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AGRONOMY JOURNAL, VOL. 93, JANUARY–FEBRUARY 2001
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Rust-Enhanced Allelopathy of Perennial Ryegrass against White Clover
Scott W. Mattner* and Douglas G. Parbery
ABSTRACT
Perennial ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) are important pasture components in the higher rainfall areas of southeastern Australia. Crown rust (Puccinia coronata
Corda f.sp. lolii Brown) is the most serious ryegrass pathogen in these
areas. In a preliminary investigation, rust reduced ryegrass biomass
by 56%. Yet, interference from rusted ryegrass suppressed the yield
of neighboring clover plants more than interference from healthy
ryegrass. The role of allelopathy in this relationship was investigated
in a greenhouse study using two bioassays. Soil previously growing
rusted ryegrass suppressed clover biomass by 36% compared with
soil previously growing healthy ryegrass. Similarly, leachate from soil
surrounding rusted ryegrass suppressed clover biomass by 27% compared with that from healthy ryegrass. This is the first demonstration
that a pathogen may influence allelopathy between plants and that
rust may enhance ryegrass allelopathy against clover. Possible implications of this in pasture ecology and the evolution of mutualism are discussed.
P
erennial ryegrass and white clover predominate
improved pastures in the higher rainfall areas of
southeastern Australia. Their growth in mixtures without added nitrogen results in greater herbage yields than
otherwise can be achieved economically (Menchaca and
S.W. Mattner, Agriculture Victoria, Institute for Horticultural Development, Private Bag 15, Scoresby Business Centre, Victoria 3176,
Australia; D.G. Parbery, Institute of Land & Food Resources, The
Univ. of Melbourne, Parkville, Victoria 3052, Australia. Received 29
Nov. 1999. *Corresponding author ([email protected]).
Published in Agron. J. 93:54–59 (2001).
Connolly, 1990). Crown rust is the most serious ryegrass
fungal pathogen in these areas (Eagling and Clark, 1993),
with epidemics regularly occurring between spring and
autumn. A preliminary study demonstrated that rust accelerates senescence and reduces ryegrass yield by 56%
(Mattner, 1998). Despite this, in ryegrass and clover mixtures, interference from rusted ryegrass suppressed clover
biomass by up to 47% compared with interference from
healthy ryegrass. This suppression did not result from
a direct effect of crown rust on clover, because rustinoculated and non-inoculated clover monocultures
yielded the same, which was expected since clover is a
nonhost of crown rust. Similarly, clover suppression is
not explained by rust increasing ryegrass competitiveness because, if this were so, the reduction in clover
yield would be greatest at high densities where resources
are most limited. Instead, clover suppression was greatest at low densities, where competition for resources
was minimal. Ryegrass allelopathy is well documented,
particularly against clovers and medics (Gussin and
Lynch, 1981; Takahashi et al., 1988, 1991, 1993; Quigley
et al., 1990; Chung and Miller, 1995). For these reasons,
we investigated the hypothesis that rusting increases
ryegrass allelopathic ability.
Both Rice (1984) and Einhellig (1995) hypothesized
that pathogens enhance their host’s allelopathic ability.
Evidence supporting this hypothesis occurs in at least
two forms. Firstly, pathogens stimulate phytoalexin (antimicrobial compounds) production by their hosts
(Smith, 1996), which can belong to similar chemical
groups and are synthesized via the same biochemical
MATTNER & PARBERY: RUST-ENHANCED ALLELOPATHY OF PERENNIAL RYEGRASS
pathways as allelochemicals. For example, isoflavonoids
are important phytoalexins (Dakora and Phillips, 1996)
and allelochemicals (Tamura et al., 1967, 1969) from
the Leguminosae. Indeed, many phytoalexins act as
allelochemicals against plants, inhibiting their germination (Chang et al., 1969), growth (Glazener and VanEtten, 1978), and cellular metabolism and function (Giannini et al., 1990; Spessard et al., 1994). Secondly, under
some conditions, the mutualistic fungus Neotyphodium
lolii (Latch, Christensen and Samuels) Glenn, Bacon
and Hanlin, increases the allelopathic ability of some
ryegrass genotypes (Sutherlund and Hoglund, 1990;
Quigley et al., 1990). Despite this, no one has previously
observed that diseased plants suppress the growth of
neighboring species (Tang et al., 1995), even though the
effect of pathogens on interference between host and
nonhost plants has been extensively investigated (Burdon, 1987; Ayres and Paul, 1990). Since competition
is the dominant process of plant interference (Tilman,
1988), competition effects often obscure any allelopathic
effects in such experiments (Trenbath, 1974). This is
particularly so at high plant densities where competition
is intense. While it is difficult to separate the processes
of competition and allelopathy in the field, separation
can be achieved using bioassays. To date, however, researchers have not used bioassays to investigate the
effects of pathogens on allelopathy.
The present investigation examined the hypothesis
that rust enhances perennial ryegrass allelopathy against
white clover using two bioassay techniques.
METHODS AND MATERIALS
Pathogen and Plant Material
In the following bioassays, perennial ryegrass (cv. Victorian) was used as the donor species and white clover (cv.
Tamar) was used as the receiver species. The perennial ryegrass cultivar Victorian is highly susceptible to crown rust
(Critchett, 1991). Crown rust urediniospores were collected
with a side-arm flask collector from naturally infected ryegrass
at the Mt. Derrimut field station, 22 km west of Melbourne,
Australia (378479 S, 1448479 E) (Mattner, 1998).
Growing Conditions and Soil Type
Bioassays were conducted in temperature-controlled glasshouses (20–238C during the day and 12–158C at night) at the
Mt. Derrimut field station. Pots were drip-irrigated at the rate
of 150 mL of water twice daily, which maintained soil at near
field capacity. The soil mixture was three parts red-brown
earth topsoil to one part sand and two parts peat moss. The
following nutrients (g/L) were added to the mixture: 0.200 N,
0.116 P, 0.140 K, 0.096 S, 0.652 Ca, 0.024 Cu, 0.012 Zn, 0.008
Mn, 0.028 Fe, 0.012 Mo, 0.360 Mg, and 0.001 B. The mixture
was then pasteurized by steam prior to planting. Following
pasteurization, soil used to grow white clover was inoculated
with the appropriate strain of Rhizobium trifolii Dangeard.
This was performed by watering soil contained in individual
pots with 150 mL of an inoculum solution (10 g of commercial
inoculum per 10 L of water).
Soil Retrieval Bioassay
Pots (15 cm diam.) containing the standard soil mix were
sown to contain an average of 10 ryegrass donor plants. In
55
total there were 108 pots, half with rusted ryegrass and half
with nonrusted ryegrass. Plants in rusted treatments were
spray inoculated (Villalta and Clarke, 1995) 52 d after sowing
with an aqueous solution containing 1 3 106 urediniospores/
mL, 0.1% soft soap as a surfactant, and 0.5% gelatine. Plants
in nonrusted treatments were sprayed with a similar solution
containing no spores. Following inoculation all plants were
placed in plastic tents for 1 wk, which maintained humidity
between 92 and 98%. Donor plants were grown for a total of
100 d.
Soil for the bioassay was gathered directly from the pots
used to grow rusted and nonrusted donor ryegrass, all of which
was removed by hand and then passed through a 5-mm sieve.
Soil from each source was bulked and thoroughly mixed prior
to further treatment. A fresh preparation of soil was also
included as a control. In addition, plus and minus nutrient
and steam-sterilization treatments were incorporated into the
design. This was done in an attempt to detect confounding
effects caused by differences in soil microflora and nutrient
content in the soils resulting from prior rusted and nonrusted
ryegrass growth. Nutrient enrichment consisted of adding concentrations of the nutrients listed previously, to ensure they
were slightly in excess of growth requirements. The soils were
then placed into 12.5-cm-diam. pots and used to grow two
clover receivers. At 70 d after sowing, receivers were washed
free of soil and dried at 808C for 4 d before total biomass was
determined. Additionally, a nodulation index was determined
by bunching individual plant roots; removing three 2-cm sections from the upper, central, and lower portion of the bunch;
counting the nodules captured in each section; drying each
sample; and recording the number of nodules per gram of root.
The bioassay was conducted as a randomized complete
factorial design with three blocks. There were four pots per
treatment in each block. Factors consisted of soil source (three
levels: soil previously growing rusted ryegrass, soil previously
growing nonrusted ryegrass, and freshly prepared soil as a
control), nutrient application (two levels: plus and minus) and
soil sterilization (two levels: plus and minus).
Soil Leachate Bioassay
Pots (15 cm in diam.) containing the standard soil mix were
sown to contain eight ryegrass donor plants. Twenty days after
sowing, irrigation lines were placed into individual pots and
calibrated to deliver 250 mL of water per pot every day. Soil
leachate was collected in plastic trays beneath the wire mesh
benches, bulked for each treatment, and thoroughly mixed
prior to application to receiver plants. Seventy days after sowing, plants in the rusted treatment were inoculated with rust
as described previously. One-half of the 64 pots contained
rusted ryegrass and the other half nonrusted plants.
The investigation was conducted in two parts. In the first,
the pre-inoculation bioassay, the bioassay was made before
donor plant inoculation to ensure that there were no intrinsic
differences in the allelopathic potential of donor plants within
the rust treatments prior to inoculation. The second, the postinoculation bioassay, was made after inoculation when rust
symptoms had fully developed. The methodology of the bioassays was the same. Pots (12.5 cm in diam.) filled with the
standard soil mix were prepared containing two clover receivers. Each was hand watered daily with 150 mL of the appropriate leachate or with water in the case of the controls.
Receivers were harvested and compared 50 d after sowing
by determining plant biomass, leaf area (with a planimeter,
Paton Industries Pty. Ltd., South Australia), leaf number, stolon number, and nodulation index. Measurements were taken
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AGRONOMY JOURNAL, VOL. 93, JANUARY–FEBRUARY 2001
Table 1. Summary of the levels of statistical significance for the
various treatments and their interactions on white clover biomass and nodulation.
Table 2. The effect of soil sterilization on the nodulation of
white clover.
Source of variation
Soil source (SS)
Nutrient (N)
Sterilization (St)
SS 3 N
SS 3 St
N 3 St
SS 3 N 3 St
Dry biomass
Nodulation
Treatment
No. nodules per
gram of root
***
NS
NS
**
NS
NS
NS
NS†
NS
**
NS
NS
NS
NS
Sterilized
554.55
Nonsterilized
752.12
191.95
Although soil sterilization did not affect clover biomass, it reduced root nodulation by 26% (see Table 2).
** Significant at the 0.01 probability level.
*** Significant at the 0.001 probability level.
† Not significant.
on one randomly selected plant per treatment in each block,
except for biomass where all plants were measured.
The bioassay was conducted as a randomized complete
block design. The treatments were the daily application of 150
mL of leachate derived from either rusted ryegrass, nonrusted
ryegrass, or irrigation water. There were eight blocks consisting of four pots per treatment.
Statistical Analysis
Data was analyzed using analysis of variance (ANOVA)
as performed on Minitab Version 12 (Minitab, 1998). Homogeneity of variance was determined by examining plots of
fitted values versus residuals, while histograms of residuals
assessed normality of distribution.
RESULTS
Soil Retrieval Bioassay
Table 1 presents statistical significance levels of main
treatments and their interactions in influencing clover
biomass and nodulation. Sterilization of the soils used
in this bioassay had no effect on the biomass of clover
growing in them. As such, sterilization treatments have
been grouped to provide a clearer demonstration of the
effects of the different soil source and nutrient treatments (see Fig. 1). The biomass of clover grown in soil
from rusted ryegrass was less than that in the control
or in soil from nonrusted ryegrass, with this effect being
particularly marked in nutrient treated soils. In contrast,
clover grown in soil from nonrusted ryegrass produced
less than the control only when no nutrients were added.
LSD ( p , 0.05)
Soil Leachate Bioassay
The pre-inoculation bioassay detected no difference
in the growth of clover watered with leachate from ryegrass in nonrusted or rusted treatments (see Table 3).
This indicates that there was no intrinsic difference in
the allelopathic ability of ryegrass plants assigned to the
treatments prior to inoculation. Growth in the control,
however, was greater than that of plants receiving soil
leachate from ryegrass.
In the post-inoculation bioassay, the growth of clover
receiving soil leachate from rusted ryegrass was less than
that of plants watered with leachate from nonrusted
ryegrass or the control, according to each parameter
measured (see Table 3). Plants exposed to leachate from
nonrusted ryegrass did not differ from the control in
any way. Nodulation did not vary between treatments.
DISCUSSION
Even though several authors have suggested that
pathogens increase their host’s allelopathic ability (Rice,
1984; Einhellig, 1995), the present study is the first to
evaluate the pathogen effect on allelopathy between
plants. Furthermore, no one has previously observed
that diseased plants suppress the growth of neighboring
species (Tang et al., 1995). Despite this, a preliminary
investigation found that rusted ryegrass suppressed the
growth of neighboring clover plants by up to 47% compared with when grown with healthy ryegrass (Mattner, 1998).
In the present study, the soil retrieval bioassay demonstrated that, overall, soil previously growing ryegrass
reduced the subsequent clover growth compared with
Table 3. Production of white clover in response to leachate derived from rusted or nonrusted perennial ryegrass or a control
of irrigation water. Bioassays were performed prior to and after
the inoculation of ryegrass with rust.
Leachate source
White clover
Nonrusted Rusted
growth parameter Control ryegrass ryegrass
Total biomass (g)
Fig. 1. The biomass of white clover grown in soil from rusted or
nonrusted perennial ryegrass. The control consisted of freshly prepared soil. Nutrients were added at concentrations described in
the text.
1.10
Total biomass (g)
2.86
203.88
Leaf area (cm2)
Stolon number
4.71
Leaf number
36.5
Nodulation index
(no. nodules
per gram root) 1222.81
† Not significant.
LSD
p , 0.05
Pre-inoculation bioassay
0.76
0.78
0.16
Post-inoculation bioassay
3.11
2.28
0.53
196.2
145.4
49.9
5.00
4.00
0.76
34.12
25.5
8.78
1121.11
1143.78
NS
p , 0.01
0.22
0.75
NS†
NS
NS
NS
MATTNER & PARBERY: RUST-ENHANCED ALLELOPATHY OF PERENNIAL RYEGRASS
a control of freshly prepared soil. The modification of
soil microflora by ryegrass to contain species antagonistic to clover growth does not explain this result because
soil sterilization had no effect on the relationship. In
contrast, nutrient addition alleviated the suppressive effect that soil previously growing healthy ryegrass had
on clover production. This suggests that the main effect
of healthy ryegrass was to deplete soil nutrients and
thereby diminish clover growth. This was not the case
for soil previously growing rusted ryegrass, however,
where nutrient application markedly increased its suppressive effect on clover growth. Under these conditions, soil previously growing rusted ryegrass suppressed
clover biomass by 36% compared with soil previously
growing healthy ryegrass. This was in similar proportions to that in the preliminary experiment where the
yield of clover grown in mixtures with rusted ryegrass
fell by an average of 37% (Mattner, 1998). Nutrient
toxicity does not explain this result because twice the
concentration of nutrients applied to soil in the rusted
treatment had no effect on clover growth in the control.
Instead, the result provides strong evidence that rust
increases ryegrass allelopathic ability against clover.
Furthermore, the above results demonstrate a potential
for rusted ryegrass to release allelochemicals into soil
at concentrations phytotoxic to clover.
Previous soil-based bioassays have not reported enhanced allelopathy following nutrient addition (Buchholtz, 1971). Despite this, there are at least three possible explanations of how nutrients might enhance clover
suppression by soil previously growing rusted ryegrass:
(i) The presence of abundant nutrients in some way
facilitates allelochemical uptake.
(ii) Nutrient addition may initiate cationic exchange.
Here, nutrient addition in the form of cations
releases allelochemicals bound to anionic colloidal or organic material into the soil solution,
where they are absorbed by receiver plants.
(iii) A reaction of allelochemicals with the added nutrients may increase their toxicity and/or stability.
Takahashi et al. (1988, 1991) established that leachate
from perennial ryegrass can be phytotoxic to white clover. In a similar manner, the present study showed that
soil leachate from developing ryegrass (up to 70 d after
sowing) suppressed clover biomass by 30% compared
with the control (i.e., in the pre-inoculation bioassay).
This highlights the potential for ryegrass to suppress
clover through allelopathy. As healthy ryegrass developed, however, the phytotoxicity of its leachate diminished and was lost, suggesting that ryegrass allelopathic
potential is greatest when it is young. Work on other
plant species has also shown a similar decrease in allelopathy as plants age (Koeppe et al., 1970; Woodhead,
1981). The hypothesis that allelopathic potential decreases with plant age may also explain why Newman
and Rovira (1975) and Newman and Miller (1977) observed no clover yield depression following exposure to
leachate from ryegrass. This is because they used lea-
57
chate only from mature ryegrass, starting at 60 d after
sowing.
In contrast to leachate from healthy ryegrass, plant
age did not affect the phytotoxicity of leachate from
rusted ryegrass, where the leachate suppressed clover
biomass by 20% compared with the control and by 27%
compared with nonrusted ryegrass. This is again in similar proportions to the preliminary investigation (Mattner, 1998). Similar reductions occurred in all parameters
measured in clover exposed to leachate from rusted
ryegrass, strongly supporting the hypothesis that rust
increases ryegrass allelopathic ability against clover. The
results also suggest that in addition to enhancing ryegrass allelopathic potential, rust prolongs allelopathy
well into maturity.
While results from each bioassay suggest that rust can
increase ryegrass allelopathy against clover, there was
no evidence of allelopathy acting against nodulation.
Furthermore, the uniformity of size and pinkness of
nodules from plants in all treatments suggested that
allelopathy did not impair nodule nitrogen-fixing capacity, although no measurement of this was made. The
only treatment that affected nodulation was soil sterilization, which reduced it by 26%. This was probably
due to sterilization reducing the resident rhizobial population in the soil prior to planting.
The ability of rust to increase ryegrass allelopathic
potential is not surprising given that infection by pathogens can induce phytoalexin production by their hosts
(Smith, 1996). Phytoalexins can belong to similar chemical groups as allelochemicals. For example, Mayama
(1981, 1982) discovered that rust resistant oat (Avena
sativa L.) produced three phytoalexins belonging to the
phenolic acid group when challenged by crown rust.
These phenolic acids inhibited rust spore germination
at concentrations as low as 200 mg/L. Although these
chemicals were not shown to suppress plant growth,
they have a great potential to do so given that many
allelochemicals are phenolic acids.
Another potential allelochemical source from rusted
ryegrass in the present experiment is from the rust itself.
It is well established that pathogens can produce a range
of phytotoxins that are important in pathogenesis (Daly
and Deverall, 1983). Despite this, a preliminary investigation demonstrated that clover monoculture inoculation with crown rust had no effect on its growth (Mattner, 1998). This suggests that the allelochemical source
from rusted ryegrass in the present experiment is more
likely to be from ryegrass than from rust. It is important
to note, however, that irrespective of the phytotoxin
source from rusted ryegrass, the relationship between
rusted ryegrass and clover remains allelopathic. This is
because rust is a biotrophic parasite of ryegrass and
because allelopathy is defined as the beneficial and detrimental chemical interaction among plant organisms,
including microorganisms (Rice, 1984).
An important next step in the confirmation of enhanced allelopathy by rusted ryegrass is to identify the
allelochemicals involved. What is their source? Are
these compounds in higher concentrations in rusted
plants or do rusted plants produce entirely different
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AGRONOMY JOURNAL, VOL. 93, JANUARY–FEBRUARY 2001
allelochemicals than healthy plants? In a related question, what is the effect of disease severity on ryegrass
allelopathic ability? The average disease severity of
rusted ryegrass at 42 d after inoculation in the soil retrieval bioassay of the present experiment was 6.6%
(as determined by a calibration method [Mattner and
Parbery, 1999]). If rust enhances allelopathy between
ryegrass and clover, heavily rusted plants should produce more allelochemicals than lightly rusted plants.
Moreover, the ryegrass cultivar used in the present experiment was highly susceptible to rust. If rust enhances
ryegrass allelopathy through phytoalexin production,
rust resistant cultivars might produce more phytoalexins, or allelochemicals, than susceptible cultivars.
The increased allelopathy of rusted ryegrass has important implications, both for pasture ecology and the
evolution of mutualism. In Australia, rust epidemics
occur in ryegrass between late spring and early autumn.
During this period, relative white clover growth is
greater than that of perennial ryegrass, due mainly to
differences in temperature optima (18–218C for ryegrass
and 248C for clover) for growth (Haynes, 1980). The
combined effects of crown rust and increased competition from clover increase ryegrass mortality. For this
reason, the increase in allelopathy by ryegrass following
rust infection may contribute to preventing its eradication from pastures during the summer–autumn period.
Although pathogens are largely detrimental to their
hosts, they may also bestow some benefits (Parbery,
1996). By conferring some benefit on its host, the pathogen maintains a self-advantage through increasing the
survival chances of its host and ultimately itself. Furthermore, Clay (1988) suggested that pathogens evolve toward a mutualistic relationship with their hosts through
the acquisition of beneficial characteristics or “new
functions.” Since biotrophic parasites such as the rusts
are heavily dependent on the continuity of their host
genotype into succeeding generations, the evolution of
interactions that enhance the chances of host survival
are important. It is well established that infections of
fodder species by biotrophs can create conditions that
either limit grazing of their hosts, limit the number of
grazing animals, or both. Morgan and Parbery (1980)
established that infection by Pseudopeziza medicagnis
(Lib.) Sacc. lowered protein content, digestibility, and
palatability of lucerne (Medicago sativa L.) as well as
increasing its oestrogenic activity. Similarly, Critchett
(1991) determined that crown rust reduces ryegrass digestibility and quality. For this reason, ruminants preferentially graze healthy ryegrass rather than rusted ryegrass (Cruickshank, 1957), indirectly benefiting rusted
ryegrass. Evidence from the present study suggests that
rust adds a further benefit to ryegrass, that of increased
allelopathy with neighboring plants. In a similar manner
to crown rust, amongst other benefits, the mutualistic
endophyte N. lolii reduces ryegrass palatability to ruminants (Fletcher and Sutherland, 1993) and reportedly
increases its allelopathic ability (Sutherland and Hoglund, 1990; Quigley et al., 1990). Is the pathogenic relationship between crown rust and ryegrass evolving toward mutualism?
CONCLUSIONS
The bioassays of the present study showed that soil
and leachate from rusted ryegrass suppressed clover
growth compared with that from nonrusted ryegrass.
Together, these bioassays provide strong evidence for
the hypothesis that rust enhances perennial ryegrass
allelopathic ability against white clover. This hypothesis
would explain why rusted ryegrass suppressed the
growth of neighboring clover plants in a preliminary
experiment (Mattner, 1998).
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Rust-Enhanced Allelopathy of Perennial Ryegrass against White Clover
Scott W. Mattner* and Douglas G. Parbery
ABSTRACT
Perennial ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) are important pasture components in the higher rainfall areas of southeastern Australia. Crown rust (Puccinia coronata
Corda f.sp. lolii Brown) is the most serious ryegrass pathogen in these
areas. In a preliminary investigation, rust reduced ryegrass biomass
by 56%. Yet, interference from rusted ryegrass suppressed the yield
of neighboring clover plants more than interference from healthy
ryegrass. The role of allelopathy in this relationship was investigated
in a greenhouse study using two bioassays. Soil previously growing
rusted ryegrass suppressed clover biomass by 36% compared with
soil previously growing healthy ryegrass. Similarly, leachate from soil
surrounding rusted ryegrass suppressed clover biomass by 27% compared with that from healthy ryegrass. This is the first demonstration
that a pathogen may influence allelopathy between plants and that
rust may enhance ryegrass allelopathy against clover. Possible implications of this in pasture ecology and the evolution of mutualism are discussed.
P
erennial ryegrass and white clover predominate
improved pastures in the higher rainfall areas of
southeastern Australia. Their growth in mixtures without added nitrogen results in greater herbage yields than
otherwise can be achieved economically (Menchaca and
S.W. Mattner, Agriculture Victoria, Institute for Horticultural Development, Private Bag 15, Scoresby Business Centre, Victoria 3176,
Australia; D.G. Parbery, Institute of Land & Food Resources, The
Univ. of Melbourne, Parkville, Victoria 3052, Australia. Received 29
Nov. 1999. *Corresponding author ([email protected]).
Published in Agron. J. 93:54–59 (2001).
Connolly, 1990). Crown rust is the most serious ryegrass
fungal pathogen in these areas (Eagling and Clark, 1993),
with epidemics regularly occurring between spring and
autumn. A preliminary study demonstrated that rust accelerates senescence and reduces ryegrass yield by 56%
(Mattner, 1998). Despite this, in ryegrass and clover mixtures, interference from rusted ryegrass suppressed clover
biomass by up to 47% compared with interference from
healthy ryegrass. This suppression did not result from
a direct effect of crown rust on clover, because rustinoculated and non-inoculated clover monocultures
yielded the same, which was expected since clover is a
nonhost of crown rust. Similarly, clover suppression is
not explained by rust increasing ryegrass competitiveness because, if this were so, the reduction in clover
yield would be greatest at high densities where resources
are most limited. Instead, clover suppression was greatest at low densities, where competition for resources
was minimal. Ryegrass allelopathy is well documented,
particularly against clovers and medics (Gussin and
Lynch, 1981; Takahashi et al., 1988, 1991, 1993; Quigley
et al., 1990; Chung and Miller, 1995). For these reasons,
we investigated the hypothesis that rusting increases
ryegrass allelopathic ability.
Both Rice (1984) and Einhellig (1995) hypothesized
that pathogens enhance their host’s allelopathic ability.
Evidence supporting this hypothesis occurs in at least
two forms. Firstly, pathogens stimulate phytoalexin (antimicrobial compounds) production by their hosts
(Smith, 1996), which can belong to similar chemical
groups and are synthesized via the same biochemical
MATTNER & PARBERY: RUST-ENHANCED ALLELOPATHY OF PERENNIAL RYEGRASS
pathways as allelochemicals. For example, isoflavonoids
are important phytoalexins (Dakora and Phillips, 1996)
and allelochemicals (Tamura et al., 1967, 1969) from
the Leguminosae. Indeed, many phytoalexins act as
allelochemicals against plants, inhibiting their germination (Chang et al., 1969), growth (Glazener and VanEtten, 1978), and cellular metabolism and function (Giannini et al., 1990; Spessard et al., 1994). Secondly, under
some conditions, the mutualistic fungus Neotyphodium
lolii (Latch, Christensen and Samuels) Glenn, Bacon
and Hanlin, increases the allelopathic ability of some
ryegrass genotypes (Sutherlund and Hoglund, 1990;
Quigley et al., 1990). Despite this, no one has previously
observed that diseased plants suppress the growth of
neighboring species (Tang et al., 1995), even though the
effect of pathogens on interference between host and
nonhost plants has been extensively investigated (Burdon, 1987; Ayres and Paul, 1990). Since competition
is the dominant process of plant interference (Tilman,
1988), competition effects often obscure any allelopathic
effects in such experiments (Trenbath, 1974). This is
particularly so at high plant densities where competition
is intense. While it is difficult to separate the processes
of competition and allelopathy in the field, separation
can be achieved using bioassays. To date, however, researchers have not used bioassays to investigate the
effects of pathogens on allelopathy.
The present investigation examined the hypothesis
that rust enhances perennial ryegrass allelopathy against
white clover using two bioassay techniques.
METHODS AND MATERIALS
Pathogen and Plant Material
In the following bioassays, perennial ryegrass (cv. Victorian) was used as the donor species and white clover (cv.
Tamar) was used as the receiver species. The perennial ryegrass cultivar Victorian is highly susceptible to crown rust
(Critchett, 1991). Crown rust urediniospores were collected
with a side-arm flask collector from naturally infected ryegrass
at the Mt. Derrimut field station, 22 km west of Melbourne,
Australia (378479 S, 1448479 E) (Mattner, 1998).
Growing Conditions and Soil Type
Bioassays were conducted in temperature-controlled glasshouses (20–238C during the day and 12–158C at night) at the
Mt. Derrimut field station. Pots were drip-irrigated at the rate
of 150 mL of water twice daily, which maintained soil at near
field capacity. The soil mixture was three parts red-brown
earth topsoil to one part sand and two parts peat moss. The
following nutrients (g/L) were added to the mixture: 0.200 N,
0.116 P, 0.140 K, 0.096 S, 0.652 Ca, 0.024 Cu, 0.012 Zn, 0.008
Mn, 0.028 Fe, 0.012 Mo, 0.360 Mg, and 0.001 B. The mixture
was then pasteurized by steam prior to planting. Following
pasteurization, soil used to grow white clover was inoculated
with the appropriate strain of Rhizobium trifolii Dangeard.
This was performed by watering soil contained in individual
pots with 150 mL of an inoculum solution (10 g of commercial
inoculum per 10 L of water).
Soil Retrieval Bioassay
Pots (15 cm diam.) containing the standard soil mix were
sown to contain an average of 10 ryegrass donor plants. In
55
total there were 108 pots, half with rusted ryegrass and half
with nonrusted ryegrass. Plants in rusted treatments were
spray inoculated (Villalta and Clarke, 1995) 52 d after sowing
with an aqueous solution containing 1 3 106 urediniospores/
mL, 0.1% soft soap as a surfactant, and 0.5% gelatine. Plants
in nonrusted treatments were sprayed with a similar solution
containing no spores. Following inoculation all plants were
placed in plastic tents for 1 wk, which maintained humidity
between 92 and 98%. Donor plants were grown for a total of
100 d.
Soil for the bioassay was gathered directly from the pots
used to grow rusted and nonrusted donor ryegrass, all of which
was removed by hand and then passed through a 5-mm sieve.
Soil from each source was bulked and thoroughly mixed prior
to further treatment. A fresh preparation of soil was also
included as a control. In addition, plus and minus nutrient
and steam-sterilization treatments were incorporated into the
design. This was done in an attempt to detect confounding
effects caused by differences in soil microflora and nutrient
content in the soils resulting from prior rusted and nonrusted
ryegrass growth. Nutrient enrichment consisted of adding concentrations of the nutrients listed previously, to ensure they
were slightly in excess of growth requirements. The soils were
then placed into 12.5-cm-diam. pots and used to grow two
clover receivers. At 70 d after sowing, receivers were washed
free of soil and dried at 808C for 4 d before total biomass was
determined. Additionally, a nodulation index was determined
by bunching individual plant roots; removing three 2-cm sections from the upper, central, and lower portion of the bunch;
counting the nodules captured in each section; drying each
sample; and recording the number of nodules per gram of root.
The bioassay was conducted as a randomized complete
factorial design with three blocks. There were four pots per
treatment in each block. Factors consisted of soil source (three
levels: soil previously growing rusted ryegrass, soil previously
growing nonrusted ryegrass, and freshly prepared soil as a
control), nutrient application (two levels: plus and minus) and
soil sterilization (two levels: plus and minus).
Soil Leachate Bioassay
Pots (15 cm in diam.) containing the standard soil mix were
sown to contain eight ryegrass donor plants. Twenty days after
sowing, irrigation lines were placed into individual pots and
calibrated to deliver 250 mL of water per pot every day. Soil
leachate was collected in plastic trays beneath the wire mesh
benches, bulked for each treatment, and thoroughly mixed
prior to application to receiver plants. Seventy days after sowing, plants in the rusted treatment were inoculated with rust
as described previously. One-half of the 64 pots contained
rusted ryegrass and the other half nonrusted plants.
The investigation was conducted in two parts. In the first,
the pre-inoculation bioassay, the bioassay was made before
donor plant inoculation to ensure that there were no intrinsic
differences in the allelopathic potential of donor plants within
the rust treatments prior to inoculation. The second, the postinoculation bioassay, was made after inoculation when rust
symptoms had fully developed. The methodology of the bioassays was the same. Pots (12.5 cm in diam.) filled with the
standard soil mix were prepared containing two clover receivers. Each was hand watered daily with 150 mL of the appropriate leachate or with water in the case of the controls.
Receivers were harvested and compared 50 d after sowing
by determining plant biomass, leaf area (with a planimeter,
Paton Industries Pty. Ltd., South Australia), leaf number, stolon number, and nodulation index. Measurements were taken
56
AGRONOMY JOURNAL, VOL. 93, JANUARY–FEBRUARY 2001
Table 1. Summary of the levels of statistical significance for the
various treatments and their interactions on white clover biomass and nodulation.
Table 2. The effect of soil sterilization on the nodulation of
white clover.
Source of variation
Soil source (SS)
Nutrient (N)
Sterilization (St)
SS 3 N
SS 3 St
N 3 St
SS 3 N 3 St
Dry biomass
Nodulation
Treatment
No. nodules per
gram of root
***
NS
NS
**
NS
NS
NS
NS†
NS
**
NS
NS
NS
NS
Sterilized
554.55
Nonsterilized
752.12
191.95
Although soil sterilization did not affect clover biomass, it reduced root nodulation by 26% (see Table 2).
** Significant at the 0.01 probability level.
*** Significant at the 0.001 probability level.
† Not significant.
on one randomly selected plant per treatment in each block,
except for biomass where all plants were measured.
The bioassay was conducted as a randomized complete
block design. The treatments were the daily application of 150
mL of leachate derived from either rusted ryegrass, nonrusted
ryegrass, or irrigation water. There were eight blocks consisting of four pots per treatment.
Statistical Analysis
Data was analyzed using analysis of variance (ANOVA)
as performed on Minitab Version 12 (Minitab, 1998). Homogeneity of variance was determined by examining plots of
fitted values versus residuals, while histograms of residuals
assessed normality of distribution.
RESULTS
Soil Retrieval Bioassay
Table 1 presents statistical significance levels of main
treatments and their interactions in influencing clover
biomass and nodulation. Sterilization of the soils used
in this bioassay had no effect on the biomass of clover
growing in them. As such, sterilization treatments have
been grouped to provide a clearer demonstration of the
effects of the different soil source and nutrient treatments (see Fig. 1). The biomass of clover grown in soil
from rusted ryegrass was less than that in the control
or in soil from nonrusted ryegrass, with this effect being
particularly marked in nutrient treated soils. In contrast,
clover grown in soil from nonrusted ryegrass produced
less than the control only when no nutrients were added.
LSD ( p , 0.05)
Soil Leachate Bioassay
The pre-inoculation bioassay detected no difference
in the growth of clover watered with leachate from ryegrass in nonrusted or rusted treatments (see Table 3).
This indicates that there was no intrinsic difference in
the allelopathic ability of ryegrass plants assigned to the
treatments prior to inoculation. Growth in the control,
however, was greater than that of plants receiving soil
leachate from ryegrass.
In the post-inoculation bioassay, the growth of clover
receiving soil leachate from rusted ryegrass was less than
that of plants watered with leachate from nonrusted
ryegrass or the control, according to each parameter
measured (see Table 3). Plants exposed to leachate from
nonrusted ryegrass did not differ from the control in
any way. Nodulation did not vary between treatments.
DISCUSSION
Even though several authors have suggested that
pathogens increase their host’s allelopathic ability (Rice,
1984; Einhellig, 1995), the present study is the first to
evaluate the pathogen effect on allelopathy between
plants. Furthermore, no one has previously observed
that diseased plants suppress the growth of neighboring
species (Tang et al., 1995). Despite this, a preliminary
investigation found that rusted ryegrass suppressed the
growth of neighboring clover plants by up to 47% compared with when grown with healthy ryegrass (Mattner, 1998).
In the present study, the soil retrieval bioassay demonstrated that, overall, soil previously growing ryegrass
reduced the subsequent clover growth compared with
Table 3. Production of white clover in response to leachate derived from rusted or nonrusted perennial ryegrass or a control
of irrigation water. Bioassays were performed prior to and after
the inoculation of ryegrass with rust.
Leachate source
White clover
Nonrusted Rusted
growth parameter Control ryegrass ryegrass
Total biomass (g)
Fig. 1. The biomass of white clover grown in soil from rusted or
nonrusted perennial ryegrass. The control consisted of freshly prepared soil. Nutrients were added at concentrations described in
the text.
1.10
Total biomass (g)
2.86
203.88
Leaf area (cm2)
Stolon number
4.71
Leaf number
36.5
Nodulation index
(no. nodules
per gram root) 1222.81
† Not significant.
LSD
p , 0.05
Pre-inoculation bioassay
0.76
0.78
0.16
Post-inoculation bioassay
3.11
2.28
0.53
196.2
145.4
49.9
5.00
4.00
0.76
34.12
25.5
8.78
1121.11
1143.78
NS
p , 0.01
0.22
0.75
NS†
NS
NS
NS
MATTNER & PARBERY: RUST-ENHANCED ALLELOPATHY OF PERENNIAL RYEGRASS
a control of freshly prepared soil. The modification of
soil microflora by ryegrass to contain species antagonistic to clover growth does not explain this result because
soil sterilization had no effect on the relationship. In
contrast, nutrient addition alleviated the suppressive effect that soil previously growing healthy ryegrass had
on clover production. This suggests that the main effect
of healthy ryegrass was to deplete soil nutrients and
thereby diminish clover growth. This was not the case
for soil previously growing rusted ryegrass, however,
where nutrient application markedly increased its suppressive effect on clover growth. Under these conditions, soil previously growing rusted ryegrass suppressed
clover biomass by 36% compared with soil previously
growing healthy ryegrass. This was in similar proportions to that in the preliminary experiment where the
yield of clover grown in mixtures with rusted ryegrass
fell by an average of 37% (Mattner, 1998). Nutrient
toxicity does not explain this result because twice the
concentration of nutrients applied to soil in the rusted
treatment had no effect on clover growth in the control.
Instead, the result provides strong evidence that rust
increases ryegrass allelopathic ability against clover.
Furthermore, the above results demonstrate a potential
for rusted ryegrass to release allelochemicals into soil
at concentrations phytotoxic to clover.
Previous soil-based bioassays have not reported enhanced allelopathy following nutrient addition (Buchholtz, 1971). Despite this, there are at least three possible explanations of how nutrients might enhance clover
suppression by soil previously growing rusted ryegrass:
(i) The presence of abundant nutrients in some way
facilitates allelochemical uptake.
(ii) Nutrient addition may initiate cationic exchange.
Here, nutrient addition in the form of cations
releases allelochemicals bound to anionic colloidal or organic material into the soil solution,
where they are absorbed by receiver plants.
(iii) A reaction of allelochemicals with the added nutrients may increase their toxicity and/or stability.
Takahashi et al. (1988, 1991) established that leachate
from perennial ryegrass can be phytotoxic to white clover. In a similar manner, the present study showed that
soil leachate from developing ryegrass (up to 70 d after
sowing) suppressed clover biomass by 30% compared
with the control (i.e., in the pre-inoculation bioassay).
This highlights the potential for ryegrass to suppress
clover through allelopathy. As healthy ryegrass developed, however, the phytotoxicity of its leachate diminished and was lost, suggesting that ryegrass allelopathic
potential is greatest when it is young. Work on other
plant species has also shown a similar decrease in allelopathy as plants age (Koeppe et al., 1970; Woodhead,
1981). The hypothesis that allelopathic potential decreases with plant age may also explain why Newman
and Rovira (1975) and Newman and Miller (1977) observed no clover yield depression following exposure to
leachate from ryegrass. This is because they used lea-
57
chate only from mature ryegrass, starting at 60 d after
sowing.
In contrast to leachate from healthy ryegrass, plant
age did not affect the phytotoxicity of leachate from
rusted ryegrass, where the leachate suppressed clover
biomass by 20% compared with the control and by 27%
compared with nonrusted ryegrass. This is again in similar proportions to the preliminary investigation (Mattner, 1998). Similar reductions occurred in all parameters
measured in clover exposed to leachate from rusted
ryegrass, strongly supporting the hypothesis that rust
increases ryegrass allelopathic ability against clover. The
results also suggest that in addition to enhancing ryegrass allelopathic potential, rust prolongs allelopathy
well into maturity.
While results from each bioassay suggest that rust can
increase ryegrass allelopathy against clover, there was
no evidence of allelopathy acting against nodulation.
Furthermore, the uniformity of size and pinkness of
nodules from plants in all treatments suggested that
allelopathy did not impair nodule nitrogen-fixing capacity, although no measurement of this was made. The
only treatment that affected nodulation was soil sterilization, which reduced it by 26%. This was probably
due to sterilization reducing the resident rhizobial population in the soil prior to planting.
The ability of rust to increase ryegrass allelopathic
potential is not surprising given that infection by pathogens can induce phytoalexin production by their hosts
(Smith, 1996). Phytoalexins can belong to similar chemical groups as allelochemicals. For example, Mayama
(1981, 1982) discovered that rust resistant oat (Avena
sativa L.) produced three phytoalexins belonging to the
phenolic acid group when challenged by crown rust.
These phenolic acids inhibited rust spore germination
at concentrations as low as 200 mg/L. Although these
chemicals were not shown to suppress plant growth,
they have a great potential to do so given that many
allelochemicals are phenolic acids.
Another potential allelochemical source from rusted
ryegrass in the present experiment is from the rust itself.
It is well established that pathogens can produce a range
of phytotoxins that are important in pathogenesis (Daly
and Deverall, 1983). Despite this, a preliminary investigation demonstrated that clover monoculture inoculation with crown rust had no effect on its growth (Mattner, 1998). This suggests that the allelochemical source
from rusted ryegrass in the present experiment is more
likely to be from ryegrass than from rust. It is important
to note, however, that irrespective of the phytotoxin
source from rusted ryegrass, the relationship between
rusted ryegrass and clover remains allelopathic. This is
because rust is a biotrophic parasite of ryegrass and
because allelopathy is defined as the beneficial and detrimental chemical interaction among plant organisms,
including microorganisms (Rice, 1984).
An important next step in the confirmation of enhanced allelopathy by rusted ryegrass is to identify the
allelochemicals involved. What is their source? Are
these compounds in higher concentrations in rusted
plants or do rusted plants produce entirely different
58
AGRONOMY JOURNAL, VOL. 93, JANUARY–FEBRUARY 2001
allelochemicals than healthy plants? In a related question, what is the effect of disease severity on ryegrass
allelopathic ability? The average disease severity of
rusted ryegrass at 42 d after inoculation in the soil retrieval bioassay of the present experiment was 6.6%
(as determined by a calibration method [Mattner and
Parbery, 1999]). If rust enhances allelopathy between
ryegrass and clover, heavily rusted plants should produce more allelochemicals than lightly rusted plants.
Moreover, the ryegrass cultivar used in the present experiment was highly susceptible to rust. If rust enhances
ryegrass allelopathy through phytoalexin production,
rust resistant cultivars might produce more phytoalexins, or allelochemicals, than susceptible cultivars.
The increased allelopathy of rusted ryegrass has important implications, both for pasture ecology and the
evolution of mutualism. In Australia, rust epidemics
occur in ryegrass between late spring and early autumn.
During this period, relative white clover growth is
greater than that of perennial ryegrass, due mainly to
differences in temperature optima (18–218C for ryegrass
and 248C for clover) for growth (Haynes, 1980). The
combined effects of crown rust and increased competition from clover increase ryegrass mortality. For this
reason, the increase in allelopathy by ryegrass following
rust infection may contribute to preventing its eradication from pastures during the summer–autumn period.
Although pathogens are largely detrimental to their
hosts, they may also bestow some benefits (Parbery,
1996). By conferring some benefit on its host, the pathogen maintains a self-advantage through increasing the
survival chances of its host and ultimately itself. Furthermore, Clay (1988) suggested that pathogens evolve toward a mutualistic relationship with their hosts through
the acquisition of beneficial characteristics or “new
functions.” Since biotrophic parasites such as the rusts
are heavily dependent on the continuity of their host
genotype into succeeding generations, the evolution of
interactions that enhance the chances of host survival
are important. It is well established that infections of
fodder species by biotrophs can create conditions that
either limit grazing of their hosts, limit the number of
grazing animals, or both. Morgan and Parbery (1980)
established that infection by Pseudopeziza medicagnis
(Lib.) Sacc. lowered protein content, digestibility, and
palatability of lucerne (Medicago sativa L.) as well as
increasing its oestrogenic activity. Similarly, Critchett
(1991) determined that crown rust reduces ryegrass digestibility and quality. For this reason, ruminants preferentially graze healthy ryegrass rather than rusted ryegrass (Cruickshank, 1957), indirectly benefiting rusted
ryegrass. Evidence from the present study suggests that
rust adds a further benefit to ryegrass, that of increased
allelopathy with neighboring plants. In a similar manner
to crown rust, amongst other benefits, the mutualistic
endophyte N. lolii reduces ryegrass palatability to ruminants (Fletcher and Sutherland, 1993) and reportedly
increases its allelopathic ability (Sutherland and Hoglund, 1990; Quigley et al., 1990). Is the pathogenic relationship between crown rust and ryegrass evolving toward mutualism?
CONCLUSIONS
The bioassays of the present study showed that soil
and leachate from rusted ryegrass suppressed clover
growth compared with that from nonrusted ryegrass.
Together, these bioassays provide strong evidence for
the hypothesis that rust enhances perennial ryegrass
allelopathic ability against white clover. This hypothesis
would explain why rusted ryegrass suppressed the
growth of neighboring clover plants in a preliminary
experiment (Mattner, 1998).
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