Content uploaded by Hua Li
Author content
All content in this area was uploaded by Hua Li on Nov 11, 2014
Content may be subject to copyright.
Taxonomic and pathogenic characteristics of a new species
Aphanomyces trifolii causing root rot of subterranean clover
(Trifolium subterraneum) in Western Australia
Tiernan A. O’Rourke
A
, Megan H. Ryan
A,D
, Hua Li
A
, Xuanli Ma
A
,
Krishnapillai Sivasithamparam
A
, Jamshid Fatehi
B
,
and Martin J. Barbetti
A,C,D,E
A
School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia,
Crawley, WA 6009, Australia.
B
The Mase Laboratories, Box 148, 751 04 Uppsala, Sweden.
C
Department of Agriculture and Food Western Australia, Baron-Hay Court, South Perth, WA 6151, Australia.
D
The UWA Institute of Agriculture, The University of Western Australia, Crawley, WA 6009, Australia.
E
Corresponding author. Email: mbarbett@cyllene.uwa.edu.au
Abstract. Subterranean clover (Trifolium subterraneum) is grown extensively as a pasture legume in agronomic regions
with Mediterranean-type climates in parts of Africa, Asia, Australia, Europe, North America and South America. Root
diseases of subterranean clover, especially those caused by oomycete pathogens including Aphanomyces,Phytophthora and
Pythium, greatly reduce productivity by significantly decreasing germination, seedling establishment, plant survival and seed
set. For this reason, experiments were conducted to determine the species of Aphanomyces causing root disease on
subterranean clover in the high-rainfall areas of south-west Western Australia. The effects of flooding, temperature and
inoculum concentration on the development of root disease on subterranean clover caused by this Aphanomyces sp. were also
investigated as was its host range. Morphological and molecular characteristics were used to identify the pathogen as a new
species Aphanomyces trifolii sp. nov. (O’Rourke et al.), which forms a distinct clade with its nearest relative being
A. cladogamus.A. trifolii caused significant lateral root pruning as well as hypocotyl collapse and tap root disease of
subterranean clover. The level of disease was greater in treatments where soil was flooded for 24 h rather than for 6 h or in
unflooded treatments. The pathogen caused more disease at 18/13
o
C than at lower (10/5
o
C) or higher (25/20
o
C) temperatures.
The pathogen caused more disease at 1% inoculum than at 0.5 or 0.2% (% inoculum : dry weight of soil). In greenhouse trials,
A. trifolii also caused root disease on annual medic (M. polymorpha and M. truncatula), dwarf beans (Phaseolus vulgaris) and
tomatoes (Solanum lycopersicum). However, the pathogen did not cause disease on peas (Pisum sativum), chickpea (Cicer
arietinum), wheat (Triticum aestivum), annual ryegrass (Lolium rigidium) or capsicum (Capsicum annuum). A. trifolii is a
serious pathogen in the high-rainfall areas of south-west Western Australia and is likely a significant cause of root disease and
subsequent decline in subterranean clover pastures across southern Australia.
Additional keywords: Aphanomyces, oomycete, root disease, subterranean clover, Trifolium.
Introduction
Subterranean clover (Trifolium subterraneum) is indigenous to
the Mediterranean regions and other parts of Western Europe
(Gladstone and Collins 1983). It is an important component of
annual pasture systems worldwide. It is grown across significant
parts of regions with Mediterranean-type climates in Africa, Asia,
Australia, Europe, North America and South America (Barbetti
et al.1986,2007).
Pasture decline manifests as a decrease in desirable species,
especially the legumes, and as an increase in weeds
(Sivasithamparam 1993). Root rots, especially those caused by
oomycetes, are a significant cause of the decline in subterranean
clover pastures in Australia (Barbetti et al.2007). They cause pre-
emergence damping off and severe root disease in seedlings
(Wong et al.1984) and mature plants (O’Rourke et al.2009),
greatly reducing pasture productivity and persistence.
As many as 35–40 species of Aphanomyces have been
described (Scott 1961). Aphanomyces euteiches is a
specialised root pathogen of Fabaceae (Papavizas and Ayers
1974) and causes significant root disease on peas (Pisum
sativum) in Australia, Europe, Japan, New Zealand and the
United States of America (Persson et al.1999).
Reliable morphological identification of species in the
oomyceteous genera, such as Aphanomyces or Pythium,is
difficult as there is often poor delineation or a lack of
distinctive anatomical structures and colony morphology
among closely related species. Furthermore, some species do
not form sexual stages and are often described based on their host
CSIRO 2010 10.1071/CP10040 1836-0947/10/090708
CSIRO PUBLISHING
www.publish.csiro.au/journals/cp Crop & Pasture Science, 2010, 61, 708–720
or their disease aetiology (Diéguez-Uribeondo et al.2009). Due to
the difficultly in identifying Aphanomyces isolates to the species
level based on morphology, molecular techniques are being
utilised with increasing frequency to characterise species from
this genus. Malvick et al.(1998) examined the genetic diversity of
A. euteiches associated with different host preferences using
random amplified polymorphic DNA analysis. They used
cluster analysis of the polymorphic DNA markers to
distinguish three pathotypes, bean, alfalfa and red clover/
alfalfa, for A. euteiches and showed that isolates of
A. euteiches not pathogenic on these hosts were genetically
distinct.
Nucleotide sequences of the internal transcribed spacer (ITS)
regions of rDNA have been used to distinguish many taxa at both
the genus and species level (e.g. Cooke et al.2000;
Constantinescu and Fatehi 2002). Levenfors and Fatehi (2004)
conducted a phylogenetic analysis of Aphanomyces species by
sequencing the total ITS region of rDNA. They found that the
sequence divergences of this region clearly separated taxa at the
species level and allowed reliable distinction between isolates of
A. astaci,A. cladogamus,A. cochlioides,A. euteiches and
A. invadans. More recently, Diéguez-Uribeondo et al.(2009)
examined the phylogenetic relationships of 12 species of
Aphanomyces, examining 108 ITS sequences of nuclear
rDNA, and identified three independent lineages: animal
parasitic, plant parasitic and saprotropic/opportunistic parasitic.
While A. euteiches is not the only species of Aphanomyces to
have been isolated from leguminous crops, it is the only one
known to be pathogenic on legumes. For instance,
A. cladogamus, also frequently isolated from diseased legume
roots (Levenfors and Fatehi 2004), is not pathogenic to the host
legume. A. euteiches is associated with reduced establishment of
lucerne (Medicago sativa) in pastures in Queensland (Othieno
Abbo and Irwin 1990), causes severe root disease in subterranean
clover pastures in Victoria (Greenhalgh et al.1985,1988), and
has been associated with root rot of bean (Phaseolus vulgaris) and
faba bean (Vicia faba) in New South Wales and of field peas
(P. sativum) in Tasmania (Geach 1936; Allen et al.1987; van Leur
et al.2008). Geach (1936) showed that an isolate of A. euteiches
pathogenic on peas was also pathogenic on subterranean clover
roots when inoculated into sterilised loam, but not when
inoculated into unsterilised loam.
The distribution of Aphanomyces on subterranean clover
across southern Australia remains to be defined. Greenhalgh
et al.(1985) surveyed 13 dryland subterranean clover pastures
at widespread locations across southern (8 pastures) and northern
(5 pastures) Victoria as well irrigated pastures (4 pastures) in
northern Victoria. A. euteiches was detected in 6 of the 8 dryland
pastures in southern Victoria, all 5 of the dryland pastures and 3
of the 4 irrigated pastures in northern Victoria. This earlier study
suggested that A. euteiches is widespread across annual
subterranean clover pastures across south-eastern Australia.
While zoosporangia and zoospores of an Aphanomyces sp.
have been previously observed on diseased subterranean
clover roots in Western Australia, neither the species identity
nor its pathogenicity were determined (Barbetti 1991a).
In the high-rainfall areas of south-west Western Australia, Ma
et al.(2008) recently surveyed the incidence of an Aphanomyces
sp. and Phytophthora clandestina. Of the 44 locations assessed
in that survey, an Aphanomyces sp. was detected at 23 locations
with 14 of these locations also containing P. clandestina.
Both Aphanomyces sp. and P. clandestina zoosporangia were
found in the diseased roots of a single plant. This occurred in
8 individual plants across 6 field sites (Ma et al.2008). In response
to these results, a study was undertaken with three aims: first, to
identify the species of Aphanomyces causing root disease on
subterranean clover in the high-rainfall areas of south-west
Western Australia; second, to determine the effects of
flooding, temperature and inoculum concentration on the
development of root disease on subterranean clover caused by
this Aphanomyces species; and third, to determine a preliminary
host range for this new Aphanomyces species.
Materials and methods
Primary isolations
Prior to isolation, ~1 kg of soil, was collected from 7 field sites, 2
sites from Bow Bridge (009, 010) as well as 1 site from Denbarker
(043), Mt Barker (041), Porongurup (040), Scotsdale (023) and
Tingledale (007), in the high-rainfall areas of south-west Western
Australia (see fig. 1 of Ma et al.2008, where bracketed numbers
correspond to locations). Soil samples from each site were air-
dried and stored for future use. The Aphanomyces sp. isolates
were baited out of the collected soil samples from regions where
Aphanomyces sp. was previously detected (Ma et al.2008), using
subterranean clover cv. Woogenellup. Baiting trials were
conducted in a controlled environment room where the air
temperature was maintained at 18/13
o
C (day/night), with a 12/
12-h photoperiod and a light intensity of 281 mmol m
–2
s
–1
. Plants
were harvested 4 weeks after sowing and the roots were washed
thoroughly under running tap water to remove soil. Whole root
systems were floated in Petri dishes containing sterile distilled
water and maintained in an incubator at 20
o
C. With the aid of a
light microscope, zoospores of Aphanomyces sp. were collected
(at 24–48 h) using fine-tip tweezers and placed directly onto
Petri dishes containing a modified metalaxyl, benomyl and
vancomycin agar (Pfender et al.1984). Single zoospore
cultures were established for each isolate of Aphanomyces sp.
The Aphanomyces sp. cultures were maintained at both 20
o
C
and at 4
o
C as conditions for optimal growth were unknown.
Four weeks before the commencement of experiments, stored
cultures were subcultured onto fresh cornmeal agar (CMA)
and grown for 1 week at 18
o
C in preparation for their use in
inoculum production.
Morphological characterisation
Five isolates of the new Aphanomyces sp. were examined.
Morphological characterisation of the isolates was performed
on subterranean clover cv. Woogenellup seedling root systems.
Seedlings were germinated in a sterile environment in the
laboratory on moist sterilised filter paper. CMA cubes 2 mm
2
were cut from the margins of 2–4-week-old cultures and floated in
sterile distilled water with 3-day-old subterranean clover
seedlings. Zoosporangia were evident on agar cubes by
1–3 days and were subsequently observed on subterranean
clover roots by 2–5 days with oogonia and oospores appearing
by 3–8 days, depending on the particular Aphanomyces sp.
isolate. The 5 isolates were assessed for a range of taxonomic
Taxonomic and pathogenic characteristics of a new species Aphanomyces trifolii Crop & Pasture Science 709
criteria including: sporangia length, zoospore size, oogonium
size, number of antheridia borne per oogonium and oospore size,
with 50 replicate observations for each aspect assessed.
DNA extraction
DNA was extracted from 24 isolates of the newly discovered
Aphanomyces sp. Mycelial plugs from 2-week-old cultures were
transferred into a 250-mL flask containing 50 mL of peptonebroth
(Levenfors and Fatehi 2004). Flasks were subsequently placed
onto a shaker (100 rpm) and incubated for 3 days at
~20
o
C. A hyphal mat was harvested from each flask and
washed with sterile distilled water. The hyphal mat was then
transferred into an Eppendorf tube and allowed to air-dry before
DNA was extracted using a standard method as described in
Wylie et al.(1993).
PCR amplification and sequencing
The ITS regions, including ITS1, ITS2 and the 5.8s rDNA, were
amplified using the universal primer set ITS1 and ITS4. PCR
fragments were amplified over 32 cycles of denaturation at 95
o
C
for 1 min, annealing at 58
o
C for 1 min and extension at 72
o
C for
2 min followed by a single 10-min extension step at 72
o
C
(Levenfors and Fatehi 2004). 5 mL of amplified PCR products
were then run through a 1% agarose gel and the remaining PCR
products sent to Macrogen Inc., Seoul, South Korea, for final
cleanup and were sequenced in both directions using the primer
pair ITS1 and ITS4.
Nucleotide sequence analysis
The sequenced products, from the primer pair ITS1 and ITS4,
were aligned using CLUSTALW available in the DNAStar
computer software package (Lasergene, Madison, WI, USA)
and minor adjustments made manually. An A. cladogamus,
sequence deposited in the GeneBank (accession number –
353290) was initially included as a reference for alignment.
A set of sequence alignments was created by incorporating
isolates sequenced in our study with available ITS sequences
representing several species of Aphanomyces parasitic on plants
and one on animals, selected based on previous studies by
Levenfors and Fatehi (2004) and Diéguez-Uribeondo et al.
(2009). The isolates sequenced in the present publication and
those used from the GenBank are listed in Table 1. Two sequences
of the homologous region from Saprolegnia parasitica and
Leptolegnia sp. (Table 1) were also included into the dataset
as outgroup taxa, on the basis of study by Diéguez-Uribeondo
et al.(2009).
Maximum parsimony (MP) analysis of the ITS nucleotide
sequences was performed with PAUP (version 4.0b10; 31)
(Swofford 2002) using 10 000 replicates of heuristic search
with random sequence addition, MulTrees option in effect, and
tree bisection-reconnection (TBR) branch swapping. Gaps were
treated as missing data and the molecular characters were given
equal weight. The robustness of the internal branches of the trees
was estimated by bootstrap analysis using 1000 replications in
heuristic search with stepwise addition of random with 100
replicates and TBR branch swapping while the use of steepest
descent was in effect. The bootstrap majority-rule (>50%)
consensus tree was obtained.
For the Bayesian analysis, first the appropriate model of DNA
substitution of the alignment file was selected by the hierarchical
likelihood ratio test in Mr Modeltest version 2 (Nylander 2004).
The best-fit selected model was the general time reversible
Table 1. The GenBank accession numbers, host and geographical origin of Aphanomyces species used for phylogenetic analysis
Scientific name GenBank accession No. Host Geographical origin
A. trifolii GQ265748 Trifolium subterraneum Australia
A. trifolii GQ267551 Trifolium subterraneum Australia
A. trifolii GQ267547 Trifolium subterraneum Australia
A. trifolii GQ267552 Trifolium subterraneum Australia
A. trifolii GQ267553 Trifolium subterraneum Australia
A. trifolii GQ267554 Trifolium subterraneum Australia
A. trifolii GQ267549 Trifolium subterraneum Australia
A. trifolii GQ267550 Trifolium subterraneum Australia
A. cladogamus AY353918 Spinacia oleracea Sweden
A. cladogamus AY353913 Medicago sativa Sweden
A. cladogamus AY353912 Melilotus officinalis Sweden
A. cladogamus AY353920 Lycopersicon esculentum USA
A. cladogamus FM999222 Chenopodium album Canada
A. cochlioides AY353911 Beta vulgaris Sweden
A. cochlioides FM999224 Beta vulgaris USA
A. euteiches AY353902 Pisum sativum USA
A. euteiches AY353906 Vicia arvense Sweden
A. euteiches AY353901 Pisum sativum Sweden
A. euteiches AY353908 Medicago sativa USA
A. euteiches AY353907 Medicago sativa USA
A. euteiches AY353909 Phaseolous vulgaris USA
A. euteiches f. sp. phaseoli AY353910 Phaseolous vulgaris USA
A. invadans FM999231 Brevoortia tyrannus USA
A. invadans FM999229 Brevoortia tyrannus USA
Leptolegnia sp. AM228851 Astacuc astacus Outgroup
Saprolegnia parasitica AY455776 Salmo trutta Outgroup
710 Crop & Pasture Science T. A. O’Rourke et al.
substitution model with gamma-distributed substitution rates of
the sites (GTR + G; Swofford et al.1996), which was used in
subsequent Bayesian analysis.
Markov chain Monte Carlo (Larget and Simon 1999; Mau
et al.1999) were performed with the computer program MrBayes
(version 3.0b4; Huelsenbeck and Ronquist 2001). Four
incrementally heated simultaneous Markov chains were run for
1 000 000 generations from which every 100th tree was sampled.
The first 1000 saved trees (10%) were discarded to be sure only
trees that were created after reaching the log-likelihood to a stable
value were included in the analyses. From the remaining trees, a
50% majority-rule consensus tree was computed and the posterior
probabilities of the groups were estimated.
Inoculum production
Inoculum was prepared using a modified procedure from Barbetti
(1984,1989). Moist sterile millet seeds (Panicum miliaceum)
were soaked in water and autoclaved at 121
o
C for 20 min on 3
consecutive days. Two-week-old colonies of the Aphanomyces
sp. growing on CMA were cut into plugs 2 mm
2
and ~20 cubes
were added to each 250-mL flask containing 200 g of the prepared
sterile moist millet seed. Flasks were vigorously shaken every
2–3 days to promote uniform colonisation. Inoculum was
incubated in the dark at 20
o
C for 3 weeks before commencing
glasshouse trials. To determine purity of inoculum, a sample of
Aphanomyces sp.-colonised millet seed inoculum for all isolates
used in glasshouse studies was plated onto CMA and also floated
in sterile distilled water and presence of the Aphanomyces sp.
confirmed.
Flooding inoculum level experiment
Six isolates of Aphanomyces sp. were examined to determine the
ideal flooding and inoculum levels. The flooding inoculum
experiment was conducted in a controlled environment room
where the air temperature was maintained at 18/13
o
C (day/night),
with a 12/12-h photoperiod and a light intensity of
281 mmol m
–2
s
–1
. These temperatures were selected to mimic
temperatures commonly seen in the field in April–June, when
seedlings emerge (Barbetti 1991b). Experiments were conducted
in pasteurised nutrient-deficient brown fluvisol soil locally
known as ‘Lancelin sand’, a nutrient-poor sandy soil of
Western Australia. The Aphanomyces sp.-colonised millet
inoculum was applied as a layer in 75-mm-diameter pots,
40 mm beneath the soil surface, at 4 different inoculum rates
depending on the treatment (viz. 0, 0.1, 0.5 and 1% of inoculum/
dry weight soil). Uninoculated sterilised millet was not used as a
control comparison as it has a ‘baiting-out’effect on any other
potential pathogens present in the soil, especially Pythium
species, when uncolonised (Barbetti and Sivasithamparam
1987). Subterranean clover cv. Woogenellup seeds were
surface sterilised in 70% ethanol for 30 s, then scarified with
sandpaper and sown at 5 per pot, at a depth of 10 mm, with 6
replicate pots per treatment. Pots were arranged in a randomised
block design. Pots were watered daily and allowed to drain to field
capacity and 5 days after sowing the flooding treatments were
administered (6 h and 24 h and 0 h flooding control). One week
after sowing, plants were fertilised at the recommended rate with a
‘complete’fertiliser treatment that provided the full range of
nutrients required for plant growth (Thrive, Yates, Padstow,
NSW, Australia). Plants were harvested 4 weeks after sowing,
washed in tap water to remove sand from roots and placed into
polyethylene zip lock bags and stored in a cool room (4
o
C) until
scored for the levels of disease over the next 6 days.
Temperature experiment
Six isolates of Aphanomyces sp. were examined to determine the
ideal temperature for disease development. The temperature
experiment was conducted across three controlled environment
rooms. Air temperature was maintained at 25/20, 18/13, or 10/5
o
C
(day/night) with a 12/12-h photoperiod and light intensities of
258, 281 and 372 mmol m
–2
s
–1
, respectively. Temperature
protocols were selected based on modified protocols from
Barbetti (1991b). This experiment was also conducted using
pasteurised Lancelin sand. The Aphanomyces sp.-colonised
millet inoculum was applied as a layer, 40 mm beneath the soil
surface, at a rate of 0.5% of dry weight of millet inoculum to dry
weight of soil. Control treatments containing 0% inoculum were
included. Subterranean clover cv. Woogenellup seeds were
surface sterilised in 70% ethanol, scarified with sandpaper and
sown in 75-mm-diameter pots at 5 per pot, at a depth of 10 mm.
Each treatment was replicated 6 times and pots were arranged in a
randomised block design. Pots were watered daily and allowed to
drain to field capacity and 5 days after sowing were flooded for
6 h. Details for fertiliser added to plants at 1 week after sowing and
for harvesting of plants at 4 weeks after sowing, were as given
above for the flooding inoculum level experiment.
Preliminary host range test
The host experiment was conducted in a controlled environment
room where air temperature was maintained at 18/13
o
C (day/
night), with a 12/12-h photoperiod and a light intensity of
281 mmol m
–2
s
–1
. Pasteurised Lancelin sand was again used.
Eleven potential hosts, as well as a susceptible (cv.
Woogenellup) and a resistant (cv. Dalkeith) subterranean
clover cultivar (T. A. O’Rourke, M. H. Ryan,
K. Sivasithamparam, M. J. Barbetti, unpubl. data), were tested
for their susceptibility to the Aphanomyces species. Alternative
host species were chosen on the basis that either they have been
grown in the high-rainfall area of south-west Western Australia,
such as annual medics (Medicago polymorpha cv. Cavalier and
M. truncatula cv. Cyprus) and annual ryegrass (Lolium rigidium
cv. Safeguard); or that they were crop legumes susceptible to
A. euteiches, such as dwarf bean (Phaseolus lunatus cv. Brown
Beauty), broad bean (V. faba cv. Early Long Pod), pea (P. sativum
cv. Dunwa) and chickpea (Cicer arietinum cv. Genesis 510); or
that they were susceptible to A. cladogamus, such as capsicum
(Capsicum annuum cv. Giant Bell) and tomato (Solanum
lycopersicum cv. Roma); or that they were a species that was
not susceptible to Aphanomyces spp., such as wheat (Triticum
aestivum cv. Wyalkatchem). The Aphanomyces sp.-colonised
millet inoculum was applied as a layer, 4 cm beneath the soil
surface, at a rate of 0.5% of dry weight. For each of the hosts,
appropriate controls were grown with 0% inoculum. Seeds of the
13 test varieties were surface sterilised with 70% ethanol, scarified
with sandpaper, when required, and sown in 75-mm-diameter
pots, 5 per pot, at a depth of 1 cm. Pots were watered daily and
Taxonomic and pathogenic characteristics of a new species Aphanomyces trifolii Crop & Pasture Science 711
allowed to drain to field capacity and 5 days after sowing were
flooded for 6 h. Details for fertiliser added to plants at 1 week after
sowing and for harvesting of plants at 4 weeks after sowing, were
as given above for the flooding inoculum level experiment.
Root disease assessment
In all instances plants were scored using the same assessment
system. Plants were floated in shallow trays of sterile deionised
water and tap and lateral roots were scored independently for root
disease using a modified scoring system described and used
earlier by Wong et al.(1984). The scoring system contained 6
disease severity categories: score 0 = root healthy, no
discolouration; 1 = <25% of root brown, no significant lesions;
2=25–<50% of root brown, lesions towards base of tap root;
3=50–<75% root brown, lesions mid-tap root; 4 = 75% root
affected, significant lesions towards crown; 5 = plant dead. The
number of plants in each disease severity category was recorded.
In all experiments Koch’s postulates were tested by
confirming that the disease symptoms observed were in fact
caused by the Aphanomyces sp. Root segments, 2 cm in
length, were dissected from diseased plants and floated in Petri
dishes containing sterile deionised water for 2–3 days at
20
o
C. Roots were examined microscopically every 12 h and
the presence of the Aphanomyces sp. zoosporangia noted.
Pathogenicity data analysis
The root disease scores were converted into percent root disease
indices using the method described by McKinney (1923). A
multiple factor ANOVA was conducted using GENSTAT (10th
edn, Lawes Agricultural Trust, Rothamsted Experimental
Station, UK). Fisher’s least significant difference at a 95%
confidence level was used to test differences between flooding,
temperature and inoculum levels within experiments. All
controls were excluded from the data analysis as they were all
zero disease. GENSTAT was also used to test the significance of
correlation coefficients between the different parameters
measured.
Results
Morphological characterisation
Hyphae were 5–10 mm in diameter, colourless, delicate in
appearance and sparingly branched. The zoosporangia were on
average 366 mm in length with zoospores elongated in the
discharge tube (Fig. 2a), forming spherical cysts, 7–9mmin
diameter. Primary zoospores were highly abundant, clumping
together and encysting at the sporangium orifice. Oogonia
(Fig. 2b) were 24–31 mm in diameter while oospores (Fig. 2c)
were 17–23 mm in diameter. Oospores were not seen germinating.
Table 2. Morphological characteristics of Aphanomyces cladogamus Dreschler (Hall 1980), Aphanomyces euteiches Dreschler (Stamps 1978;
Greenhalgh et al.1985) and a new species Aphanomyces trifolii (O’Rourke et al.) from Western Australia
Pathogen
characteristics
A. cladogamus(G. Hall CMI) A. euteiches (Stamps CMI) A. euteiches (Greenhalgh) A. trifolii sp. nov.
(Western Australia)
Hyphae 4–10 mm diameter, sparingly
branched, with a delicate
appearance on agar and
water
4–10 mm diameter Data not provided 5–10 mm in diameter. Delicate
appearance on CMA and
water
Zoosporangia 200–300 mm long 7–9um
diameter, with many lateral
branches 150–200 mm long,
not tapering towards apex
Data not provided Zoosporangia only slightly
tapering towards apex,
6.2 mm wide at base
5.1 mm at tip
366 mm long, 5–9mm diameter.
No lateral branches tapering
slightly towards apex
Primary zoospores Elongate, highly abundant,
encysting at sporangium
orifice on emergence
Elongate highly abundant,
encysting at sporangium
orifice on emergence
Data not provided Elongate highly abundant,
encysting at sporangium
orifice on emergence
Primary cysts 7–10 mm in diameter. Release
biflagellate secondary
zoospores
8–11 mm in diameter. Release
biflagellate secondary
zoospores
Data not provided 7–9mm in diameter. Release
biflagellate secondary
zoospores
Antheridia Born on branching stalks
10–20 mm long. Usually
joined to oogonium.
Clavate/cylindrical, with an
apical prolongation. Can be
monoclinous or diclinous
1–5 large, curved-clavate often
conspicuously arched, stalk
simple or branched,
diclinous
Antheridial stalks enveloped
the oogonia
Antheridial stalks enveloped
the oogonia
Oogonia Subspherical 25 mm
(20–33 mm) diameter, borne
terminally, winding around
2 or 3 antheridia, externally
smooth
Spherical or sub-spherical
(25–35 mm). Wall
irregularly thickened
30 mm diameter, internal wall
smoother than expected
Subspherical 26 mm
(24–31 mm) diameter
Oospores Spherical, colourless, 21 mm
(15–25 mm) diameter, wall
3mm thick, granular
contents, large central oil
globule
18–25 mm, with a uniformly
thickened wall (1–5mm)
Spherical, colourless, 23 mm
diameter
Spherical 20 mm (17–23 mm) in
diameter, granular contents,
large central oil globule,
germination not observed
712 Crop & Pasture Science T. A. O’Rourke et al.
The Aphanomyces sp. isolates from Western Australia were
morphologically similar to both A. euteiches and A. cladogamus,
as described in the literature. The zoosporangia were much shorter
in the Aphanomyces sp. (366 mm) than those seen in
A. cladogamus (200–300 mm). The diameters of oogonia of the
Aphanomyces sp. (24–31 mm) encompassed those of both
A. euteiches (25–35 mm) and A. cladogamus (20–33 mm).
Likewise the diameter of the Aphanomyces sp. oospores
(17–23 mm) fitted the size range for both A. euteiches
(18–25 mm) and A. cladogamus (15–25 mm) (Table 2). The
Aphanomyces sp. from Western Australia was morphologically
similar to A. euteiches isolates from subterranean clover in
Victoria. Both the Aphanomyces sp. isolates from Western
Australia and the A. euteiches isolates from Victoria showed
similar morphological differences to other recognised
A. euteiches isolates as the oogonia were enveloped by
stalks of the antheridia and the inner lining of the oogonial
wall was not sculptured as prominently as that for A. euteiches
(Table 2).
Molecular characterisation
The ITS region of the Aphanomyces sp. isolates were amplified
and sequenced. The ITS regions of all isolates were 650 base
pairs in length with identical nucleotide sequences, with the
exception of three isolates, one UWA041-1 that had a deletion
of 60 nucleotides in the ITS1 region and two other isolates
UWA007-03 and UWA007-05 that had a single ‘G’
nucleotide insertion in ITS1.
The species of Aphanomyces closest to the isolates from
Western Australia was A. cladogamus when their sequences
were compared (using BLAST) to available data in the GenBank.
Subsequently, the ITS sequences of Western Australian
isolates were aligned and compared with ITS sequences of the
representative plant associated/parasitic species of Aphanomyces
including A. euteiches,A. cochlioides and A. cladogamus
originating from diverse host plants and locations. MP analysis
of the complete ITS dataset revealed two most parsimonious
trees of score 312, in which the Western Australian isolates
formed a distinct clade, separated from the three other plant-
associated/parasitic Aphanomyces species, supported by a
high bootstrap value of 98%. The two parsimonious trees
differed only in the position of the A. cochlioides clade
relative to A. euteiches and A. cladogamus. One of these trees
is shown in Fig. 1;A. euteiches,A. cladogamus and A. cochlioides
were each grouped in three separate clades with high
bootstrap supports. The Bayesian tree was fully in agreement
with MP tree as the Western Australian isolates formed a
distinct clade with posterior probability of 100%. The closest
group to the Australian isolates was A. cladogamus, comprising
non-pathogenic isolates obtained from a variety of legumes
in Sweden (Levenfors and Fatehi 2004) and isolates
pathogenic to spinach and tomato. The genetic relatedness
between these two clades was high (93%) in MrBayes tree
compared to a relatively low bootstrap value (74%) obtained
in parsimony analysis.
On the basis of the molecular characteristics, the
Aphanomyces sp. causing root disease on subterranean clover
in the high-rainfall region of south-west Western Australia is
considered to be a new species and we propose the name
Aphanomyces trifolii sp. nov. (O’Rourke et al.). A culture of
this new species A. trifolii (WAC 13269) has been lodged in the
culture collection of the Department of Agriculture and Food
Western Australia. A Latin diagnosis describing the new species
is given below.
Disease symptoms
A. trifolii caused a light-brown to dark-brown discolouration,
darkening as symptoms developed, of the cortical tissue along
both tap and lateral roots. This discolouration frequently ascended
into the hypocotyl and in some seedlings resulted in hypocotyl
collapse. However, A. trifolii principally caused severe disease on
lateral roots and severely affected subterranean clover seedlings
had very few if any lateral roots (Fig. 2d).
Flooding inoculum level experiment
Tap root disease and lateral root disease were more severe as
flooding duration increased, with a flooding time of 24 h leading
UWA009-1 GQ267548
A. euteiches AY353902
A. euteiches AY353906
A. euteiches AY353901
A. euteiches AY353908
A. euteiches AY353907
A. euteiches AY353909
A. euteiches f. sp. phaseoli AY353910
A. cochlioides AY353911
A. cochlioides FM999224
A. cladogamus AY353918
A. cladogamus AY353913
A. cladogamus AY353912
A. cladogamus AY353920
A. cladogamus FM999222
UWA010-6 GQ267551
UWA042-13 GQ267547
UWA041-6 GQ267552
UWA040-8 GQ267553
UWA040-5 GQ267554
UWA007-5 GQ267549
UWA007-3 GQ267550
A. invadans FM999231
A. invadans FM999229
Leptolegnia sp. AM228851
Saprolegnia parasitica AY455776
A. trifolii
10 changes
100
100
100
100
100
100
100
100
100
99
98
98
100
100
93
74
70
50
63
87
63
60
Fig. 1. One of two most parsimonious trees (length 312) resulting from
analysis of the ITS sequence data including ITS1, ITS2 and 5.8s rRNA gene.
Branch lengths correspond to the number of substitutions, numbers above
branches represent maximum parsimony bootstrap values (>50%) and
numbers below represent posterior probabilities obtained from a Bayesian
analysis of 1M generations
Taxonomic and pathogenic characteristics of a new species Aphanomyces trifolii Crop & Pasture Science 713
to the most severe tap and lateral root disease (Table 3). There was
no difference between the free-draining and the 6-h flooding
treatments. There was a significant increase in both tap and lateral
root disease with increasing level of inoculum with 1% inoculum
causing more disease than either 0.5 or 0.1% inoculum
(Table 3). There was no tap or lateral root disease in the
controls (data not presented). Meaned across tap and lateral
root disease, UWA043-4 and UWA043-11 were overall the
two most pathogenic isolates of A. trifolii in the flooding
experiment, followed by isolate UWA043-13. In contrast
UWA043-3 caused the least severe root disease of the isolates.
There were no two-way interactions between flooding, inoculum
level and isolate for either tap or lateral root rot. However, there
was a significant three-way interaction between flooding,
inoculum level and isolate in relation to both tap and lateral
root disease (Table 3).
Temperature experiment
There was a strong relationship between temperature and the
severity of root disease, with significantly more disease present on
both lateral and tap roots at 18/13
o
C compared with the other two
temperature regimes (Table 4). There was substantial variation in
the pathogenicity of A. trifolii isolates with, for both tap and lateral
root disease, UWA043-13 and UWA043-11 being overall the
most pathogenic and causing the most severe disease, followed by
UWA043-4. In contrast, UWA043-2 and UWA043-3 were only
slightly pathogenic and caused very little disease on subterranean
clover (Table 4). The lateral root disease levels observed at the
10/5
o
C regime may not be truly reflective of the potential damage
that A. trifolii does to lateral roots of subterranean clover as many
of the plants did not form lateral roots during the 4 weeks’growing
time at this temperature regime. There was no tap or lateral root
disease in the controls (data not presented).
Preliminary host range test
Aside from being pathogenic on subterranean clover, A. trifolii
was slightly pathogenic on annual medic (M. polymorpha and
M. truncatula), dwarf beans and tomato (Table 5). A. trifolii was
not pathogenic on peas, chickpea, wheat, ryegrass or capsicum
(Table 5). There was no tap or lateral root disease in the controls
for any of the host species (data not presented).
Taxonomy
Aphanomyces trifolii sp. nov. O’Rourke, Ryan, Li, Ma,
Sivasithamparam, Fatehi & Barbetti (Figs 1and 2).
Etym: Referring to Trifolium the host plant from which the
pathogen was isolated.
Coloniae: Hyphae incolores, tenues, 5–10 mm diam., parce
ramosae. Zoosporangia ~366 mm longa, 7–10 mm lata. Zoosporae
tubo dimisso elongatae, 7–9mm diam., cystis globosis, 7–9mm
(a)
100 µm
20.0 µm
50.0 µm
i ii iii iv
(c)
(b)
(d)
Fig. 2. Morphological structures and disease symptoms of A. trifolii sp. nov.; (a) sporangium with encysted primary zoospores, (b) oogonia
with a visible antheridium, (c) oospores, (d) symptoms of root disease on subterranean clover: (i) uninfested control, (ii–iv) diseased plants
displaying typical lateral root pruning.
714 Crop & Pasture Science T. A. O’Rourke et al.
Table 3. Levels of root disease on subterranean clover (Trifolium subterraneum) cv. Woogenellup caused by 6 isolates of
Aphanomyces trifolii sp. nov. and the response to 3 flooding treatments (free draining, 6 h and 24 h) and 3 different rates of
inoculum (0.2, 0.5 and 1.0% inoculum :dry weight of soil)
Flooding Inoculum rate Isolate Percent disease index
(% dry weight) Tap Lateral
Free draining 0.2 UWA-043-001 52.6 56.7
UWA-043-002 9.4 10.4
UWA-043-003 10.8 19
UWA-043-004 24.9 38.2
UWA-043-011 30.8 41.7
UWA-043-013 12 17
0.5 UWA-043-001 25.2 30.7
UWA-043-002 20.5 31.8
UWA-043-003 12.4 18.2
UWA-043-004 51.2 52.2
UWA-043-011 56.8 65.2
UWA-043-013 39.7 51.3
1 UWA-043-001 34.8 38.1
UWA-043-002 42 48.8
UWA-043-003 25.3 44.2
UWA-043-004 46.4 55.2
UWA-043-011 43.7 57.5
UWA-043-013 43.6 63.2
6hflooding 0.2 UWA-043-001 16.9 25.3
UWA-043-002 26.8 26.7
UWA-043-003 12.2 15.3
UWA-043-004 32.1 41.2
UWA-043-011 38.2 55.3
UWA-043-013 37.4 54.8
0.5 UWA-043-001 25.7 16
UWA-043-002 36.5 39.8
UWA-043-003 10 13.7
UWA-043-004 50.6 60.6
UWA-043-011 50 55.8
UWA-043-013 24.7 32
1 UWA-043-001 60.7 67.2
UWA-043-002 52 59.3
UWA-043-003 39.7 42.6
UWA-043-004 76.1 78
UWA-043-011 76.7 76.3
UWA-043-013 20.6 22.2
24 h flooding 0.2 UWA-043-001 33.4 42.1
UWA-043-002 30.3 34.5
UWA-043-003 23.7 24.8
UWA-043-004 62.2 66.2
UWA-043-011 66 78
UWA-043-013 21.3 23
0.5 UWA-043-001 42.2 48.8
UWA-043-002 62.8 67.3
UWA-043-003 21.8 29.2
UWA-043-004 52.7 58.1
UWA-043-011 77.3 84.7
UWA-043-013 66.9 60.3
1 UWA-043-001 53.8 66.5
UWA-043-002 39.7 42.7
UWA-043-003 40.2 41.2
UWA-043-004 76.1 85.6
UWA-043-011 68.1 76.6
UWA-043-013 74 64.2
Tap roots
Significance of flooding, P<0.001; l.s.d. at P= 0.05 = 5.4
Free draining 6h flooding 24 h flooding
27.9 32.8 43.5 –
Taxonomic and pathogenic characteristics of a new species Aphanomyces trifolii Crop & Pasture Science 715
diam., ejectis formantibus. Primo cystae abundantissimae, ad
sporangium aperturam aggregatae. Antheridia clavata ad
cylindracea, 10–14 05–7mm, oogonia involvens. Oogonia
subsphaerica, incolora, 24–31 mm diam. Oosporae 17–23 mm
diam., contento granulari, pariete 200–300 mm crasso;
germinatio non visa.
Typus: Australia: Western Australia: on tap and lateral roots
of Trifolium subterraneum at 7 field sites in the high-rainfall
region of south-west Western Australia.
Discussion
Isolates belonging to the genus Aphanomyces were obtained from
diseased subterranean clover roots from 7 sites in the high-rainfall
region of south-west Western Australia. Initially, we attempted to
identify these isolates based on their morphological features, as
traditionally used for the identification and taxonomy of
Aphanomyces species. However, reliable species identification
in oomyceteous genera such as Aphanomyces or Pythium is often
constrained by poor delineation or the lack of distinctive
anatomical structures and colony morphology among closely
related species. For example, spore sizes from Western
Australian Aphanomyces isolates for the most part overlapped
key diagnostic criteria for both A. cladogamus and A. euteiches.
These isolates were very similar to A. euteiches in appearance
with overlapping size ranges for oospores, zoospores and oogonia
as well as causing disease on legumes.
A. euteiches caused significant root disease on subterranean
clover in Victoria (Greenhalgh et al.1985,1988). However, it was
noted at the time that the Victorian isolates differed slightly from
A. euteiches as oogonia were enveloped by stalks of the
antheridia, the inner lining of the oogonial wall was not
sculptured as prominently as that for A. euteiches and that the
pathogen grew better on agar at 20
o
C than 28
o
C (Greenhalgh et al.
1985). These differences were not considered to be of sufficient
taxonomic importance to distinguish the Victorian isolates from
A. euteiches. However, Victorian isolates designated to
A. euteiches and Western Australian isolates described in this
study as a new species, A. trifolii, are morphologically very
similar and may well be the same species. Unfortunately, there
are no living cultures of isolates previously reported as
A. euteiches on subterranean clover in Victoria and therefore it
was not possible to include them as comparisons in this study.
Due to the morphological similarities, Western Australian
isolates of A. trifolii were originally thought to be A. euteiches
(Barbetti 1991b). To determine definitively whether the isolates
from Western Australia were in fact a new species, it was
necessary, in our study, to use molecular techniques. The ITS
region was amplified using ITS1 and ITS4 and the PCR products
were purified and sequenced. Analyses of the nucleotide
sequences and comparison with the homologous region from
known plant parasitic Aphanomyces species showed that Western
Australian isolates formed a distinct clade with very high
bootstrap support, separate from A. euteiches,A. cladogamus
and A. cochlioides (Fig. 1). The closest related species was
A. cladogamus, a pathogen frequently isolated from diseased
legume roots that has never been shown to be pathogenic on the
Table 3. (continued )
Significance of inoculum, P<0.001; l.s.d. at P= 0.05 = 5.4
0.2 0.5 1 –
26 34.9 43.4 –
Significance of isolate, P<0.001; l.s.d. at P= 0.05 = 8.35
UWA-043-001 UWA-043-002 UWA-043-003 –
38.4 35.6 21.8 –
UWA-043-004 UWA-043-011 UWA-043-013 –
51.7 56.4 37.8 –
Significance of flooding inoculum, P>0.05, n.s.
Significance of flooding isolate, P>0.05, n.s.
Significance of inoculum isolate, P>0.05, n.s.
Significance of flooding inoculum isolate, P<0.005; l.s.d. at P= 0.05 = 24.74
Lateral roots
Significance of flooding, P<0.001; l.s.d. at P= 0.05 = 5.94
Free draining 6h flooding 24 h flooding
35.2 37.2 47.4 –
Significance of inoculum, P<0.001; l.s.d. at P= 0.05 = 5.94
0.2 0.5 1 –
31.9 38.9 49 –
Significance of isolate, P<0.001; l.s.d. at P= 0.05 = 9.07
UWA-043-001 UWA-043-002 UWA-043-003 –
43.5 40.1 27.6 –
UWA-043-004 UWA-043-011 UWA-043-013 –
59.5 65.7 43.1 –
Significance of flooding inoculum, P>0.05, n.s.
Significance of flooding isolate, P>0.05, n.s.
Significance of inoculum isolate, P>0.05, n.s.
Significance of flooding inoculum isolate, P<0.005; l.s.d. at P= 0.05 = 27.2
716 Crop & Pasture Science T. A. O’Rourke et al.
legume host of origin (Levenfors et al.2003). In contrast,
A. trifolii is highly pathogenic on subterranean clover cv.
Woogenellup causing significant root disease particularly to
the lateral root system.
Isolates of A. trifolii examined generally caused more root
disease as the level of inoculum increased, probably as a
consequence of more zoosporangia being present, increasing
the loci of infection thus leading to greater levels of root
disease. This is similar to other oomycete pathogens that cause
root disease on subterranean clover in Australia such as Pythium
irregulare (Wong et al.1984) and P. clandestina (Wong et al.
1985).
An increase in the duration of soil saturation promoted the
development of severe root disease caused by A. trifolii with the
most severe disease associated with 24 h of flooding. There was
no difference in disease levels between treatments that received
6hofflooding compared to soils watered to field capacity. This
result is consistent with the findings of Greenhalgh et al.(1988)
that A. euteiches caused more disease after 24 h flooding
compared with 4 h flooding. The duration of soil saturation has
been previously shown to be an important factor affecting the
levels of root disease caused by other oomycetes such as
P. clandestina (Greenhalgh and Taylor 1985; Taylor and
Greenhalgh 1987; Greenhalgh et al.1988). Our study showed
that A. trifolii is capable of causing moderate damage on
subterranean clover root systems in moist soils at field
capacity. However, A. trifolii is likely to cause more damage
and be more prevalent in areas that receive higher levels of
rainfall, especially if this is associated with prolonged periods
of soil flooding.
Table 4. Levels of root disease caused by 6 isolates of Aphanomyces trifolii sp. nov. on subterranean clover
(Trifolium subterraneum) cv. Woogenellup under 3 temperature regimes (10/5, 18/13 and 25/20
o
C)
Temperature (8C) Isolate Percent disease index
Day/night Tap Lateral
10/5 UWA-043-001 30 0
UWA-043-002 12.8 0
UWA-043-003 13.3 10
UWA-043-004 25 27.2
UWA-043-011 44.4 50.5
UWA-043-013 73.3 61.2
18/13 UWA-043-001 29.3 35.6
UWA-043-002 10 23.3
UWA-043-003 10.3 7.3
UWA-043-004 47.6 27.7
UWA-043-011 61.3 70.7
UWA-043-013 68.9 84.9
25/20 UWA-043-001 18.7 16
UWA-043-002 5.3 8
UWA-043-003 9.7 7.3
UWA-043-004 40 48.3
UWA-043-011 33.3 53.3
UWA-043-013 39.3 61
Tap roots
Significance of temperature, P<0.01; l.s.d. at P= 0.05 = 9.67
Means 10/58C 18/138C 25/208C
28.7 32.5 20.9
Significance of isolate, P<0.001; l.s.d. at P= 0.05 = 14.77
Means UWA-043-001 UWA-043-002 UWA-043-003
26 9.4 11.1
UWA-043-004 UWA-043-011 UWA-043-013
37.5 46.4 60.5
Significance of temperature isolate, P>0.05; n.s.
Lateral roots
Significance of temperature, P<0.05; l.s.d. at P= 0.05 = 9.59
Means 10/58C 18/138C 25/208C
21.3 35.6 27.7
Significance of isolate, P<0.001; l.s.d. at P= 0.05 = 14.65
Means UWA-043-001 UWA-043-002 UWA-043-003
17.2 10.4 8.2
UWA-043-004 UWA-043-011 UWA-043-013
34.4 58.2 69
Significance of temperature isolate, P>0.05; n.s.
Taxonomic and pathogenic characteristics of a new species Aphanomyces trifolii Crop & Pasture Science 717
A. trifolii was pathogenic over all three temperature regimes
tested, but caused most disease when day/night temperatures were
18/13
o
C. This temperature range is consistent with temperatures
most commonly seen in the field in the high-rainfall areas of
south-west Western Australia during the early part of the growing
season (Barbetti 1991b), a period likely to coincide with A. trifolii
inoculum levels being at their highest. A. trifolii still caused
significant disease at the cooler temperature regime of 10/5
o
C, but
disease was noticeably concentrated on tap roots, with the
presence of only a few lateral roots after 4 weeks of growth
under such conditions. At the 25/20
o
C regime, plants were less
diseased than at the other two temperature regimes and this was
probably related to rapid plant growth at this temperature regime
allowing the plants to better cope with and compensate for
diseased roots (Barbetti 1984). Greenhalgh et al.(1988) found
that there were no differences in the level of disease from his
Aphanomyces at 10
o
C compared with 20
o
C. Variable, early
seasonal conditions such as the duration of autumn rains and
soil temperatures which coincide with the germination and early
establishment of subterranean clover each year are likely to be
major influences on the severity of Aphanomyces root disease.
The host range of root disease pathogens has significant
implications for disease management, especially where
pastures are not permanent, but rather a component of a
broader cropping rotation including one or more cereal crops
such as wheat, oats (Avena sativa) and lupins (Lupinus spp.) as
well as other grain legumes. A preliminary screening of disease
resistance to A. trifolii found that in addition to causing significant
root disease on subterranean clover, A. trifolii was moderately
pathogenic on two annual medic species (M. polymorpha and
M. truncatula) and mildly pathogenic on dwarf beans and tomato.
Our findings for annual Medicago spp. confirm those for
A. euteiches isolates in France that were also pathogenic on
Medicago spp. (Moussart et al.2007). However, as there is a
high level of variability in populations of Aphanomyces such as
A. euteiches (Malvick and Percich 1999), and as we only used one
isolate for the host screening of A. trifolii in our studies, it is
possible that the host range depicted in our studies could be
an incomplete representation of the A. trifolii host range.
Furthermore, it is possible for variation of susceptibility/
resistance to exist between cultivars belonging to the one host
(Wicker et al.2003; Moussart et al.2008). As our study only
utilised one cultivar of each host species, it is also possible that
the performance within the host genus/species may not be fully
indicative from our results. It is clear that as annual medics are
widely grown across the grainbelt and in some high-rainfall areas
where A. trifolii is prevalent, annual medics could maintain or
even increase inoculum build-up of A. trifolii in the absence of
subterranean clover. This is not entirely unexpected as
subterranean clover and annual medic species are similarly
affected by several necrotrophic root rot pathogens (Tivoli
et al.2006; Barbetti et al.2007). A. trifolii was not pathogenic
on other legumes such as pea and chickpea, or on wheat or annual
ryegrass which are commonly grown in the grainbelt and high-
rainfall regions of south-west Western Australia. As such,
utilisation of these particular species offers opportunity to
impede both the spread and inoculum build-up of A. trifolii.
A. trifolii is widespread and has potential to be a major root
pathogen in the high-rainfall areas of Western Australia, in
particular causing damage to the root systems of subterranean
clover seedlings during establishment. Other significant
pathogens on subterranean clover in these areas of Western
Australia include P. clandestina,P. irregulare,Rhizoctonia
solani and Cylindrocarpon didymum. The role of A. trifolii in
the root pathogen complexes occurring in these areas is yet to be
defined. A. trifolii is likely to be a widespread, severe pathogen of
subterranean clover in the Mediterranean-type environments of
southern Australia. The impoverished and nutrient-deficient soils
across much of this region predispose the plant host to such
pathogens as there is often little microbial competition, which is
particularly beneficial for oomycete pathogens (Sivasithamparam
1993,1996).
Our study has established A. trifolii as a serious root rot
pathogen of subterranean clover in the high-rainfall areas of
south-west Western Australia. It is likely that A. trifolii has
been present in Western Australia for many years, however, as
it is a difficult pathogen to isolate, identify and maintain, it has not
been characterised previously. While A. trifolii causes root
disease on both the tap root and hypocotyl in seedlings of
subterranean clover, it primarily damages lateral roots. These
results show that Western Australian isolates of Aphanomyces are
in fact a new species, A. trifolii. However, it is possible that the
A. euteiches reported causing root disease on subterranean clover
in Victoria was also A. trifolii and Victorian sites should be re-
sampled. It is likely that A. trifolii is a major root pathogen across
the higher-rainfall areas of southern Australia, particularly where
periodic flooding occurs that predisposes subterranean clover to
severe root disease from this pathogen.
Acknowledgments
We thank the Australian Wool Innovation Ltd and the late Mr Frank Ford,
Western Australia, for a bequest that funded this research, which is a part of
Table 5. Level of root disease expressed as percent disease indices
caused by Aphanomyces trifolii sp. nov. (isolate UWA040-13) on a
range of possible hosts including; pea, chickpea, broad bean, dwarf
bean, subterranean clover (Dalkeith, Woogenellup), annual medic
(Medicago polymorpha and M. truncatula), wheat, annual ryegrass,
tomato and capsicum
n.a., No tap roots available to assess
Host Cultivar Tap root
rot
Lateral
root rot
Pisum sativum Dunwa 0 0
Cicer arietinum Genesis 510 0 0
Vicia faba Early Long Pod 0 0
Phaseolous lunatus Brown Beauty 18.4 15.6
Trifolium subterraneum Dalkieth 18.5 31.8
Trifolium subterraneum Woogenellup 34.5 62.7
Ornithopus compressus Santorini 9.8 16.7
Medicago polymorpha Cavalier 37.3 40.9
Medicago truncatula Cyprus 15.8 24.7
Triticum aestivum Wyalkatchem n.a. 0
Lolium ridgidum Safeguard n.a. 0
Lycopersicum esculantum Roma 13.3 10.3
Capsicum annuum Giant Bell 0 0
Significance –P<0.01 P<0.01
l.s.d. P= 0.05 –8.1 10.2
718 Crop & Pasture Science T. A. O’Rourke et al.
the primary author’s PhD. We also thank Dr Ovidiu Constantinescu for
performing the Latin translation.
References
Allen RN, Letham DB, Akehurst AA, Say MM (1987) Aphanomyces root rot
of bean at Valla, New South Wales. Australasian Plant Pathology 16,
82–84. doi:10.1071/APP9870082
Barbetti MJ (1984) Subterranean clover foliage fungi as root pathogens.
Australasian Plant Pathology 13,38–40. doi:10.1071/APP9840038
Barbetti MJ (1989) Response of subterranean clover cultivars to
Phytophthora clandestina.Phytophylactica 21,65–67.
Barbetti MJ (1991a) Effects of clover free rotations upon the severity
of root rot in regenerating subterranean clover pastures. Australian
Journal of Agricultural Research 42, 1195–1204. doi:10.1071/
AR9911195
Barbetti MJ (1991b) Effects of temperature and humidity on diseases caused
by Phoma medicaginis and Leptosphaerulina trifolii in lucerne
(Medicago sativa). Plant Pathology 40, 296–301. doi:10.1111/j.1365-
3059.1991.tb02379.x
Barbetti MJ, Sivasithamparam K (1987) Effects of soil pasteurization on root
rot, seedling survival and plant dry weight of subterranean clover
inoculated with six fungal root pathogens. Australian Journal of
Agricultural Research 38, 317–327. doi:10.1071/AR9870317
Barbetti MJ, Sivasithamparam K, Wong DH (1986) Root rot of subterranean
clover. Review of Plant Pathology 65, 287–295.
Barbetti MJ, You MP, Li H, Ma X, Sivasithamparam K (2007)
Management of root diseases of annual pasture legumes in
Mediterranean ecosystems –a case study of subterranean clover root
diseases in the south-west of Western Australia. Phytopathologia
Mediterranea 46, 239–258.
Constantinescu O, Fatehi J (2002) Peronospora-like fungi (Chromista,
Peronsoporales) parasitic on Brassicaceae and related hosts. Nova
Hedwigia 74, 291–338. doi:10.1127/0029-5035/2002/0074-0291
Cooke DEL, Drenth A, Duncan JM, Wagels G, Brasier CM (2000) A
molecular phylogeny of Phytophthora and related Oomycetes. Fungal
Genetics and Biology 30,17–32. doi:10.1006/fgbi.2000.1202
Diéguez-Uribeondo J, García MA, Cernius L, Kozubíková E, Ballesteros I,
Windels C, Weiland J, Kator H, Söderhäll K, Martín MP (2009)
Phylogenetic relationships among plant and animal parasites, and
saprotrophs in Aphanomyces (Oomycetes). Fungal Genetics and
Biology 46, 365–376. doi:10.1016/j.fgb.2009.02.004
Geach WL (1936) Root rot of gray peas in Tasmania. Journal of the Council
for Scientific and Industrial Research 9,77–87.
Gladstone JS, Collins WJ (1983) Subterranean clover as a naturalized plant
in Australia. Journal of the Australian Institute of Agricultural Science
49, 191–202.
Greenhalgh FC, Merriman PR, Keane PJ (1985) Aphanomyces euteiches,a
cause of root rot of subterranean clover in Victoria. Australasian Plant
Pathology 14,34–37. doi:10.1071/APP9850034
Greenhalgh FC, Merriman PR, Keane PJ (1988) Relative importance of root
rots of subterranean clover caused by Aphanomyces euteiches and
Phytophthora clandestina. Plant Pathology 37, 344–350. doi:10.1111/
j.1365-3059.1988.tb02084.x
Greenhalgh FC, Taylor PA (1985) Phytophthora clandestina, cause of
severe taproot rot of subterranean clover in Victoria, Australia. Plant
Disease 69, 1002–1004. doi:10.1094/PD-69-1002
Hall G (1980) ‘Aphanomyces cladogamus Drechsler.’CMI Descriptions of
Pathogenic Fungi and Bacteria No. 971. (CAB International Mycological
Institute: Kew, England)
Huelsenbeck JP, Ronquist F (2001) MRBAYES: Bayesian inference of
phylogenetic trees. Bioinformatics 17, 754–755. doi:10.1093/
bioinformatics/17.8.754
Larget B, Simon DL (1999) Markov chain Monte Carlo algorithms for
the Bayesian analysis of phylogenetic trees. Molecular Biology and
Evolution 16, 750–759.
Levenfors JP, Fatehi J (2004) Molecular characterization of Aphanomyces
species associated with legumes. Mycological Research 108, 682–689.
doi:10.1017/S0953756204009931
Levenfors JP, Wickstrom M, Persson L, Gerhardson B (2003) Pathogenicity
of Aphanomyces spp. from different leguminous crops in Sweden.
European Journal of Plant Pathology 109, 535–543. doi:10.1023/
A:1024711428760
Ma X, Hua L, O’Rourke TA, Sivasithamparam K, Barbetti MJ (2008) Co-
occurrence of an Aphanomyces sp. and Phytophthora clandestina in
subterranean clover pastures in the high rainfall areas of the lower
south-west of Western Australia. Australasian Plant Pathology 37,
74–78. doi:10.1071/AP07086
Malvick DK, Grau CR, Percich JA (1998) Characterization of Aphanomyces
euteiches strains based on pathogenicity tests and random amplified
polymorphic DNA analyses. Mycological Research 102, 465–475.
doi:10.1017/S0953756297005029
Malvick DK, Percich JA (1999) Identification of Pisum sativum germplasm
with resistance to root rot caused by multiple strains of Aphanomyces
euteiches.Plant Disease 83,51–54. doi:10.1094/PDIS.1999.83.1.51
Mau B, Newton M, Larget B (1999) Bayesian phylogenetic inference via
Markov chain Monte Carlo methods. Biometrics 55,1–12. doi:10.1111/
j.0006-341X.1999.00001.x
McKinney HA (1923) Influence of soil temperatures and moisture on
infection of wheat seedlings by Helminthosporium sativum.The
Journal of Agricultural Science 26, 195–218.
Moussart A, Even MN, Tivoli B (2008) Reaction of genotypes from species
of grain and forage legumes to infection with a French pea isolate of the
oomycete Aphanomyces euteiches.European Journal of Plant Pathology
122, 321–333. doi:10.1007/s10658-008-9297-y
Moussart A, Onfroy C, Lesne A, Esquibet M, Grenier E, Tivoli B (2007) Host
status and reaction Medicago truncatula accessions to infection by three
major pathogens of pea (Pisum sativum) and alfalfa (Medicago sativa).
European Journal of Plant Pathology 117,57–69. doi:10.1007/s10658-
006-9071-y
Nylander JA (2004) Bayesian phylogenetics and the evolution of gall
wasps. Acta Universitatis Uppsaliensis. Comprehensive summaries
of Uppsala Dissertations from the Faculty of Science and Technology
937, 43.
O’Rourke TA, Scanlon TT, Ryan MH, Wade LJ, McKay AC, Riley IT, Li H,
Sivasithamparam K, Barbetti MJ (2009) Severity of root rot in mature
subterranean clover and associated fungal pathogens in the wheatbelt of
Western Australia. Crop & Pasture Science 60,43–50. doi:10.1071/
CP08187
Othieno Abbo E, Irwin JAG (1990) Aphanomyces euteiches, a possible cause
of poor lucerne establishment in the Lockyer Valley, Queensland.
Australian Journal of Experimental Agriculture 30, 361–364.
doi:10.1071/EA9900361
Papavizas CG, Ayers WA (1974) Aphanomyces species and their root
diseases in peas and sugarbeet –a review. Technical Bulletin 1485,
United States Department of Agriculture.
Persson L, Larsoon-Wikstrom M, Gerhardson B (1999) Assessment of soil
suppressiveness to Aphanomyces root rot of pea. Plant Disease 83,
1108–1112. doi:10.1094/PDIS.1999.83.12.1108
Pfender WF, Delwiche PA, Grau CR, Hagedorn DJ (1984) A medium to
enhance recovery of Aphanomyces from infected plant tissue. Plant
Disease 68, 845–847. doi:10.1094/PD-69-845
Scott WW (1961) ‘A monograph of the genus Aphanomyces.’
Technical Bulletin 151. (Virginia Agricultural Experiment Station:
Blacksburg, VA)
Sivasithamparam K (1993) Ecology of root-infecting pathogenic fungi in
Mediterranean environments. Advances in Plant Pathology 10, 245–279.
Taxonomic and pathogenic characteristics of a new species Aphanomyces trifolii Crop & Pasture Science 719
Sivasithamparam K (1996) The effect of soil nutrients on microbial
suppression of soil-borne diseases. In ‘Management of soil-borne
diseases’. (Eds RS Utkhede, VK Gupta) pp. 123–145. (Kalyani
Publishers: Ludhiana, India)
Stamps J (1978) ‘Aphanomyces euteiches Drechsler.’CMI Descriptions
of Pathogenic Fungi and Bacteria No. 600. (CAB International
Mycological Institute: Kew, England)
Swofford DL (2002) ‘PAUP*. Phylogenetic Analysis Using Parsimony (*and
Other Methods).’(Sinauer Associates: Sunderland, MA)
Swofford DL, Olsen GJ, Waddell PJ, Hillis DM (1996) Phylogenetic
inference. In ‘Molecular systematics’. (Eds DM Hiillis, D Moritz,
BK Mable) pp. 407–514. (Sinauer Associates: Sunderland, MA)
Taylor PA, Greenhalgh FC (1987) Significance, causes and control of root
rots of subterranean clover. In ‘Temperate pastures: their production, use
and management’. Australian Wool Corporation Technical Publication
Series. (Eds JL Wheeler, CJ Pearson, GE Robards) pp. 249–251. (CSIRO
Publishing: Melbourne)
Tivoli B, Baranger A, Sivasithamparam K, Barbetti MJ (2006) Annual
Medicago –from a model crop challenged by a spectrum of
necrotrophic pathogens to a model plant to explore the nature of
disease resistance. Annals of Botany 98, 1117–1128. doi:10.1093/aob/
mcl132
van Leur JAG, Southwell RJ, Mackie JM (2008) Aphanomyces root rot on
faba bean in northern NSW. Australasian Plant Disease Notes 3,8–9.
doi:10.1071/DN08004
Wicker E, Moussart A, Duparque M, Rouxel F (2003) Further contributions to
the development of a differential set of pea cultivars (Pisum sativum)to
investigate the virulence of Aphanomyces euteiches.European Journal
of Plant Pathology 109,47–60. doi:10.1023/A:1022020312157
Wong DH, Barbetti MJ, Sivasithamparam K (1984) Effects of soil
temperature and moisture on the pathogenicity of fungi associated with
root rot of subterranean clover. Australian Journal of Agricultural
Research 35, 675–684. doi:10.1071/AR9840675
Wong DH, Barbetti MJ, Sivasithamparam K (1985) Fungi associated with
root rots of subterranean clover in Western Australia. Australian Journal
of Experimental Agriculture 25, 574–579. doi:10.1071/EA9850574
Wylie S, Wilson CR, Jones RAC, Jones MGK (1993) A polymerase chain
reaction assay for cucumber mosaic virus in lupin seeds. Australian
Journal of Agricultural Research 44,41–45. doi:10.1071/AR9930041
Manuscript received 2 February 2010, accepted 13 July 2010
720 Crop & Pasture Science T. A. O’Rourke et al.
http://www.publish.csiro.au/journals/cp
