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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 INTRODUCTION Grapevine leafroll disease (GLD) is the most economically destructive virus disease of wine grapes (Vitis vinifera), severely affecting vine health and fruit yield and quality. It continues to threaten the sustainability of premium wine grape production in Washington State. Grapevine leafroll-associated viruses (GLRaVs) are a group of viruses documented in GLD-affected vines. Among them, Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widespread and insidious in Washington State vineyards (Rayapati et al. 2008; Naidu et al. 2015). GLRaVs have an exceptionally complex genome organization. Studies on the genetic variability of GLRaV-3 across grapevine-growing regions in the United States and abroad have reported the existence of multiple genetic variants. Based on examination of critical virus-encoded genes, ten distinct GLRaV-3 genetic variant groups, named I through X, have been reported thus far (Naidu et al. 2015; Burger et al. 2017; Diaz-Lara et al. 2018; Thompson et al. 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 with Hsp70h-based primers (Donda et al. 2016). 4. Cloning and Sanger sequencing of RT-PCR amplicons. 5. Sequence alignment with globally reported GLRaV-3 sequences and phylogenetic analysis (MEGA7 software package). RESULTS Sampling 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 0 10 20 30 40 50 60 70 80 90 100 Mixed infection Single variant infection Percentage of vines tested 9% 91% OBJECTIVE The goal of this project was to explore the genetic diversity landscape of GLRaV-3 in Washington State vineyards, gain research-based insight into GLD epidemiology, and ultimately apply that knowledge to area-wide clean plant programs 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 founder GLRaV-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). The red 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 the RT-PCR diagnostic assay. M: 1 Kb+ DNA molecular size marker, (+): GLRaV-3 positive control, (-): GLRaV-3 negative control, 1-14: samples tested, (C) Summary of 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 genetic variant groups across Washington State vineyards, (B) Proportion of GLRaV-3-infected grapevines harboring a single GLRaV-3 variant group or a mixture of variant groups. CONCLUSIONS Fig. 3: Maximum likelihood phylogenetic tree of partial Hsp70h sequences (~580 bp) of representative GenBank-sourced GLRaV-3 isolates and corresponding GLRaV-3 sequences obtained in this study (adapted from Diaz-Lara et al. 2018). The bootstrap numbers at the branches are >80% confidence values derived from 1,000 replications of phylogenetic reconstruction of the tree. The partial Hsp70h sequence from GLRaV-2 was used as an outgroup. Horizontal branch length is proportional to genetic distance. Red boxes indicate divergent groups of GLRaV-3 isolates identified in this study in Washington vineyards. Red asterisks indicate the five reported GLRaV-3 variant groups which were also identified to be present in Washington vineyards from this study. REFERENCES ACKNOWLEDGEMENTS This is the first comprehensive report demonstrating the presence of several distinct genetic 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 belonging to other reported GLRaV-3 variant groups. Although majority of the GLRaV-3 isolate sequences aligned with reported variant groups, ~4% of the isolates were ‘divergent’ and did not align with the established classification system of GLRaV-3 genetic variants. Occurrence of these distinct variants warrant further research to understand their overall diversity and spread across vineyards in the state. The data from this study will be used in improving the currently used laboratory-based diagnostic methods for detecting all variants of GLRaV-3 in planting materials. Therefore, knowledge of the genetic diversity of GLRaV-3 will provide opportunities to resolve the complex epidemiology of GLD for implementing disease management strategies and improving grapevine planting material supply chain for healthy vineyards. This research was funded by the Northwest Center for Small Fruits Research, the WSU Agricultural Research Center, the Wine Research 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 Study of 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 bp 550 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 Washington vineyards were compared with corresponding virus sequences reported from other grapevine-growing regions worldwide to profile genetic diversity of the virus. Phylogenetic analysis of Hsp70h gene sequences showed that GLRaV-3 isolates 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 variant group I (Fig. 3). Overall, GLRaV-3 isolates belonging to five reported genetic variant groups were identified. 52% of the GLRaV-3 isolates identified in this study clustered with GLRaV-3 variant group I, 20% aligned with variant group III, 16% aligned with variant group VI, 5% aligned with variant group V, and 3% aligned with group II. This suggests that GLRaV-3 isolates belonging to variant group I are predominant compared to virus isolates belonging to other variant groups in Washington vineyards. In addition, 4% of the GLRaV-3 isolates did not align with any of the reported variant groups and appeared to be divergent (Fig. 4A). Further analysis showed that majority (91%) of GLRaV-3-positive samples from individual vines contained sequences belonging to a single variant group and 9% of the samples were found to have sequences belonging to two 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_WA KX756669_TRAJ-BR_Brz 13931X34_cg1_SB_WA 13931X19_cg1_Sy_WA 13931X18_Sy_WA EU344893_Cl-766_Chl 11820x12_Ch_WA 11979x09_cg1_Ch_WA 14206X11_MB_WA 13931X13_Ch_WA JX559645_3138-07_Can 13931X32_SB_WA GU983863_WAMR_USA AF037268_NY_USA 13850x59_Ch_WA GQ352631_621_SA 13850x60_cg1_Ch_WA 13850x03_Ng_WA 13850x01_Ng_WA 13931x05_Rg_WA 13446x10_SB_WA 13446x11_SB_WA 13931X24_CS_WA 13931X14_CB_WA 14206X08_MB_WA 13850x17_CB_WA KX756668_TC-BR_Brz 13931X19_cg2_Sy_WA GQ352632_623_SA KX701860_IASAB-BR_Brz EU259806_GP18_SA KJ174518_Isr GQ352633_PL20_SA 13850x61_Ch_WA 13850x58_Ch_WA 13931X22_Mo_WA JQ423939_LN_Chi 13931X34_cg2_SB_WA 13850x60_cg2_Ch_WA 13931X15_cg1_Sy_WA 12093x05_Ch_WA 11979x19_cg1_Ch_WA 11979x09_cg2_Ch_WA 11820x08_Ch_WA KY886362_GLRaV-3-I-LR101_Cro 13931X15_cg3_Sy_WA KY073324_8415B_Can 14206X10_MB_WA KY764333_Trc138_USA KY707824_Pro95_USA 11979x19-cg2_Ch_WA KY707825_Rod96_USA MH796136_ID45_USA 14977x2_MB_WA 14977x4_MB_WA JX220899_NZ2_NZ KY764332_Trc139_USA 13931x29_SB_WA MB_WA_2018 13931X15_cg2_Sy_WA 13931x03_CS_WA JX220900_NZ1B_NZ JQ796828_clone3_USA JQ655295_GH11_SA JQ655296_GH30_SA KM058745_GH24_SA KY707826_NdA121_USA FJ436234_GLRaV2 (OUTGROUP) 99 99 99 92 96 99 99 98 99 94 91 95 91 90 86 99 80 83 99 93 99 99 99 91 97

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Page 1: Elucidating the genetic makeup of Grapevine leafroll ... · Elucidating the genetic makeup of Grapevine leafroll-associated virus 3 for managing leafroll disease in Washington State

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

0

10

20

30

40

50

60

70

80

90

100

Mixed infection Single variantinfection

Pe

rce

nta

ge o

f vi

ne

s te

ste

d

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)

99

99

99

9296

99

99

98

99

94

91

95

91

90

86

99

80

8399

9399

99

9991

97