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A
ProMED-mail post
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International Society for Infectious Diseases
June 19, 2003
Source: DL Hailstones <deborah.hailstones@agric.nsw.gov.au
>
Surveys of greenhouse grown tomato plants in New South Wales
(NSW) have recently been finalised, with about 500 000
greenhouse tomato plants on 15 properties being surveyed over
the past 12 months. No evidence of PSTVd symptoms were observed.
PSTVd was detected in 2 plants on one farm in 2001, and that
farm has been inspected approximately every month since that
time, with no evidence of disease symptoms in successive crops.
Eradication of the pathogen from that farm (and from the state)
therefore appears to have been successful and NSW is still
considered to be free of PSTVd.
[I am indebted to Deb Hailstones (molecular biologist, Plant
Protection, Elizabeth Macarthur Agricultural Institute, NSW
Agriculture) for providing this information. Continued vigilance
will be required to maintain freedom from the viroid. Kudos to
staff in NSW Agriculture for a job well done! - Mod.DH]
June 17, 2003
Source: Australasian Plant Pathology, Disease Notes, Vol
2, 2003 [edited]
Detection and eradication of potato
spindle tuber viroid in tomatoes in commercial production in New
South Wales, Australia
DL Hailstones
<deborah.hailstones@agric.nsw.gov.au>, LA Tesoriero, MA
Terras, C Dephoff, NSW Agriculture, Australia. Australasian
Plant Pathology Journal 2003; 32(2): 317-8.
Australia is currently free of potato spindle tuber pospoviroid
(PSTVd), which causes serious yield reductions in infected
potatoes and tomatoes. Outbreaks in potato breeding programs in
Victoria and New South Wales (NSW) in 1982 were managed by
eradication (1). PSTVd was also detected and eradicated in
breeding tomatoes in the Northern Territory (2) and more
recently was detected in greenhouse-grown tomatoes in New
Zealand (3). In Western Australia (WA) the pathogen was detected
and subsequently eradicated from the affected property (4).
Given that PSTVd can be transmitted via seed and pollen (5), a
national strategy was initiated to determine the extent of the
PSTVd infection. In June 2001, about 100 000 greenhouse-grown
tomato plants were surveyed on 15 properties in Western Sydney,
NSW. Symptoms associated with PSTVd infection were observed on 2
plants of tomato (_Lycopersicon esculentum_, cultivar La Belle,
from a single property.
These plants were removed and their PSTVd status evaluated by 3
methods. Sap from 1 plant was inoculated onto cotyledons of 6
seedlings of tomato cv. Grosse Lisse (6). Uninoculated plants
were maintained as controls. Twisted and bunched shoot symptoms
typical of PSTVd infection were visible
after 19 days. These plants tested positive for the presence of
PSTVd in molecular tests. Leaf lysates were prepared from 1 of
the symptomatic greenhouse-grown plants and extracted viroids
were analysed using sequential PAGE (7).
Total nucleic acids were separated by size on a native 5 per
cent polyacrylamide gel, stained with EtBr and the "viroid band"
excised and applied to a denaturing 5 per cent polyacrylamide
gel (containing 8 M
urea). Viroids in the second dimension gels were visualised by
silver staining and compared in size with Citrus exocortis
viroid (CEV, genome size 378 bp).
Extracts from symptomatic (PSTVd-infected) plants displayed a
single band at a size smaller than CEV, consistent with the size
expected for PSTVd. No viroid band was found in non-infected
tomato plants. Identity of the viroid as PSTVd was confirmed by
reverse transcriptase polymerase chain reaction
(RT-PCR) of total nucleic acids from the 2 symptomatic plants
identified in the original survey, the 6 inoculated bioassay
plants and 4 other potentially symptomatic plants, using a One
Step RT-PCR kit (Gibco Life Technologies) and PSTVd-specific
primers (nts 95-113 for the homologous primer and 87-68 for the
complementary primer).
All reaction sets included negative controls (water-only and
nucleic acids extracted from healthy tomato plants). Reaction
products from the 2 symptomatic plants from the original survey,
and the 6 seedlings inoculated with sap from 1 of the original
plants, produced a band at about 350 bp corresponding to the
predicted size of the PSTVd amplicon.No fragment was amplified
from the 4 plants collected in later surveys, or in uninfected
control plants.
To confirm the identity of the viroid as PSTVd, cDNAs amplified
from the 2 original plants were subjected to direct nucleotide
sequence analysis using the primers from the initial
amplification. The resulting sequence (Genbank accession number
AF536193) predicts a total size of 357 bp for the viroid species
present in these plants and when used as the query sequence in a
BlastN search, the amplicon was identified as PSTVd. The
sequence most closely matched that of isolate PTVCGA (Genbank
Accession number M25199), originally derived from potato (8), to
which it shows more than 97 per cent
identity. In contrast, the isolate(s) sequenced from recent
infections in tomato in New Zealand and Western Australia were
most similar to the "Naaldwijk" strain (4).
The 3 distinct detection methods confirmed the presence of PSTVd
in tomato plants showing typical symptoms. Nucleotide sequence
data suggests that this variant was introduced from a source
distinct from that responsible for other recent incursions. To
eradicate the pathogen, plants on the affected property were
deep-buried and greenhouses were sanitised with sodium
hypochlorite. Subsequent tomato crops were monitored every 3 to
4 weeks, but so far no symptomatic plants have been detected.
Greenhouse-grown tomato plants in NSW were being extensively
surveyed for PSTVd infection during 2002. Given the risk that
PSTVd can be seed-borne and that Australia remains free of this
pathogen, these results suggest import regulations pertaining to
tomato seed may need to be more stringent.
References
(1) Constable F, Moran J (1996) PCR protocols for the detection
of chrysanthemum stunt and potato spindle tuber viroids. Final
report HRDC project no PT410. ISBN No: 07306 6557 7. 23 pp.
(2) Conde B, Connelly M, Pitkethley R. Potato spindle tuber
viroid, Darwin strain -- further investigations in Darwin.
Pacific Association of Tropical Phytopathology Newsletter 1996;
14: 7-9.
(3) Elliot DR, Alexander BJR, Smales TE, Tang Z, Clover GRG
(2001) Infection of glasshouse tomatoes by Potato spindle tuber
viroid in New Zealand. In "Proceedings of the 13th Biennial
Australasian Plant Pathology Conference, Cairns". p224.
(4) Mackie AE, McKirdy SJ, Rodoni B, Kumar S. Potato spindle
tuber viroid eradicated in Western Australia. Australasian Plant
Pathology 2002; 31: 311-2.
(5) Kryczynski S, Paduch-Cichal E, Skrzeczkowski LJ.
Transmission of three viroids through seed and pollen of tomato
plants. Journal of Phytopathology 1988; 121: 51-7.
(6) Fernow KH. Tomato as a test plant for detecting mild strains
of potato spindle tuber virus. Phytopathology 1967; 57: 1347-52.
(7) Semancik JS. Viroid purification and charcterization. In:
Graft-transmissible diseases of Citrus: a handbook for detection
and diagnosis. Rome: IOCV/FAO, 1991: pp 233-41.
(8) van Wezenbeek P, Vos P, van Boom J, van Kammen A. Molecular
cloning and characterization of a complete DNA copy of potato
spindle tuber viroid RNA. Nucleic Acids Research 1982; 10:
7947-57.
[PSTVd causes significant crop losses in potato and tomato
with yield losses of up to 65 and 50 per cent, respectively. It
occurs in many countries, including the Americas, Asia, and
Oceania. It also occurs in
western Europe (France, Netherlands, and eastern Europe). There
are no resistant cultivars of either host. Eradication appears
to be the only management option. PSTVd-infected potatoes are a
trade issue, hence the need for constant awareness of possible
outbreaks. Surveys for PSTVd in tomato were to be done in 2002.
Do any of our readers in NSW have information on the results of
that survey? If you have information, please let me know. -
Mod.DH]
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