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