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Severity of root rot in mature subterranean clover
and associated fungal pathogens in the wheatbelt
of Western Australia
Tiernan A. O’Rourke
A,G
, Tim T. Scanlon
A,B
, Megan H. Ryan
A
, Len J. Wade
A,E
,
Alan C. McKay
C
, Ian T. Riley
C,D
, Hua Li
A
, Krishnapillai Sivasithamparam
A
,
and Martin J. Barbetti
A,F
A
School of Plant Biology, Faculty of Natural Agricultural Sciences, The University of Western Australia,
Crawley, WA 6009, Australia.
B
Department of Agriculture and Food Western Australia, GPO Box 432, Merredin, WA 6415, Australia.
C
Plant and Soil Health, South Australian Research and Development Institute, GPO Box 397,
Adelaide, SA 5001, Australia.
D
School of Agriculture Food and Wine, Faculty of Sciences, The University of Adelaide, SA 5005, Australia.
E
Charles Sturt University, E.H. Graham Centre for Agricultural Innovation, Locked Bag 588,
Wagga Wagga, NSW 2678, Australia.
F
Department of Agriculture and Food Western Australia, Baron-Hay Court, South Perth,
WA 6151, Australia.
G
Corresponding author. Email: orourt02@Student.uwa.edu.au
Abstract. Pasture decline is considered to be a serious challenge to agricultural productivity of subterranean clover across
southern Australia. Root disease is a significant contributing factor to pasture decline. However, root disease assessments are
generally carried out in the early part of the growing season and in areas predominantly sown to permanent pastures. For this
reason, in spring 2004, a survey was undertaken to determine the severity of root disease in mature subterranean clover plants
in pastures located in the wheatbelt of Western Australia. DNA-based soil assays were used to estimate population density in
the soil of a variety of soil-borne pathogens known to commonly occur in the Mediterranean-type environments of southern
Australia. The relationships between severity of disease on tap and lateral roots and root diameter, root length, nodulation, and
total rainfall were determined. The survey showed, for the first time, that severe root disease is widespread in spring across the
wheatbelt of Western Australia. There was a positive correlation between rainfall and tap root disease, and between tap root
disease and average root diameter of the entire root system. Despite the high levels of root disease present across the sites, the
DNA of most root disease pathogens assayed was detected in trace concentrations. Only Pythium Clade F showed high DNA
concentrations in the soil. DNA concentrations in the soil, in particular for Phytophthora clandestina and Rhizoctonia solani
AG 2.1 and AG 2.2, were higher in the smaller autumn sampling in 2006. This study suggests that the productivity of
subterranean clover-based pastures is severely compromised by root rot diseases throughout the growing season in the
wheatbelt of Western Australia.
Introduction
In Australia, pasture legumes provide an important source of
biologically fixed nitrogen for subsequent use in cropping
rotations as well as high-quality livestock fodder. Subterranean
clover (Trifolium subterraneum) is an important component of
pastures, such as those present across southern and eastern
Australia (Barbetti et al. 1986). There are an estimated
22 million ha of subterranean clover in Australia (Sandral
et al. 1997). Historically, subterranean clover has been the
dominant pasture legume, in the wheatbelt of Western
Australia, with some 6 million ha sown by the mid 1970s
(Gladstone 1975). Pasture decline manifests as an increase in
weeds and a decrease in desirable legumes, grasses, and
perennials (Reeve et al. 2000). Pasture decline is a
multifaceted widespread disorder of subterranean clover-based
pastures and has been attributed to a combination of biotic and
abiotic stresses, including inappropriate cultural practices,
poor nutrition, and high occurrence of root disease (Barbetti
et al. 2007).
Pasture decline, as a consequence of root disease, was first
reported in Western Australia during the late 1960s (Shipton
1967). Necrotrophic pathogens tend to be prevalent in the
Mediterranean-type environments of southern Australia
(Sivasithamparam 1993). The impoverished and nutrient-
deficient soils across much of this region predispose the plant
host to these pathogens as there is often little microbial
competition (Sivasithamparam 1996). Root diseases have been
repeatedly shown to be a significant cause of pasture decline in
CSIRO 2009 10.1071/CP08187 1836-0947/09/010043
CSIRO PUBLISHING
www.publish.csiro.au/journals/cp Crop & Pasture Science, 2009, 60,43–50
Australia, greatly reducing seedling establishment and pasture
productivity of annual legumes. In disease-prone areas, extreme
cases have involved greater than 90% failure in seedling
emergence (Wong et al. 1985b).
Necrotrophic pathogens that have been implicated as causal
agents of root damage of subterranean clover, in approximate
order of importance, include: Phytophthora clandestina
(Greenhalgh and Taylor 1985; Taylor et al. 1985a, 1985b),
Pythium irregulare (Barbetti and MacNish 1978; Greenhalgh
and Lucas 1984; Wong et al. 1984, 1985b, 1986a), Aphanomyces
spp. (Greenhalgh et al. 1985, Ma et al. 2008), Fusarium
avenaceum (Shipton 1967; Kollmorgen 1974; McGee and
Kellock 1974; Wong et al. 1984), Rhizoctonia solani (Wong
et al. 1985a, 1986a), and Cylindrocarpon didymum
(Barbetti 2005). However, pathogens known to cause root
disease frequently form complexes that greatly increase the
severity of root disease (Wong et al. 1984, 1985b). For
example, neither F. oxysporum nor Phoma medicaginis is
highly pathogenic in subterranean clover when inoculated
singly, yet when applied jointly they can cause severe disease
(Wong et al. 1984).
Several means could be used to reduce the effect of root
disease in disease-prone areas, including fungicide treatments,
nutrient manipulation, other cultural practices, and the
development of pasture cultivars with greater resistance
(Barbetti et al. 2007). Several subterranean clover cultivars
have been developed that have useful resistance to one or
more of the major root-rot pathogens, including Coolamon
(Nichols and Barbetti 2005a), Denmark (Nichols and Barbetti
2005b), Gosse (Nichols and Barbetti 2005c), Goulburn (Nichols
and Barbetti 2005d), Napier (Nichols and Barbetti 2005e),
Riverina (Nichols and Barbetti 2005f) and York (Nichols and
Barbetti 2005g).
While pasture decline is known to be a serious challenge to
agricultural productivity of subterranean clover across southern
Australia, until now the extent of root disease has mainly been
assessed in the early part of the growing season. With the
exception of annual Medicago spp. (You et al. 1999, 2000),
all studies previously conducted on root-rot and/or pasture
decline of pasture legumes in Western Australia have been
undertaken in areas predominantly sown to permanent
subterranean clover pastures. For this reason a survey was
undertaken to determine root disease levels in mature
subterranean clover pastures located in the wheatbelt of
Western Australia. This survey included sites with crop
rotations that included non-cereal crops such as lupins and
other grain legumes and canola. The relatively recent
introduction of grain legumes may have resulted in a build-up
of pathogens, which may not have dominated in the traditional
pasture–wheat rotations or the permanent pasture swards
previously studied. As routine isolations on agar often ignore
slow-growing or fastidious pathogen taxa, DNA-based
assays (Ophel-Keller et al. 2008) were used to estimate
pathogen population density of a variety of soil-borne
pathogens known to commonly occur in the Mediterranean-
type environments of Australia. Attempts were also made to
relate the severity of diseases on tap and lateral roots of
subterranean clover to root diameter, root length, nodulation,
and total rainfall.
Materials and methods
Field sampling
2004 survey
Field sampling was conducted across the Western Australian
wheatbelt from September to October 2004 to coincide with the
seasonal peak of pasture growth. Eighty-five sites from 15 farms
were sampled (Fig. 1). Sites were selected in areas where annual
pastures were based on subterranean clover. At each site,
5 replicate 100 mm long by 100 mm wide by 200 mm deep
intact sods of soil were carefully removed along a transect of
100 m. Approximately 500 g of soil was removed from each site,
freeze-dried, and later used for DNA analysis.
2006 sampling
Field trials were established 4 weeks after the break of season
(20 mm or more rain), in mid May 2006, at field sites located at
Denmark, Denbarker, and Mount Barker, in southern Western
Australia (Fig. 1). Location of sites did not correspond to sites in
the 2004 survey, with different farms sampled at Denmark and
Mount Barker. These 3 sites were located in areas known
(historically) to suffer from severe root disease of subterranean
clover and had not been resown in the past 10 years. At each site,
10 replicate strips were prepared for the sowing of subterranean
clover cv. Woogenellup. A flat sharpened spade was used to
North
Perth
Darkan
Boyup Brook Kojonup
Wagin
Narembeen
Mount Barker
Denmark
Albany
Average annual rainfall (mm)
<300
301–400
Cities and towns
Sample sites 0 50 100 200 km
401–500
501–600
601–700
701–800
801–900
>900
Fig. 1. Location in the wheatbelt of Western Australia of the 85 sites across
15 farms sampled in the spring 2004 survey and the 3 sites across 3 farms in the
2006 sampling. Average annual rainfall zones in the wheatbelt of Western
Australia are indicated.
44 Crop & Pasture Science T. A. O’Rourke et al.
scrape away the top 5–10 mm of soil to form strips 1m long and
250 mm wide. The edge of a clean 1-m-long star picket was
pressed into the soil to form a furrow (4 mm wide and 5 mm deep)
and 100 germinable seeds were sown evenly along the row and the
furrow closed over. Prior to sowing, seeds were surface-sterilised
by washing in 70% ethanol for 30 s. To protect seedlings from
redlegged earthmites the strips and the immediately surrounding
areas (2 m radius) were sprayed with bifenthrin (Talstar, FMC Pty
Ltd) at 100 mL/ha.
Plant root assessment
The same root disease assessment methodology was used in the
2004 survey and 2006 sampling. Plant roots were thoroughly
washed under running tap water to remove soil. Subterranean
clover plants were then floated in shallow trays of water and
both tap and lateral roots were scored independently using a
6-step rating scheme where: score 0 = root healthy, no
discoloration; 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. This
rating scheme was a modification of a scoring system described
and used earlier by Wong et al. (1984). The number of plants in
each disease severity category was recorded. In the 2004 survey,
nodulation was also assessed, based on nodule positioning, using
a 6-step rating scheme where: score 0 = no nodules on crown or
elsewhere; 1 = no nodules on crown, few (1–10) elsewhere;
2 = few crown nodules, no nodules elsewhere; 3 = many crown
nodules (>10), no nodules elsewhere; 4 = many crown nodules,
few nodules elsewhere; 5 = many crown nodules, many nodules
elsewhere. This rating scheme was a modification from a scoring
system used by Corbin et al. (1977). Nodule counts were also
conducted to determine the average number of nodules per plant.
Total root length and the percentage of root length in 3 root
diameter classes (fine 0–0.2 mm, medium 0.2–0.5 mm, and thick
>0.5 mm) of individual plants were calculated using the
WhinRHIZO (Version 3.9, Reagent Instruments; Québec)
computerised root scanning analysis. Plants were placed into
paper envelopes, dried at 708C for 24 h, and weighed.
Detection of common soil-borne plant pathogens
using DNA assays
DNA assays, as described by Ophel-Keller et al. (2008), for
several the major fungal and oomycete subterranean clover root
pathogens, were used to estimate the pathogen population
density. These involved forward and reverse primers and
probes for target DNA, all previously designed for a suite of
plants and soil organisms (I. T. Riley, S. Wiebkin, D. Hartley,
A. C. McKay, unpublished) based on rDNA sequences (ITS)
available through GenBank or obtained specifically for the
purpose. Pathogens targeted using these DNA assays in our
2004 survey and the 2006 sampling included R. solani
anastomosis groups AG-2.1, 2.2, 4, and 8; Pythium Clade F
(consisting of P. kunmingense,P. spinosum,P. cylindrosporum,
P. irregulare,P. mamillatum,P. paroecandrum,P. sylvaticum,
P. debaryanum,P. macrosporum, and P. intermedium [Lévesque
and de Cock 2004]); P. clandestina; and the ‘blackspot complex’
(viz. Mycosphaerella pinodes/Phoma medicaginis var. pinodella
and a new Phoma species associated with blackspot of peas
[Ophel-Keller et al. 2008]).
Data analysis
A single-factor analysis of variance was conducted using GENSTAT
(10th edn, Lawes Agricultural Trust). Fisher’s least significant
difference (l.s.d.) at a 95% significance level was used to test
differences between sites for DNA concentrations of various
pathogens, tap and lateral root rot, and nodule number.
GENSTAT was also used to test the significance of correlation
coefficients between the different parameters measured.
Results
Root disease
2004 survey
High levels of both tap and lateral root disease were present on
mature subterranean clover plants harvested from the Western
Australian wheatbelt in October 2004. Plants from 6 of the 85 sites
had severe tap root rot with scores >3, while plants from 45 sites
had scores >3 for lateral root disease. All of the sites examined had
plants that were affected by both tap and lateral root disease.
2006 sampling
In the 2006 field sampling, tap root disease was greater than
lateral root disease at all 3 sites, with the Denmark site showing the
greatest level of tap root disease (Table 1).
Detection of necrotrophic plant pathogens
using the DNA assay: oomycetes
2004 survey
Of the oomycetes screened, Pythium Clade F was most
abundant, being present at 82 of the 85 sites surveyed, with
8 of the 85 sites having greater than 200 pg DNA/g soil.
P. clandestina was detected at trace levels at only 12 of the 85
sites and none of these sites contained greater than 10 pg DNA/g
soil (Fig. 2a).
2006 sampling
DNA concentrations of Pythium Clade F, although not directly
comparable, were similar to those found in the October 2004
survey, with sites having 10–200 pg DNA/g soil. There was
a 2-fold increase in Pythium Clade F DNA concentrations at
1 of the 3 sites between 15 May and 26 June, although
DNA concentrations were still comparably low at this site
(Table 1). P. clandestina was detected at 2 of the 3 sites, with
DNA concentrations around 300–450 pg DNA/g soil. The DNA
concentration of P. clandestina greatly decreased at these 2 sites,
by as much as 9-fold, between 15 May and 26 June (Fig. 2a).
Pathogenic fungi
2004 survey
R. solani anastomosis groups and the blackspot complex were
the only pathogenic fungi assayed in 2004. R. solani anastomosis
groups 2.1, 2.2, 4, and 8 were assayed, although DNA
concentrations were low (Fig. 2b). Of the R. solani
anastomosis groups, AG 2.1 was detected at 20 sites, AG 2.2
Root rot severity in subterranean clover Crop & Pasture Science 45
at 6 sites, and AG 8 at 2 sites. AG4 was not detected at any of the 85
sites surveyed. The blackspot complex had very low DNA
concentrations at the 47 sites where it was detected.
2006 sampling
R. solani AG 2.2 was detected at all 3 sites, with DNA
concentrations of 4000–9000 pg DNA/g soil, on 15 May.
However, this was reduced by as much as 11-fold by 26 June
(Fig. 2b). R. solani AG 2.1 was also detected at all 3 sites but at the
much lower DNA concentrations of 1–15 pg DNA/g soil. Neither
R. solani AG 4 nor AG 8 was detected at the 3 sites during the 2006
sampling.
Correlations
2004 survey
There was a positive correlation between tap and lateral root
disease (P<0.001, n= 391; Fig. 3a) and a negative correlation
between tap root disease and percentage fine roots (0–0.2 mm)
(P<0.005, n= 80; Fig. 4a). Tap root disease was positively
correlated with both percentage medium (0.2–0.5 mm)
(P<0.001, n= 80) and percentage thick roots (>0.5 mm)
(P<0.001, n= 80; Fig. 4band c, respectively). Tap root
disease was also positively correlated with average rainfall
(P<0.005, n= 80; Fig. 5). Nodule number was positively
correlated with average dry weight per plant (P<0.001,
n= 391; Fig. 6). However, neither nodule number nor nodule
score was correlated with tap or lateral root disease or with the
DNA concentrations of any necrotrophic pathogens assayed.
Likewise, root length was not correlated with either tap or
lateral root disease or with the DNA concentrations of any
necrotrophic pathogens assayed and neither tap nor lateral root
disease was correlated with average plant dry weight.
Furthermore, none of the percentage root diameters was
correlated with lateral root disease.
2006 sampling
There was a positive correlation between tap and lateral root
disease in the 2006 sampling (P<0.001, n= 60; Fig. 3b). The
DNA concentration of the blackspot complex was positively
correlated with both tap and lateral root disease (P<0.001,
n= 60; P<0.005, n= 60; respectively; Fig. 7aand b). There
were no other correlations between either tap or lateral root
disease and the necrotrophic pathogens as detected by the
DNA assays.
Discussion
The survey conducted in 2004 was the first of its kind in Australia
to examine the extent of root disease in subterranean clover
pastures late in the growing season. The field sampling was
conducted from September to October 2004 and was
0
200
400
600
800
1000
May 2006
DNA concentration (pg/g soil)
0
5000
10 000
15 000
20 000
(a)
(b)
June 2006 Oct. 2004
Fig. 2. Box plots showing the effect of sampling date on (a)Phytophthora
clandestina DNA concentrations within the soil and (b)Rhizoctonia solani
AG2.2 DNA concentrations within the soil.
Table 1. Pathogen DNA concentrations (DNA/g soil) in soil and tap and lateral root-rot scores (disease index) from subterranean clover plants
in the 2006 autumn sampling
Pathogens assayed were Pythium Clade F (Py), Phytophthora clandestina (Ph), blackspot complex (Bsp), and Rhizoctonia solani anastomosis groups AG-2.1
and 2.2 (R). –, No score taken for this date; n.a., not applicable
Sites Date of Py pg Ph pg Bsp pg R AG2.1 pg R AG2.2 pg Tap root- Lateral root-
sampling rot score rot score
Denbarker 15 May 158.3 293 –1.3 4062 ––
Mt Barker 15 May 163.6 456 –16.7 8803 ––
Denmark 15 May 18.2 0 –0.4 5082 ––
Denbarker 26 June 133.7 27 9.3 0 127 2.18 1.59
Mt Barker 26 June 189.9 52 133.6 15.5 736 1.37 0.91
Denmark 26 June 40.7 0 44.2 5.2 36 3.26 2.07
Significance <0.001 <0.001 <0.001 0.037 0.102 <0.001 0.004
l.s.d. (P<0.05) 42.83 108.4 32.92 13.57 n.a. 0.58 0.63
46 Crop & Pasture Science T. A. O’Rourke et al.
deliberately timed to coincide with the ‘peak of season’in terms of
pasture growth. Previous samplings of subterranean clover have
focussed on evaluating the levels of root disease over the first
few weeks during pasture establishment (Barbetti et al. 2007).
The 2004 survey showed, for the first time, that widespread and
severe root disease occurs in subterranean clover late in the
growing season.
Although the effect of disease on pasture productivity in
the 2004 survey was not measured, it is likely that
productivity was greatly reduced during the peak of season.
For instance, in a fungicide trial conducted at a single site in
Victoria, Taylor et al. (1985b) showed that the application of the
fungicides metalaxyl and benomyl in early May increased dry
matter herbage production by 95% at an October harvest. The
large reductions in late-season productivity caused by severe root
disease likely inhibit root function during the time of peak
demand for water and nutrient uptake. Also, plants with severe
root disease would be likely to have a shallower and less extensive
root system and thus would experience an earlier end to the
growing season, with a consequent reduction in seed set.
There was a positive correlation in the 2004 survey between
tap root disease and root diameter. This trend could reflect a plant
y = 0.3485x + 0.9168
R2 = 0.1105
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
012345
Lateral root rot (disease index)
Tap root rot (disease index)
y = 1.0785x + 0.6251
R2 = 0.746
012345
(a)(b)
Fig. 3. Correlation between tap root disease and lateral root disease for subterranean clover harvested (a) from 85 sites in
October 2004 (P<0.001) and (b) across 3 sites in June 2006 (P<0.001).
y = –0.0236x + 3.1734
R2 = 0.0945
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
020406080
% Root diameter 0–0.2
Tap root rot (disease index)
y = 0.0471x + 0.3596
R2 = 0.1634
0 1020304050
% Root diameter 0.2–0.5
y = 0.0443x + 1.1247
R2 = 0.1165
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
01020304050
% Root diameter >0.5
(a)(b)
(c)
Fig. 4. Correlation between tap root disease and percentage of total plant root length with a diameter of (a)0–0.2 mm
(P<0.005), (b) 0.2–0.5 mm (P<0.001), and (c)>0.5 mm (P<0.001), for subterranean clover harvested from 85 sites
in October 2004.
Root rot severity in subterranean clover Crop & Pasture Science 47
adaptation to the loss of tap root function. That is, as tap root
disease increases, lateral roots compensate for the loss of tap
root function and replace it as conduits for water and nutrient
transfer, thus becoming thicker. However, plants appear limited
in their ability to adequately compensate for loss of tap root
function, as extensive tap root disease eventually results in
plant death (Barbetti 1984a). Severe tap root rot has a greater
effect on pasture productivity than lateral root rot (Barbetti
1984a). Hence, it is probably only plants with lower levels of
tap root rot that are able to rapidly produce new lateral roots
to offset the effects of roots damaged or lost due to severe tap root
disease.
In the 2004 survey, there was a positive correlation between
rainfall and tap root disease as the higher levels of disease (>2.5)
occurred far more frequently in the high-rainfall areas. Root
diseases are more severe at sites where rainfall is significantly
heavier and more frequent (MacNish et al. 1976). The oomycete
pathogens P. clandestina (Wong et al. 1986c; Taylor and
Greenhalgh 1987), P. irregulare (Wong et al. 1984), and
Aphanomyces euteiches (Greenhalgh et al. 1988) are favoured
by wetter soils. In particular, P. clandestina has been shown to
have greater genetic diversity, in terms of the number of different
races, in higher rainfall areas (700–1000 mm) (You et al. 2006).
Thus it seems probable that the occurrence of more races of one or
more oomycete pathogens, and/or wetter soils, contributed to the
higher levels of tap root disease observed in the high-rainfall areas
during the 2004 survey. Additionally, increased root disease in
high-rainfall areas may also be partly attributed to pastures in
these areas being permanent or semi-permanent (Nichols et al.
2007). Permanent pastures favour build-up of root pathogen
inoculum as there are no ‘disease breaks’.
Individual plant dry weights and dry weights per sod both
increased as nodule number and nodule score increased. Despite
this, nodule number and nodule score did not correlate with the
level of either tap or lateral root disease. The lack of correlation
between root disease and nodulation by the time plants were
harvested, at approximately 6 months of age, may be a result of
nodule recruitment being outpaced by nodule senescence. Nodule
numbers may have already been significantly lowered in healthy
plants late in the growing season. Rhizobial application has been
shown to reduce root disease in subterranean clover (Barbetti
1984b; Smiley et al. 1986; Wong 1986), perhaps due to root-rot
pathogens being unable to enter root cells previously infected by
rhizobia (Brown et al. 1994). It is unlikely that plants suffering
from severe root disease would have an adequate number of
healthy, productive nodules.
Despite the high levels of root disease present across the range
of sites in the 2004 survey, the DNA of most root disease
y = 0.0014x + 1.1695
R2 = 0.1301
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
200 400 600 800 1000
Rainfall (mm)
Tap root rot (disease index)
Fig. 5. Correlation between tap root disease and 5-year average annual
rainfall (2002–06) for subterranean clover harvested from 85 sites in October
2004 (P<0.005).
y = 28.822x + 12.591
R2 = 0.3353
0
10
20
30
40
50
60
70
80
90
100
0123
Dry weight (g)
No. of nodules (average)
Fig. 6. Correlation between average nodule number per plant and average
plant dry weight for subterranean clover harvested across 85 sites in October
2004 (P<0.001).
y = 0.0123x + 1.5354
R2 = 0.5873
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 50 100 150 200 250
Blackspot complex (pg DNA/g soil)
Tap root rot (disease index)
y = 0.0073x + 1.088
R2 = 0.3245
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 50 100 150 200 250
Lateral root rot (disease index)
(a)(b)
Fig. 7. Correlation between blackspot complex and (a) tap root disease (P<0.001) and (b) lateral root disease (P<0.005) for
subterranean clover harvested from 3 sites in June 2006.
48 Crop & Pasture Science T. A. O’Rourke et al.
pathogens assayed from the soil was present only in trace
concentrations. Pythium Clade F was the only pathogen group
assayed to show high concentrations of DNA in the soil. Although
Pythium spp. such as P. irregulare have been recorded on
subterranean clover previously, they were not thought to be as
important on well established plants (M. J. Barbetti,
unpublished), being particularly damaging at pre-germination
and immediately after germination (Wong et al. 1985a). Our
DNA assay results suggest that one or more Pythium spp. are
associated with the severe root disease observed in the 2004
spring survey.
Although not directly comparable, as studies were conducted
in different years at different sites within the Western Australian
wheatbelt, it is notable that DNA concentrations were higher in
the sampling conducted in autumn 2006. In particular,
P. clandestina and R. solani AG-2.1 and AG-2.2 were found
in higher concentrations in the autumn 2006 sampling than in the
spring survey of 2004. Furthermore, DNA concentrations of
P. clandestina were much higher at the establishment of the
2006 sampling on 15 May, decreasing 9-fold by 26 June (Fig. 7a).
This may reflect the preference of P. clandestina for warm, wet
soils, which are more prevalent earlier in the growing season
(Wong et al. 1986c). The peak in P. clandestina DNA
concentration is associated with plant germination and
emergence. This is consistent with previous observations
(Wong et al. 1986b) that P. clandestina inoculum levels in soil
peak during May, coinciding with subterranean clover
germination. R. solani AG-2.2 showed similar trends, with
high initial DNA concentration on 15 May, which
subsequently decreased 11-fold by 26 June (Fig. 7b). The
rapid decrease in R. solani inoculum levels in soil from the
start of the season may be attributed to the unfavourable wet,
cold conditions (Wong et al. 1984), which become prevalent as
the season progresses. The spike in DNA concentrations followed
by the rapid decrease observed in the key root-rot pathogens
P. clandestina and R. solani AG-2.2 suggests that the majority of
root damage may occur very early in the season.
In contrast there were no major differences between the
Pythium Clade F DNA concentrations found in the autumn
2006 and spring 2004 sampling times. However, other data
(R. J. Simpson et al., unpublished) indicate a peak in Pythium
DNA concentrations early in the growing season. To reliably
determine how inoculum levels fluctuate between autumn and
spring and relate it to the occurrence of severe root rot it would be
necessary to analyse the same sites at 1–2-week intervals
throughout the growing season, preferably over several years.
As plant density was not recorded in the 2004 survey, results
may not reflect the heavy production losses that occur due to
damping-off, which not only greatly reduces seedling vigour but
results in seedling death before emergence. However, it is
probable that severe disease seen on plants in spring was a
consequence of damage caused at the beginning of the season,
but this has yet to be confirmed. The studies of Barbetti (1984a)
showed that it is likely that plants which survive the first
6–7 weeks after emergence, even with severe tap root disease,
will persist until the end of the season. This suggestion is
supported by the trace concentrations of pathogen DNA
observed in the 2004 spring survey, with the exception of
Pythium Clade F, and by the peaks in DNA levels in the first
6 weeks of the 2006 sampling, especially of P. clandestina and
R. solani AG-2.2. However, the root system disease index
found in the spring survey suggests that the productivity of
subterranean clover-based pastures remains severely
compromised by root disease throughout the growing season
in Western Australia.
Acknowledgments
We thank the Australian Wool Innovation Ltd and the late Mr Frank Ford,
Western Australia, for a bequest that funded this research. We also thank
Susanne Ehrenberg and Madeleine Wouterlood for assistance in the
laboratory and with WhinRHIZO, respectively.
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Manuscript received 2 June 2008, accepted 7 November 2008
50 Crop & Pasture Science T. A. O’Rourke et al.
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