• To better understand evolution we have studied aggressiveness of the anthracnose pathogen, Colletotrichum gloeosporioides, collected from Stylosanthes scabra pastures between 1978 and 2000 and by inoculating two isolates onto two cultivars over 25 sequential infection cycles at ambient (350 ppm) and twice-ambient atmospheric CO2 in controlled environments.
• Regression analysis of the field population showed that aggressiveness increased towards a resistant cultivar, but not towards a susceptible cultivar, that is no longer grown commercially.
• Here we report for the first time that aggressiveness increased on both cultivars after a few initial infection cycles at twice-ambient CO2 as isolates adapted to combat enhanced host resistance, while at ambient CO2 this increased steadily for most cycles as both cultivars selected for increased aggressiveness. Genetic fingerprint and karyotype of isolates changed for some CO2-cultivar combinations, but these were not related to changed aggressiveness.
• At 700 ppm fecundity increased for both isolates, and this increased population size, in combination with a conducive microclimate for anthracnose from an enlarged plant canopy under elevated CO2, could accelerate pathogen evolution.
In agricultural systems, where the deployment of host resistance genes is largely determined by plant breeders, pathogen fitness determines how quickly a pathogen population responds to genetic changes in the host. Virulence, defined as the genetic ability of a pathogen race to overcome genetically determined host resistance, that is effective against other races; and aggressiveness, a property of the pathogen reflecting the relative amount of damage caused to the host without regard to resistance genes (Shaner et al., 1992), together determine the overall fitness of pathogen population (Burdon, 1987). There are many examples in the literature of host resistance being overcome by matching pathogen virulence, including those involving gene–for–gene interactions (Staskawicz et al., 1995; McDonald et al., 1996; Brown et al., 1997; Kolmer, 1997). Adaptation to some cultivars has occurred after only five asexual cycles in Uromyces appendiculatus (Alexander et al., 1985) and after 30 asexual cycles in Erysiphe graminis (Newton & McGurk, 1991).
In comparison, there has been limited research on pathogen adaptation for increased aggressiveness. It is a heritable quantitative trait (Caten et al., 1984; Mundt et al., 2002) and in one of the first examples, aggressiveness was shown to increase over three sexual generations of the pathogen Cochliobolus heterostrophus infecting maize (Kolmer & Leonard, 1986). Pathogen strains often show enhanced aggressiveness towards cultivars from which they originate (Caten, 1974). For instance, the ability of Mycosphaerella graminicola to adapt to quantitative resistance of winter wheat cultivars is influenced strongly by the host (Mundt et al., 1999). In one study, susceptible cultivars have been shown to simultaneously select for increased aggressiveness and virulence within a single growing season in the field (Ahmed et al., 1996), while a more recent study has demonstrated that resistant cultivars are more likely to select for more aggressive isolates (Cowger & Mundt, 2002). The physical environment is another important variable that influences aggressiveness (Asher & Thomas, 1984; Newton, 1989) and strains with increased aggressiveness may arise more frequently under conditions of favorable weather for pathogen growth (Mundt et al., 1999). In addition, a favorable environment may accelerate selection for increased aggressiveness by increasing pathogen reproduction rate, and changes in environmental conditions may modify host resistance to indirectly influence pathogen aggressiveness; but these have not been addressed in the published literature.
Rising atmospheric CO2 is a major environmental change that influences the physiology, morphology and biomass of plants (Kimball, 1985). CO2 concentration has risen by 31% since preindustrial times and carbon cycle models project concentrations of 540–970 ppm by 2100 (Intergovernmental Panel on Climate Change, 2001). Limited information on the influence of elevated CO2 on pathogens and diseases suggests that higher carbohydrate concentration within host tissue promotes the development of some biotrophic pathogens such as rusts (Manning & Tiedemann, 1995) but inhibits others such as mildews (Hibberd et al., 1996a). Significant impacts of elevated CO2 are manifested through changes in host physiology and resistance. In addition, increases in canopy size and the amount of usable host tissue at elevated CO2 with associated changes in microclimate can influence the growth, sporulation, spread and severity of some leaf infecting fungi (Coakley et al., 1999; Chakraborty et al., 2000). At high CO2 increased resistance in some plants slows host invasion but some fungal pathogens produce more spores on host tissue because of increased fecundity (Chakraborty et al., 2000; Hibberd et al., 1996a). How these changes may influence pathogen evolution is not known. Pathogen aggressiveness comprises components that include the ability to invade, damage and reproduce within a host (Shaner et al., 1992) and isolates with the same virulence can have substantially different reproductive fitness (Johnson & Taylor, 1972). Of particular concern is whether the increased pathogen fecundity at elevated CO2 could rapidly erode the usefulness of disease resistance in crop plants.
The fungal pathogen Colletotrichum gloeosporioides (Penz.) Penz. & Sacc, which causes anthracnose disease of the tropical pasture legume Stylosanthes scabra, has established in Australia from a single accidental introduction (Manners et al., 2000). The pathogen predominantly reproduces asexually and genetic changes are associated with horizontal transfer of genetic materials such as retrotransposon and supernumerary chromosomes (Manners et al., 2000). Despite this, races with novel virulence have arisen to devastate many previously resistant commercial cultivars in Australia (Chakraborty, 1998). S. scabra cultivar Fitzroy, once productive over a large area of central Queensland was discarded within 5 yr of its release after race 1 was discovered to be severely damaging stands in the early 1970s. Subsequent work has shown that Fitzroy is susceptible to all four races that have adapted to infect S. scabra cultivars in Australia (Chakraborty et al., 1996) and this cultivar has completely disappeared from commercial pastures and seeds are no longer produced commercially. S. scabra cultivar Seca was released in 1976 with two resistance genes: one conferred qualitative resistance to race 1 and the second gene offers quantitative resistance (Cameron et al., 1988). At the time of its release Seca only suffered minor anthracnose damage but a new race (race 3) virulent on Seca was discovered within 6 yrs of its release (Davis et al., 1984). Race 3 isolates with increased aggressiveness, some with new genotypes, have been reported in recent years (Chakraborty et al., 1999) but a systematic analysis of the pathogen population has not been carried out.
Previously we have shown that both Seca and Fitzroy develop large and dense canopy when grown in an environment of 700 ppm CO2. Anthracnose severity is reduced at elevated CO2 because of delayed and reduced germination, germtube growth and appressoria production by the pathogen. Despite this resulting extended incubation period, C. gloeosporioides developed sporulating lesions faster and produced more spores per day in high CO2 within the same latent period as in ambient CO2 (Chakraborty et al., 2000). In this study we address three specific objectives: first, to document how aggressiveness towards the dominant commercial cultivar Seca has changed over the last 22 yr; second, experimentally determine how two cultivars, with different levels of resistance to race 3, influence aggressiveness of two race 3 isolates of C. gloeosporioides over 25 sequential infection cycles; and third, whether a change in the atmospheric composition of CO2, predicted under the global climate change scenario, modifies the influence of these cultivars on aggressiveness. We have used disease severity, assessed either as the percentage of leaf area diseased or as the number of lesions per leaf, as a measure of pathogen aggressiveness in this work.
Materials and Methods
Aggressiveness of field isolates
Since 1986, every 2–3 yr we have collected samples of infected plant materials from surveys of commercial Stylosanthes pastures in Queensland, Australia. More than 20 field sites in eastern and northern Queensland between Southedge (17°0′ S, 145°20′ E) in the north and Samford (27°22′ S, 152°53′ E) in the south were sampled. With 111 cm annual average rainfall and a granitic sandy-loam soil Southedge is typical of large areas of Cape York Peninsula where Stylosanthes spp. are well adapted, while Samford has 105 cm average annual rainfall with a subtropical climate and is located at the southern edge of the climatic zone for Stylosanthes-growing regions in Australia. Each site was delineated into 1–2 transects depending on its size and samples were collected from a number of points along each transect. The length of transect was varied to suit each site and several samples were usually collected from each point along each transect. To determine whether less aggressive isolates have survived in the Seca-dominant pastures, in 2000 we used the susceptible cultivar Fitzroy as a trap plant. Disease-free Fitzroy seedlings were grown in a glasshouse for 6 wk and exposed to natural anthracnose inoculum for 48 h within a commercial pasture at the Cedarvale site (24°18′ S, 151°38′ E) that had been regularly sampled. Plants were brought back and incubated in a dew chamber to allow disease development. The pathogen was isolated by plating small pieces of surface sterilized infected plant tissue on Oat Meal Agar (OMA) and monoconidial cultures were obtained by streaking a spore suspension onto water agar plates and picking up single conidia using a sterile needle. A small number of monoconidial isolates collected before 1986 were obtained from culture collections of Mr Robert Davis of the Queensland Department of Primary Industries and Professor John Irwin of the University of Queensland. In this study we have used 1774 isolates collected in the following years: 1978 (4), 1982 (10), 1984 (6), 1986 (11), 1987 (42), 1991 (143), 1992 (119), 1993 (509), 1994 (395), 1998 (454), 1999 (51) and 2000 (30).
Aggressiveness of isolates was tested on cultivars Fitzroy and Seca and accessions, Q10042 and 93116. Fitzroy is susceptible to all races; Q10042 is partially resistant to all races; Seca is resistant to race 1 but partially resistant to race 3 and 93116 is resistant to all races. Of these, Seca is the most widely grown in commercial large-scale pastures in Queensland and advanced lines derived from 93116 and Q10042 form a small component of a synthetic variety, Siran, which occupies a small area. 6-wk-old-seedlings of each host were raised in a sandy loam soil in 5 × 5 cm square plastic pots (Kwik pots, Arthur Yates & Co, Australia) in a glasshouse. Each isolate was grown on OMA for 7 d and 3–4 replicate seedlings of each host was inoculated with 106 conidia ml−1 suspension. Details on culturing and inoculation methods have appeared before (Chakraborty & Jones, 1993). Seedlings were maintained in a dew chamber for 48 h and visually examined for disease symptoms and percentage leaf area diseased per seedling 10 d after inoculation was rated using a 0–9 scale (Chakraborty et al., 1999).
Changing aggressiveness at ambient and twice-ambient CO2
To examine if both susceptible and partially resistant cultivars select for increased aggressiveness and whether a change in atmospheric CO2 composition modifies the influence of these cultivars on aggressiveness, batches of Fitzroy and Seca plants were raised in a controlled environment facility (CEF) at CSIRO Plant Industry in Brisbane for 6 wk. Single seedlings were raised in a loamy soil in pots and fertilized as necessary with a 0.8-g l−1 solution of a mineral fertilizer (‘Aquasol’, Hortico, Sydney, Australia). Two separate rooms in the CEF provided 30/25°C day/night temperature, 65/95% day/night relative humidity and a 14-h photoperiod with 500 µmol m−2 s−1 photon flux density at the level of the plant canopy. CO2 concentration was maintained at 350 (ambient) or 700 ppm in the two rooms. CO2 level was regulated and monitored using a CO2 gas analyzer (ADC 2000 series, The Analytical Development Company, Hoddesdon, UK), which uses a nondispersive infrared absorption technique. In addition, temperature and relative humidity (rh) were monitored during the entire experiment and according to sensor outputs; temperature fluctuated by ±0.5°C, rh by ±5% and CO2 level by ±60 ppm largely because of opening of the CEF doors to perform tasks such as inoculation and assessment. For a given CO2 level, each generation cycle for all isolate-cultivar combinations were maintained in one CEF room. The experiment took more than 2 yr to complete and used six different CEF rooms, but no systematic switching of rooms was followed.
Two monoconidial isolates of C. gloeosporioides were used in this study. SR24 was isolated in 1984 from a field of Seca in north Queensland and CS1571 was isolated in 1996 from a commercial Seca seed crop in north Queensland. Both isolates belong to race 3 with specificity towards Seca and have the same genotype according to their RAPD fingerprinting profile. Isolate CS1571 is generally more aggressive on Seca than SR24 (Chakraborty et al., 1999). The isolates were grown on OMA to inoculate five replicate seedlings of each cultivar at each CO2 level. Control seedlings were sprayed with sterilized distilled water. All inoculated plants were incubated inside Perspex tents to provide near-saturated rh at their respective CO2 environment. The tent was removed after 48 h and the youngest fully expanded leaf of each replicate plant was assessed for lesions per leaf as a measure of aggressiveness 10 d after inoculation.
Three infected leaves from each replicate were assessed for pathogen fecundity. Conidia were removed by shaking leaves in 5 ml sterile distilled water for 2 h in an orbital shaker and spores in the suspension were counted using a hemacytometer. Leaves were removed after shaking and blotted dry to determine leaf and lesion area by superimposing a transparent acetate sheet divided into 1 mm3 grids. Fecundity was expressed as spore production per unit lesion area.
Pieces of infected stem and leaf tissue were collected from each of the five replicates for each isolate-cultivar combination at each CO2 treatment and plated out on OMA to obtain pure cultures. The inoculum for the subsequent infection cycle was obtained by scraping the surface of five colonies, each originating from a different piece of tissue from the respective isolate-cultivar-CO2 combination. The suspension was adjusted to 106 conidia ml−1 before inoculation. Pure cultures from selected infection cycles were stored in liquid nitrogen for future use.
To detect genetic changes, Random Amplified Polymorphic DNA (RAPD) fingerprints and electrophoretic karyotype of the two isolates were established before and after the 25 infection cycles on the two cultivars at each CO2 level. Six arbitrary primers (Operon Technologies Inc. Alameda, CA, USA) used for the RAPD analysis were selected from a previous study, which provides further details (Chakraborty et al., 1999). Cultures were grown on OMA and DNA from duplicate cultures of each isolate before and after the 25 infection cycles on each of the two cultivars at the two CO2 levels was extracted using the method of Edwards et al. (1991) with minor modifications. Mycelia and conidia were ground in an extraction buffer (250 mm Tris HCl, 200 mm NaCl, 25 mm EDTA and 5% SDS), centrifuged, cleaned with phenol chloroform, followed by a chloroform wash and DNA was precipitated in ethanol. DNA was amplified by polymerase chain reaction in a programmable thermocycler (MJ Research, USA). An initial 5-min denaturation step at 94°C was followed by 40 cycles of 1-min denaturation at 94°C, 1-min annealing at 37°C and 2-min primer extension at 72°C. 30 µl amplification product was electrophoresed in 1.5% agarose gel with 40 µg ethidium bromide in 0.5 TBE (45 mm tris-borate, 0.127 mm EDTA at pH 8.0) buffer.
For Electrophoretic Karyotype analysis conidia were obtained from cultures grown in V8 juice for 7 d with shaking. Spore blocks were prepared for pulse field electrophoresis according to Masel et al. (1990). Conidia were centrifuged for 10 min at 2150 g, pellet was washed with 50 mm EDTA and resuspended in 1% low melting point agarose in 125 mm EDTA and 50 mm Na Citrate (pH 5.7) with a lysing enzyme (Sigma-Aldrich Pty. Ltd., Sydney, Australia). Agar blocks were set in a commercial sample mould (Biorad Laboratories, Inc., CA, USA). Conidia were lysed in 50 mm Na Citrate with 0.4 m EDTA and 7.5% mercaptoethanol (pH 5.7) at 45°C for 24 h and in proteinase K (mg/mL) in NDS buffer at 50°C for 24 h.
A contour-clamped homogeneous electric field dynamically regulated system (CHEF-DR II, Biorad Laboratory, Inc., USA) was used for gel electrophoresis in 0.5 TBE buffer pH 8.0 maintained at 14°C with constant buffer circulation. Spore blocks were placed in wells in a 0.8% agarose gel (14 × 12.7 × 1 cm, Pharmacia Australia Pty. Ltd., Rydalmere, NSN, Australia) and run with 20 h, 30 s followed by 9 h, 90 s switch times. Saccharomyces cerevisiae chromosomes (Biorad Laboratory Inc., USA) were used as size markers. Gels were stained with ethidium bromide and destained in distilled water before examination using an UV-transilluminator.
Data on disease severity rating of field isolates were log(y + 1) transformed and examined using regression analysis. Analysis of CEF data was performed using the S-Plus statistical software (Venables & Ripley, 1999). The influence of CO2, infection cycle, isolate and cultivar on aggressiveness was determined using the total number of lesions per leaf as a measure of aggressiveness. Aggressiveness is sensitive to small changes in physical factors such as temperature (Newton, 1989), consequently data are generally variable and our results on aggressiveness were no exception. In presenting results, we have included original data wherever possible to illustrate the inherent variability in the data. A log-transformation was used to stabilize variance. However, because of the presence of zeros a positive constant term was added before applying the log transformation, log(y +α), where a value of 3.5 for α maximized the profile likelihood function. For the fecundity data, a Box-Cox transformation of the form y′ = ((1 + y)λ−1)/λ was used to stabilize variance. An optimal value of λ= 0.15 maximized the profile likelihood function. Procedures from the MASS library (Venables & Ripley, 1999) were used to obtain optimal log and Box-Cox transformations of the aggressiveness and fecundity data, respectively.
Changing aggressiveness in field population
Aggressiveness of the pathogen population, mainly originating from commercial Seca pastures, showed a significant increase on Seca over the 22-yr period, with a fourfold difference between isolates with the lowest and highest severity levels. As indicated by large standard errors of mean for aggressiveness data on Seca (Fig. 1), the pathogen population before 1987 consisted of both weakly (severity < 0.6) and moderately aggressive (severity 0.6–0.8) isolates. By comparison, populations sampled between 1991 and 1999 were more uniform with isolates that were aggressive (severity 0.8–1.2) to highly aggressive (severity > 1.2) towards Seca. An exception was the isolates originating from exposed Fitzroy plants in 2000. These were not highly aggressive towards Seca, indicating that less aggressive isolates were still present in the pathogen population. Despite the small sample size in some years, the trend in aggressiveness is generally consistent over the entire period. Aggressiveness on the resistant line 93116 increased more modestly but there was no increase on the partially resistant Q10042 or the susceptible Fitzroy (Fig. 1).
Changing aggressiveness at ambient and twice-ambient CO2
A summary ANOVA using all data on the number of lesions per leaf showed that aggressiveness increased significantly with infection cycle and decreased at twice-ambient CO2 (Table 1). Also the negative estimated coefficient of the CO2 main effect suggests that aggressiveness is reduced at the twice-ambient level. However, this is modified by the significant two–way interactions. The effect of infection cycle was significantly nonlinear. A full second order model was used and a smooth term, determined using nonparametric estimation techniques, was used for the nonlinear cycle effect. The overall isolate effect was not significant but its significant interaction with CO2 indicated that the difference in the degree of aggressiveness changes with the CO2 environment. As both cycle × CO2 and isolate × CO2 effects were highly significant we examined the cycle effect separately for the two CO2 environments. Separate models with a smooth nonlinear cycle term were fitted to the aggressiveness data using the S-Plus procedure ‘gam’.
Table 1. Significant terms from an overall ANOVA of the aggressivenessa data for two isolates of Colletotrichum gloeosporioides after 25 sequential infection cycles on two cultivars of Stylosanthes scabra at ambient and twice-ambient CO2
Fitting a separate regression model with smooth nonparametric terms for the cycle effect to the ambient CO2 data showed that aggressiveness increased for most of the infection cycles (Fig. 2). An ANOVA of the ambient CO2 data showed that both cycle and cultivar effects were highly significant (Table 2). Overall, aggressiveness was higher on Seca (Table 2), indicating a possible stronger selection for increased aggressiveness on this cultivar than on the susceptible Fitzroy. However, aggressiveness increased at similar rates on both cultivars (Fig. 2) and the difference in the overall mean aggressiveness between the cultivars was relatively small. There was no significant difference between isolates, as aggressiveness increased in both isolates with increasing infection cycles at ambient CO2.
Table 2. Mean aggressivenessa at ambient and twice-ambient CO2 of two Colletotrichum gloeosporioides isolates after 25 sequential infection cycles on two Stylosanthes scabra cultivars Fitzroy and Seca
A separate ANOVA of the twice-ambient CO2 data showed that, in contrast to ambient CO2, the cultivar effect was not significant (Table 2). Aggressiveness remained largely unchanged for both isolate-cultivar combinations during the first 10 cycles but increased thereafter (Fig. 2). This initial lag points to the number of asexual generations before selection for increased aggressiveness becomes apparent against an enhanced host resistance at twice-ambient CO2. After the first few cycles, both isolates were able to adapt to the increased host resistance and aggressiveness increased on both Seca and Fitzroy.
To ascertain if genetic changes were associated with changes in aggressiveness, we determined RAPD fingerprints from duplicate DNA samples of each isolate before and after the 25 infection cycles. Polymorphism was detected with two (AN10 and D18) of the six primers. Isolate CS1571 developed new RAPD fingerprints after 25 cycles on Seca at ambient CO2 and on Fitzroy at twice-ambient CO2 and SR24 developed new fingerprint on Fitzroy at twice-ambient CO2 (Fig. 4). Electrophoretic karyotype changed for CS1571 with an extra 285 kb putative chromosome after 25 cycles on Fitzroy at ambient CO2 (data not shown).
Changes in Fecundity
Changes in fecundity were measured at the end of each infection cycle and a summary analysis of the fecundity data from all isolate, cultivar and CO2 levels showed that the cycle effect was not significantly nonlinear. Therefore fecundity data were analyzed using a linear regression model with a stepwise procedure for variable selection (Table 3). The overall fecundity increased significantly at twice-ambient CO2. Fecundity increased over successive infection cycles at twice-ambient CO2 but not at ambient CO2. The cycle effect is not significant but the cycle–CO2 interaction is significant. This indicates that fecundity does not increase simply as a result of infection cycles. Of the two isolates, fecundity of the aggressive isolate CS1571 increased on both cultivars at twice-ambient CO2, but in the less aggressive SR24 fecundity at high CO2 only increased on the partially resistant cultivar Seca (Fig. 3). The increased fecundity of CS1571 was most pronounced on Seca at high CO2.
Table 3. Mean fecunditya of two isolates of Colletotrichum gloeosporioides after 25 sequential infection cycles on two cultivars of Stylosanthes scabra at ambient and twice-ambient CO2 and significant terms in an ANOVA
CO2 Level (ppm)
Fecundity = ((1 + spores per unit lesion area)0.15 − 1)/0.15.
CO2 × Cycle
CO2 × Isolate
Cycle × Cultivar
Cultivar × Isolate
CO2 × Cycle × Isolate
CO2 × Cultivar × Isolate
Changing aggressiveness in field population
Our results of field monitoring over the past 22 yr show that aggressiveness of C. gloeosporioides has increased towards the partially resistant cultivar Seca and to a lesser extent towards the resistant 93116 but not for the susceptible Fitzroy or the partially resistant Q10042. We have previously shown that, during the same period, the pathogen has developed new genotypes, but these changes are not directly linked to changes in aggressiveness (Chakraborty et al., 1999). The literature on the selective effect of host resistance on aggressiveness is very limited, and confusion over the concept and nomenclature of terms relating to pathogenicity (Shaner et al., 1992) has contributed to this. For instance, aggressiveness is the nonspecific disease causing ability of a pathogen (Vanderplank, 1968) and should not be influenced by host resistance genes (Shaner et al., 1992). But in the handful of studies on fungal pathogens, host resistance has always significantly influenced aggressiveness, although the relationship between resistance and aggressiveness has not been consistent even for the same host-pathogen combination. For instance, in Mycosphaerella graminicola some studies have shown an association between host susceptibility and aggressiveness (Ahmed et al., 1996; Mundt et al., 1999), while a more recent study (Cowger & Mundt, 2002) has shown that resistant hosts selected for more aggressive isolates. In our field study, because of the dominance of Seca in commercial pastures, the pathogen population mainly originated from Seca and a lack of commercial Fitzroy pastures did not allow us to study pathogen evolution on a susceptible host. This question was examined in the experiment in controlled environment.
Changing aggressiveness at ambient and twice-ambient CO2
Our study is the first to document changing pathogen aggressiveness in elevated CO2 projected under a climate change scenario. We have demonstrated that selection for increased aggressiveness by both cultivars is influenced by a change in the atmospheric CO2 concentration. At twice-ambient CO2 the overall level of aggressiveness of the two isolates was significantly reduced on both cultivars. Aggressiveness increased on both cultivars after an initial lag period, lasting the first 10 infection cycles. As shown previously (Chakraborty et al., 2000), the susceptible Fitzroy develops a level of resistance to anthracnose at elevated CO2, but resistance in Seca remains largely unchanged. Consequently, at high CO2 the impact of anthracnose is more pronounced on Seca than on Fitzroy (Chakraborty et al., 2000). A lack of significant difference between the two cultivars at elevated CO2 in the current work points to a similar induction of resistance in Fitzroy. Some of the changes in plant physiology, anatomy and morphology that have been implicated in increased resistance or can potentially enhance host resist-ance at elevated CO2 include: increased net photosynthesis allowing mobilization of resources into host resistance (Hibberd et al., 1996a); reduced stomatal density and conductance (Hibberd et al., 1996b); greater accumulation of carbohydrates in leaves; more waxes, extra layers of epidermal cells and increased fibre content (Owensby, 1994); production of papillae and accumulation of silicon at penetration sites (Hibberd et al., 1996a); greater number of mesophyll cells (Bowes, 1993); and increased biosynthesis of phenolics (Hartley et al., 2000), among others. C. gloeosporioides predominantly penetrates through stomata and decreased stomatal density at high CO2 reduces the germination and penetration rates of spores on S. scabra leaves (Chakraborty et al., 2000). The increase in aggressiveness after an initial lag suggests that the enhanced host resistance at elevated CO2 may not arrest the development of highly aggressive pathogen strains in the long term. However, host resistance itself will evolve with increasing generations of plants at elevated CO2 to modify pathogen aggressiveness.
At ambient CO2 aggressiveness of both isolates increased over the 25 infection cycles on both resistant and susceptible cultivars. This is among a growing number of studies that demonstrate selection for increased aggressiveness in asexually reproducing fungal pathogens (Caten, 1974; Alexander et al., 1985; Newton, 1989; Newton & McGurk, 1991; Ahmed et al., 1996; Cowger & Mundt, 2002). Although the overall level of aggressiveness was significantly higher on Seca in our study, this may not indicate a stronger selection by the resistant cultivar for increased aggressiveness, as both susceptible and resistant cultivars are equally capable of selecting for increased aggressiveness. This is consistent with the notion that aggressiveness is an additive trait that ideally does not interact differentially with host genotypes (Mundt et al., 1999; Shaner et al., 1992).
Being sensitive to small changes in physical factors such as temperature, aggressiveness data are generally variable (Newton, 1989) and our results are no exception, despite the highly reproducible environment of the CEF rooms. In this study aggressiveness either declined or did not increase after cycle 22. This may have been caused by minor variation in the inoculation and assessment techniques or pathogen and plant growth conditions. Increasing the number of infection cycles and replications can help reduce the coefficient of variation (Chakraborty & Jones, 1993) in future studies.
Changes in pathogen genotype
RAPD genotype and electrophoretic karyotype of some isolate-cultivar combinations changed after the 25 infection cycles, but these were not linked to changes in aggressiveness phenotype. These provide additional evidence of genetic change in this asexual pathogen following the 25 infection cycles. Our results are consistent with published literature from Australia, that demonstrate hyper-variable chromosomes and retrotransposons are the main contributors to the genetic variation in this pathogen (Manners et al., 2000). There is evidence that chromosomes are transferable across strains and such transfer occurs under field conditions (Masel et al., 1996). These molecular mechanisms, in addition to the usual mutation and parasexual recombination, explain the rapid evolution in the pathogen following its introduction to Australia.
Changes in RAPD genotype or karyotype did not match increased aggressiveness. For instance, RAPD fingerprint of CS1571 did not change after 25 cycles on Seca at high CO2. This is to be expected because in many pathogens (Leung et al., 1993) some genotypes contain more than one race and the same race can have different genotypes. In populations of C. gloeosporioides infecting Stylosanthes aggressiveness groups can arise convergently from different genetic lineages (Chakraborty et al., 1999) and there is no strong association between genetic and aggressiveness grouping. However, given the inherent variability in aggressiveness data, the use of molecular markers can provide a more concrete evidence of genetic change and a combination of aggressiveness and selectively neutral markers offers the best strategy.
Changes in fecundity
Our results concur with the handful of studies that have demonstrated increased pathogen fecundity at elevated CO2 (Hibberd et al., 1996a; Klironomos et al., 1997; Chakraborty et al., 2000). Previously we have shown that the 30% larger S. scabra plants at high CO2 (Chakraborty et al., 2000) makes the canopy microclimate more conducive to anthracnose development and an overall increase in fecundity at high CO2 in the current study is a reflection of the altered canopy environment. Of the two isolates, fecundity of CS1571 was significantly higher than SR24 at high CO2 and its fecundity on Seca increased with each cycle. This indicates that high reproductive fitness of CS1571 is an important component of its high aggressiveness. Increasing fecundity, together with a favorable microclimate, could accelerate pathogen evolution under a changing climate. By using a constant number of spores to initiate each sequential infection we have ignored the effect of fecundity on aggressiveness. Field studies under free to air CO2 enrichment are necessary to determine whether strains with increased aggressiveness and large population size can quickly overcome host resistance under elevated CO2.
This work was supported by the Australian Rural Industries Research and Development Corporation, the Australian Centre for International Agricultural Research and CSIRO. We thank Peter Wilson and Ross Perrott for technical assistance.