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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 Fisheries2 CSIRO3 Northern Australian Quarantine Strategy4 Department of Agriculture, Thailand5 International Centre for Agricultural Reseach in the Dry Areas, Tunisia6 CIRAD, Brazil7 EMBRAPA, Brazil8 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
• 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
Healthy
CBTV
Cotton bunchy top virus (CBTV)
CBTV-1 and -2
Edge infection and spread
Severe widespread damage
ORF 0
ORF 1 + ORF 2 ORF 3
ORF 4ORF 3+5
Coat protein
RNA dependent RNA polymerase
Polerovirus genome• ssRNA genome (~5900nt). Several open reading frames (ORF) code for
proteins that allow virus to infect, replicate and transmit
Movement protein
Suppressor of RNA silencing Aphid transmission
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 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
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
Isolate-3092 Emerald CBTV-B
Isolate 3103 Dalby CBTV-B
WHS23 Mungindi CBTV-B
WHS58 Moree CBTV-B
WHS66 Narromine CBTV-B
20131216ms-49 Clermont sturtianum CBTV-B
20110608ms-20 Emerald CBTV-B
Isolate-3093 Emerald asthma CBTV-B
20130114ms-2 Chinchilla CBTV-B
GU167940 CLRDV Arg complete
HQ827780 CLRDV-PV1 Brazil
NC 008249 CpCSV complete
20120917ms-56 Hillston CBTV-A
20131216ms-49 Clermont sturtianum CBTV-A
20131216ms-48 Clermont sturtianum CBTV-A
JF803842 CBTV-A
20110608ms-20 Emerald CBTV-A
20131216ms-98 Clermont CBTV-A
20130301ms-9 St George CBTV-A
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
99
78
99
58
80
41
99
67
76
0.1
CBTV-1 and –2 have little variation within each species
• Phylogenetic tree of 201aa complete coat protein (ML analysis)
CBTV-2
CBTV-1
70 % identity
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 leafAnoda cristata Spurred anodaGossypium australe Desert roseGossypium hirsutum CottonHibiscus sabdariffa RosellaHibiscus trionum Bladder ketmiaMalva parviflora MarshmallowMalva sp. unknownMalvastrum coromandelianum
Malvastrum
Sida rhombifolia Paddy’s lucerneGossypium sturtianum Sturt’s desert rose
Euphorbiaceae Euphorbia hirta Asthma plantLamiaceae Lamium amplexicaule DeadnettleFabaceae Cicer arietinum Chickpea
Medicago polymorpha Burr MedicVicia 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.
• 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
Cotton blue disease – Cotton leafroll dwarf virus (exotic)
• Wide geographical distribution
• Confirmed by PCR in G. hirsutum, G. barbadense, chickpea and hibiscus
Surveys for CLRDV in Timor-Leste
• 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).
30 miles
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)
CLRDV-Q3513
CLRDV-Q3514
Y1153ZCpSDaV chickpea
JN033875CLRDV cotton
JR2843CLRDV East Timor
GU167940CLRDV Argentina
HQ827780CLRDV Brazil
AF473561BWYV USA
CBTV-2
X76931CABYV
CBTV-1
NC 008249CpCSV complete
NC 003056SbDV
74
76
57
99
99
100
100
83
89
98
0.1
Thailand
IndiaCLRDV
Q3513
Q3514
M
Q3515
Q3518
Q3519
Q3523
Q3524
Q3526
Q4625
Q4631
M
CBTV 1/2
CBTV 1/2
Healthy cotton
Specific detection of CLRDV
Thailand CLRDV
Brazil CLRDV
CLRDV3675F
Pol3982R
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
Detection of CLRDV
• Non-specific reactions are a problem with species other than cotton
1st PCR 2nd (nested) PCR
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
Improving detection of CLRDV
CLRDV3675F
Pol3982R
• A nested PCR greatly increases sensitivity and reduces non-specific
reactions
Pol4021R
Pol3628F
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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