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MULTIGENE PHYLOGENY AND PATHOGENICITY OF VERTICILLIUM SPP.
FROM POTATOES IN SOUTH EAST AUSTRALIA
V.R. Prakash A , T.J. Wiechel B , N.S. Crump C and P.W.J. Taylor A
A Department of Agriculture and Food Systems, Melbourne School of Land and Environment, The University of Melbourne, Parkville,
Victoria, Australia, Email: prakasvr@gmail.com
B Biosciences Research Division, DPI Victoria, Australia
C Victorian Certified Seed Potato Authority, Healesville, Victoria 3777, Australia.
ABSTRACT. Validation of the taxonomy of the Verticillium spp. involved in Verticillium wilt of potato in South East
Australia confirmed that V. dahliae was the major Verticillium species. V. albo-atrum was also identified from several potato
tubers from Tasmania and Victoria. Multigene phylogeny based on gene sequence comparison of ITS, β-tub and EF-1,
showed that there was very little gene diversity within V. dahliae potato isolates, V. albo-atrum was phylogeneticaly related
to V. dahliae, and that the isolates of V. dahliae from tomato and strawberry were genetically similar to isolates from potato.
Pathogenicity studies on potato (susceptible cv Shepody and moderately resistant cv Ranger Russet) and eggplant (cv Black
Beauty) showed that the majority of V. dahliae isolates were highly aggressive in potato and eggplants especially Tasmanian
V. dahliae isolate Vd25. Disease severity and the effect on plant growth varied between isolates with most isolates generally
being more pathogenic on potato cv Shepody.
INTRODUCTION
Verticillium wilt (Vw), caused by the soilborne fungi
Verticillium dahliae or Verticillium albo-atrum, is a serious
disease of potato as well as many other crops. Vw has been
recorded in potatoes in Australia (1, 2, 3) and has been
shown to be an interaction between V. dahliae and nematode
Pratylenchus crenatus to cause potato early dying (PED)
disease. Comprehensive studies have not been carried out on
multigene phylogenetic analysis and pathogenicity of V.
dahliae in Australia. The main objective of this research was
therefore to (i) validate the taxonomy of Verticillium species
in potatoes using multigene phylogeny; and (ii) study the
pathogenicity of the Australian isolates of V. dahliae.
MATERIALS AND METHODS
Multigene phylogenetic analysis: DNA was extracted from
twenty seven single spored isolates (21 isolates of V.
dahliae, two of V. albo-atrum, V. tricorpus, one of V.
tenerum and V. nigrescens). These isolates had been
collected from the petiole and tubers of infected potato from
Victoria, Tasmania and South Australia. Two isolates of V.
dahliae from tomato and strawberry were also included in
the study. DNA amplification and sequencing were
performed by PCR using three gene sequences (ITS, partial
β-tubulin and translation elongation factor 1-alpha) and sent
for sequencing to Australian Genome Research Facility,
Melbourne. Sequences were aligned in MEGA 4 using
Clustal W and phylogenetic trees constructed.
Pathogenicity test: Pathogenicity and virulence of nine
isolates of V. dahliae from different geographical locations
were determined by root dip inoculation experiment into
potato (susceptible cv Shepody & moderately resistant cv
Ranger Russet) and eggplant cv Black Beauty in 2011 and
2012. Disease severity was assessed using 0-5 scale.
Eggplants were destructively sampled seven weeks after
planting and height, aerial biomass (fresh & dry) and root
weight (fresh and dry) were measured.
RESULTS AND DISCUSSION
Multigene phylogenetic analysis revealed five monophyletic
groups corresponding to the five species in the investigation.
V. dahliae and V. albo-atrum were the two closely related
species with all isolates of V. dahliae clustering in one tight
cluster. Molecular identity using three gene sequences
validated taxonomy based on traditional morphological
characters. V. dahliae from tomato and strawberry were
genetically similar to isolates from potato. Sequences from
ITS PCR were adequate to separate species.
Pathogenicity results showed that the majority of V.
dahliae isolates from potato were highly aggressive in
potato and eggplants especially Tasmanian V. dahliae
isolates (Vd25) (Table 1). Isolates varied in pathogenicity in
terms of disease severity and the effect on plant growth.
Table 1. Vw severity (0-5 scale) recorded on potatoes cv.
Shepody (SH) and Ranger Russet (RR) and eggplant (EG)
inoculated with different V. dahliae isolates compared with
the non inoculated control.
Disease severity*
Treat 2011 2012 EG
SH RR SH RR 2011 2012
Vd36 4 2 3 2 3 2
Vd54 2 1 2 1 2 1
Vd85 4 3 2 2 2 2
Vd7 4 3 2 2 2 2
Vd16 3 3 2 2 2 2
Vd12 4 2 3 2 3 2
Vd24 5 4 3 2 3 2
Vd25 5 5 4 4 4 4
Vd27 5 3 4 2 2 2
Control 0 0 0 0 0 0
*Mean disease severity of 4 plants rounded off to the nearest
whole number.
ACKNOWLEDGEMENT
We thank Fran Richardson, Justin Verstraten, Mark
Wardzynski and Dolf de Boer (DPI, Knoxfield) for support.
The primary author is the recipient of Australian
Postgraduate Award. The research is funded by Horticulture
Australia Limited using the processing potato levy and
voluntary contributions. The University of Melbourne and
the Victorian Seed Potato Authority have provided in-kind
support.
REFERENCES
1. Powney, R. A., Hay, F. and Crump, N. S. (2005). In
The 5 th Biennial Australasian Plant Pathology Society
Conference Handbook, 278.
2. Harding, R. B. and Wicks, T. J. (2007). Australasian
Plant Pathology 36: 62-67.
3. Prakash, V. R., Wiechel, T. J., Crump, N. S., Porter, I.
J. and Taylor, P. W. J. (2011). Inaugural joint 4th Asian
Conference on Plant Pathology and the 18th Biennial
Australasian Plant Pathology Society Conference,
Darwin, Australia. P. 46.
7th Australasian Soilborne Diseases Symposium 68