineyards
Arunabha Mitra, Sridhar Jarugula, Bhanupriya Donda, Emily Jordan, and Naidu Rayapati*Department of Plant Pathology, Washington State University-Irrigated Agriculture Research and Extension Center, Prosser, WA 99350.
*Corresponding author: [email protected]
VITICULTURE & ENOLOGY
INTRODUCTIONGrapevine leafroll disease (GLD) is the most economically destructive virusdisease of wine grapes (Vitis vinifera), severely affecting vine health and fruityield and quality. It continues to threaten the sustainability of premium winegrape production in Washington State. Grapevine leafroll-associated viruses(GLRaVs) are a group of viruses documented in GLD-affected vines. Amongthem, Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widespreadand insidious in Washington State vineyards (Rayapati et al. 2008; Naidu et al.2015). GLRaVs have an exceptionally complex genome organization. Studieson the genetic variability of GLRaV-3 across grapevine-growing regions in theUnited States and abroad have reported the existence of multiple geneticvariants. Based on examination of critical virus-encoded genes, ten distinctGLRaV-3 genetic variant groups, named I through X, have been reported thusfar (Naidu et al. 2015; Burger et al. 2017; Diaz-Lara et al. 2018; Thompson etal. 2018).
METHODOLOGY
1. Sample collection from commercial WA vineyards.
2. Sample processing (Rowhani et al. 2000).
3. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assay withHsp70h-based primers (Donda et al. 2016).4. Cloning and Sanger sequencing of RT-PCR amplicons.5. Sequence alignment with globally reported GLRaV-3 sequences andphylogenetic analysis (MEGA7 software package).
RESULTSSampling details (2016 to 2018):
• Total samples collected : 1260
• Red-fruited cultivars sampled : 11
• White-fruited cultivars sampled : 11
• Juice grape cultivars sampled : 2
• Number of vineyards : 13
• GLRaV-3-positive samples : 1146
Screening samples for the presence of GLRaV-3 by RT-PCR:
52%
3%20%
5%
16%4% Group I
Group II
Group III
Group V
Group VI
DIVERGENT
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Mixed infection Single variantinfection
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f vi
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9%
91%
OBJECTIVEThe goal of this project was to explore the genetic diversity landscape ofGLRaV-3 in Washington State vineyards, gain research-based insight into GLDepidemiology, and ultimately apply that knowledge to area-wide clean plantprograms for managing grapevine leafroll disease in vineyards.
Fig. 1: (A) GLRaV-3 particle visualized under Transmission Electron Microscope(TEM), (B) Emergence of diverse GLRaV-3 genetic variants from a single founderGLRaV-3 genome molecule replicating within an infected host grapevine.
(A) (B)
Fig. 2: (A) Genome organization of GLRaV-3 (adapted from Naidu et al. 2015). Thered circle highlights the Hsp70h gene targeted for RT-PCR amplification with gene-specific primers, (B) Agarose gel electrophoresis image of the ~580 base pair (bp)RT-PCR amplicons (indicated by red arrow-head on the right) generated from theRT-PCR diagnostic assay. M: 1 Kb+ DNA molecular size marker, (+): GLRaV-3positive control, (-): GLRaV-3 negative control, 1-14: samples tested, (C) Summaryof RT-PCR results of the samples tested for GLRaV-3 in this study.
Fig. 4: (A) Proportion of GLRaV-3 isolates belonging to different reported geneticvariant groups across Washington State vineyards, (B) Proportion of GLRaV-3-infectedgrapevines harboring a single GLRaV-3 variant group or a mixture of variant groups.
CONCLUSIONS
Fig. 3: Maximum likelihoodphylogenetic tree of partialHsp70h sequences (~580 bp) ofrepresentative GenBank-sourcedGLRaV-3 isolates andcorresponding GLRaV-3sequences obtained in this study(adapted from Diaz-Lara et al.2018). The bootstrap numbers atthe branches are >80%confidence values derived from1,000 replications ofphylogenetic reconstruction ofthe tree. The partial Hsp70hsequence from GLRaV-2 wasused as an outgroup. Horizontalbranch length is proportional togenetic distance.Red boxes indicate divergentgroups of GLRaV-3 isolatesidentified in this study inWashington vineyards.Red asterisks indicate the fivereported GLRaV-3 variant groupswhich were also identified to bepresent in Washington vineyardsfrom this study.
REFERENCES
ACKNOWLEDGEMENTSThis is the first comprehensive report demonstrating the presence of several distinctgenetic variant groups of GLRaV-3 in Washington State vineyards.GLRaV-3 isolates belonging to five reported variant groups were identified in this study.Isolates belonging to variant group I were predominant compared to isolates belongingto other reported GLRaV-3 variant groups.Although majority of the GLRaV-3 isolate sequences aligned with reported variantgroups, ~4% of the isolates were ‘divergent’ and did not align with the establishedclassification system of GLRaV-3 genetic variants. Occurrence of these distinct variantswarrant further research to understand their overall diversity and spread acrossvineyards in the state.The data from this study will be used in improving the currently used laboratory-baseddiagnostic methods for detecting all variants of GLRaV-3 in planting materials.Therefore, knowledge of the genetic diversity of GLRaV-3 will provide opportunities toresolve the complex epidemiology of GLD for implementing disease managementstrategies and improving grapevine planting material supply chain for healthy vineyards.
This research was funded by the Northwest Center for Small FruitsResearch, the WSU Agricultural Research Center, the WineResearch Advisory Committee, the Washington Wine Commission,and the Washington State Grape and Wine Research Program.
Rowhani et al. 2000. Proceedings of XIII International Council for the Studyof Viruses and Virus-Like Diseases of the Grapevine, Adelaide, p. 148.
Rayapati et al. 2008. WSU Extension Bulletin EB2027E, p. 20. Naidu et al. 2015. Annu. Rev. Phytopathol. (53): 613-634. Donda, B. P. 2016. Molecular Biology And Epidemiology of Grapevine
Leafroll-Associated Viruses. Burger et al. 2017. Grapevine viruses: Molecular Biology, diagnostics and
Management. Diaz-Lara et al. 2018. PloS one 13.12: e0208862. Thompson et al. 2018. Plant Disease, (ja).
91%
9%
GLRaV-3-positive vines
GLRaV-3-negative vines
(A)
(B)
600 bp550 bp
M (+) (-) 1 2 3 4 5 6 7 8 9 10 11 12 13 14
(C)
MET HEL
L-Pro AlkB
p6
p5 p20A
p20B
p4
p7
3’5’RdRp HSP70h
HSP90h
CP
CPm
p21
The partial Hsp70h gene sequences of GLRaV-3 isolates found in Washingtonvineyards were compared with corresponding virus sequences reported fromother grapevine-growing regions worldwide to profile genetic diversity of thevirus. Phylogenetic analysis of Hsp70h gene sequences showed that GLRaV-3isolates from Washington State fell into 5 reported variant groups of GLRaV-3:groups I, II, III, V, and VI, and the majority of GLRaV-3 isolates belonged to variantgroup I (Fig. 3).
Overall, GLRaV-3 isolates belonging to five reported genetic variant groupswere identified. 52% of the GLRaV-3 isolates identified in this studyclustered with GLRaV-3 variant group I, 20% aligned with variant group III,16% aligned with variant group VI, 5% aligned with variant group V, and3% aligned with group II. This suggests that GLRaV-3 isolates belonging tovariant group I are predominant compared to virus isolates belonging toother variant groups in Washington vineyards. In addition, 4% of theGLRaV-3 isolates did not align with any of the reported variant groups andappeared to be divergent (Fig. 4A).Further analysis showed that majority (91%) of GLRaV-3-positive samplesfrom individual vines contained sequences belonging to a single variantgroup and 9% of the samples were found to have sequences belonging totwo or more variant groups of GLRaV-3 (Fig. 4B).
(A) (B)
Group I*
Group V*
Group II*
Group III*
Group IX
Group X
Group VI*
Group VII
13850x22_Ng_WAKX756669_TRAJ-BR_Brz13931X34_cg1_SB_WA13931X19_cg1_Sy_WA13931X18_Sy_WAEU344893_Cl-766_Chl11820x12_Ch_WA11979x09_cg1_Ch_WA14206X11_MB_WA13931X13_Ch_WAJX559645_3138-07_Can13931X32_SB_WAGU983863_WAMR_USAAF037268_NY_USA
13850x59_Ch_WAGQ352631_621_SA13850x60_cg1_Ch_WA13850x03_Ng_WA13850x01_Ng_WA13931x05_Rg_WA13446x10_SB_WA13446x11_SB_WA13931X24_CS_WA13931X14_CB_WA14206X08_MB_WA13850x17_CB_WAKX756668_TC-BR_Brz13931X19_cg2_Sy_WAGQ352632_623_SAKX701860_IASAB-BR_Brz
EU259806_GP18_SAKJ174518_IsrGQ352633_PL20_SA13850x61_Ch_WA13850x58_Ch_WA13931X22_Mo_WAJQ423939_LN_Chi13931X34_cg2_SB_WA13850x60_cg2_Ch_WA13931X15_cg1_Sy_WA12093x05_Ch_WA11979x19_cg1_Ch_WA11979x09_cg2_Ch_WA11820x08_Ch_WAKY886362_GLRaV-3-I-LR101_Cro
13931X15_cg3_Sy_WAKY073324_8415B_Can14206X10_MB_WAKY764333_Trc138_USAKY707824_Pro95_USA11979x19-cg2_Ch_WAKY707825_Rod96_USAMH796136_ID45_USA14977x2_MB_WA14977x4_MB_WAJX220899_NZ2_NZKY764332_Trc139_USA13931x29_SB_WAMB_WA_201813931X15_cg2_Sy_WA
13931x03_CS_WAJX220900_NZ1B_NZJQ796828_clone3_USAJQ655295_GH11_SAJQ655296_GH30_SAKM058745_GH24_SAKY707826_NdA121_USAFJ436234_GLRaV2 (OUTGROUP)
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