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Endemic cotton-infecting viruses from Australia, and detection and studies of the exotic Cotton leafroll dwarf virus Murray Sharman1, Lewis Wilson2, Tanya Smith2, Matt Webb1, Fiona Filardo1, Jane Ray3, Siwilai Lapbanjob4, Safaa Kumari5, Marc Giband6, Nelson Suassuna7, Jean-Louis Belot8 Valente Quintao9 1 Queensland Department of Agriculture and Fisheries 2 CSIRO 3 Northern Australian Quarantine Strategy 4 Department of Agriculture, Thailand 5 International Centre for Agricultural Reseach in the Dry Areas, Tunisia 6 CIRAD, Brazil 7 EMBRAPA, Brazil 8 Mato Grosso Institute of Cotton (IMAmt) 9 National Directorate of Quarantine and Biosecurity, Timor Leste Virus diseases in Australian cotton Cotton bunchy top virus (CBTV) – Sporadic outbreaks cause localized losses – Major outbreaks in 1998-9 and 2010-11 Tobacco streak virus (TSV) – Common in Central Queensland but no yield or quality affect Cotton bunchy top virus (CBTV) CBTV-1 and -2 CBTV Healthy • Transmitted by cotton aphid (semi-persistant) • Pale green angular patterns on leaves with dark green centres • Small leathery, brittle leaves • Reduced internodes and petioles – bunchy appearance • Reduced boll size, number and shorter fibres Edge infection and spread Severe widespread damage Polerovirus genome • ssRNA genome (~5900nt). Several open reading frames (ORF) code for proteins that allow virus to infect, replicate and transmit Coat protein RNA dependent RNA polymerase ORF 1 + ORF 2 ORF 3 ORF 3+5 ORF 0 ORF 4 Movement protein Suppressor of RNA silencing Aphid transmission CBTV-1 and –2 are distinct species • Distinct virus species have less than 90 % aa identity for one or more open reading frames (ORFs). CBTV-1 and –2 have less than 90 % aa identity for all ORFs • Distinct species don’t cross protect. CBTV-1 and -2 can co-infect same plant • Distinct species have differences in host range CBTV-1 and -2 appear to have some unique hosts. CBTV-2 is causing CBT. CBTV-1 compared to CBTV-2 ORF # Amino acid % identity ORF 0 51 % ORF 1 65 % ORF 1+2 75 % ORF 3 70 % ORF 4 59 % ORF 3+5 60 % CBTV-1 and –2 have little variation within each species • Phylogenetic tree of 201aa complete coat protein (ML analysis) 20131216ms-98 Clermont CBTV-B Isolate-2747 Murgon CBTV-B 20130427ms-1 Theodore CBTV-B 20130318ms-77 Breeza CBTV-B 20130314ms-1 Wee Waa CBTV-B 20130114ms-7 Goondiwindi CBTV-B 76 Isolate-3092 Emerald CBTV-B Isolate 3103 Dalby CBTV-B WHS23 Mungindi CBTV-B CBTV-2 WHS58 Moree CBTV-B 99 WHS66 Narromine CBTV-B 20131216ms-49 Clermont sturtianum CBTV-B 20110608ms-20 Emerald CBTV-B 41 67 Isolate-3093 Emerald asthma CBTV-B 20130114ms-2 Chinchilla CBTV-B GU167940 CLRDV Arg complete 80 99 HQ827780 CLRDV-PV1 Brazil NC 008249 CpCSV complete 20120917ms-56 Hillston CBTV-A 20131216ms-49 Clermont sturtianum CBTV-A 58 20131216ms-48 Clermont sturtianum CBTV-A 70 % identity JF803842 CBTV-A 99 20110608ms-20 Emerald CBTV-A 20131216ms-98 Clermont CBTV-A 20130301ms-9 St George CBTV-A CBTV-1 78 20130312ms-1 Wee Waa CBTV-A 20140206ms-11 Brookstead CBTV-A 20140206ms-3 Brookstead CBTV-A Isolate-2747 Murgon CBTV-A WHS58 Moree CBTV-A X76931 CABYV NC 003056 SbDV 0.1 Alternative hosts for cotton bunchy top • Host range determined by field surveys, field trials or lab tests Plant Family Host species Common name Malvaceae Abutilon theophrasti Velvet leaf Anoda cristata Spurred anoda Gossypium australe Desert rose Gossypium hirsutum Cotton Hibiscus sabdariffa Rosella Hibiscus trionum Bladder ketmia Malva parviflora Marshmallow Malva sp. unknown Malvastrum Malvastrum coromandelianum Sida rhombifolia Paddy’s lucerne Gossypium sturtianum Sturt’s desert rose Euphorbiaceae Euphorbia hirta Asthma plant Lamiaceae Lamium amplexicaule Deadnettle Fabaceae Cicer arietinum Chickpea Medicago polymorpha Burr Medic Vicia faba Faba bean Aizoaceae Trianthema portulacastrum Black or Giant pigweed Localised CBT disease outbreak, 2018 Neighbours fallow paddock upwind with CBTV-infected volunteers. Sprayed with herbicide. Aphids moved into crop nearby, downwind. Four transect counts shown in graph. Cotton blue disease – Cotton leafroll dwarf virus (exotic) • Similar life cycle to cotton bunchy top but probably more easily transmitted by cotton aphid and more severe disease than CBTV • Symptoms include severe stunting, down curling of leaves and intense green colour CBD - Brazil CBD - Thailand • Wide geographical distribution • Confirmed by PCR in G. hirsutum, G. barbadense, chickpea and hibiscus Surveys for CLRDV in Timor-Leste 30 miles • 69 cotton plants tested from >20 locations • CLRDV in 10/32 G. barbadense, 7/19 G. arboreum and 4/18 G. hirsutum • >400 samples from about 30 host species collected for host range studies (continuing). Timor-Leste surveillance Gossypium arboreum G. barbadense G. hirsutum (with CLRDV) CLRDV diversity • Phylogenetic tree of 560 bp genome section, partial ORF-3 (ML analysis) 100 CLRDV-Q3513 Thailand 89 CLRDV-Q3514 Y1153ZCpSDaV chickpea 83 98 India JN033875CLRDV cotton CLRDV 100 JR2843CLRDV East Timor 99 GU167940CLRDV Argentina 99 HQ827780CLRDV Brazil AF473561BWYV USA CBTV-2 57 X76931CABYV 76 CBTV-1 74 NC 008249CpCSV complete NC 003056SbDV 0.1 Specific detection of CLRDV CLRDV3675F Pol3982R Q3513 Q3514 M Thailand CLRDV Q3515 Q3518 Q3519 Q3523 Q3524 Q3526 Brazil CLRDV Q4625 Q4631 M CBTV 1/2 CBTV 1/2 Healthy cotton Detection of CLRDV • Non-specific reactions are a problem with species other than cotton Malvaceae sample 29 M Malvaceae sample 30 Malvaceae sample 31 Malvaceae sample 32 CLRDV East Timor M CLRDV East Timor CLRDV East Timor CLRDV Brazil Q4620 Healthy cotton CLRDV Brazil Q4620 PCR negative Improving detection of CLRDV • A nested PCR greatly increases sensitivity and reduces non-specific reactions Pol3628F CLRDV3675F Pol3982R Pol4021R Malvaceae sample 29 Malvaceae sample 30 Malvaceae sample 31 Malvaceae sample 32 CLRDV + Malvaceae sample 30 CLRDV Brazil isolate Q4620 CLRDV East Timor JR2843 Healthy cotton PCR negative 1st PCR 2nd (nested) PCR Acknowledgments • Cotton Research and Development Corporation (CRDC) • Grains Research and Development Corporation (GRDC) • CSIRO • Department of Agriculture and Water Resources • National Directorate of Quarantine and Biosecurity, Timor Leste • Cotton and Grain growers and agronomists for samples and access to crops.
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