Effects of planting density and the composition of wheat cultivar mixtures on stripe rust: an analysis taking into account limits to the replication of controls

ABSTRACT The effect of plant density on disease is not well understood in populations of a single host plant genotype and has been studied even less in mixtures of host genotypes. We performed an experiment to evaluate the effect of wheat planting density on infection by Puccinia striiformis in experimental plots with a single wheat genotype and in plots with two genotypes making up a range of frequencies. Stripe rust severity in single-genotype plots increased with planting density in 1997 but decreased with planting density in 1998. Disease in host mixtures was compared to the weighted mean of disease levels in the corresponding single-genotype plots. The design of the field experiment included limited replication of these reference treatments (that is, there was not a unique pair of single-genotype plots for each mixture plot); therefore, we devised an analysis based on collapsing the data into independent mean observations. Disease reduction due to host diversity was less when one genotype predominated than when both host genotypes were present at nearly equal frequencies. The greatest mean host-diversity effect for reduced disease was at the intermediate planting density of 250 seeds per m(2).     

Influence of spatial structure on pathogen colonization and extinction: a test using an experimental metapopulation

The Linum marginale–Melampsora lini plant–pathogen interaction has been studied extensively with regard to its epidemiology and population genetic structure (host resistance and pathogen virulence) in a natural metapopulation. In this study, this system was used in an experimental metapopulation approach to investigate explicitly how the distance (degree of isolation) between local population patches influences disease dynamics within a growing season, as well as the genetic structure of pathogen populations through stochastic colonization and extinction processes. The experimental design centred on four replicate sets of populations, within which patches were spaced at increasingly greater distances apart. Each patch consisted of an identical set of host and pathogen genotypes, with each pathogen genotype having the ability to attack only one of four host-resistance types. Over the 2 years of the experiment, the results showed clear 'boom-and-bust' epidemic patterns, with the strongest determinant of disease dynamics within a growing season being the identity of particular host–pathogen genotypic combinations. However, there were also significant effects of spatial structure, in that more isolated patches tended to exhibit lower levels of disease during epidemic peaks than patches that were close together. Extinction of pathogen genotypes from individual populations was positively related to the severity of disease during preceding epidemic peaks, but negatively related to the level of disease present at the final census prior to overwintering. The probability of recolonization of pathotypes into populations during the second growing season was most strongly related to the distance to the nearest neighbouring source population in which a given pathotype was present. Overall, these results highlight the importance of spatial scale in influencing the numerical and genetical dynamics of pathogen populations.     

Evolution of gene-for-gene systems in metapopulations: the effect of spatial scale of host and pathogen dispersal

The concept of gene-for-gene coevolution is a major model for research on disease resistance in crop plants. However, few theoretical or empirical studies have examined such systems in natural situations, and as a consequence, there is little knowledge of how spatial effects are likely to influence the evolution of host resistance and pathogen virulence in gene-for-gene interactions. In this work, a simulation approach was used to investigate the epidemiological and genetic consequences of varying host and pathogen dispersal in metapopulation situations. The results demonstrate clear impacts of dispersal distance on the total number of host and pathogen genotypes that are maintained, as well as on genetic variation at individual host resistance and pathogen virulence loci. Several other important results also emerged from this study. In contrast to the predictions of many earlier nonspatial models, so-called 'super-races' of pathogens do not always evolve and dominate, indicating that it is not necessary to assume costs of resistance or virulence to maintain high levels of polymorphism in biologically realistic situations. The rate of evolution of both resistance and virulence depend on the scale of dispersal, with greater mixing (as a function of dispersal scale) resulting in a faster approach to a dynamic endpoint. The model in this paper also predicts that, despite the greater total genotypic diversity of pathogens across the metapopulation, variation in host resistance will generally be greater than variation in pathogen virulence within local populations.     

Ecological Genomics and Epidemiology

The huge amount of genomic data now becoming available offers both opportunities and challenges for epidemiologists. In this “preview” of likely developments as the field of ecological genomics evolves and merges with epidemiology, we discuss how epidemiology can use new information about genetic sequences and gene expression to form predictions about epidemic features and outcomes and for understanding host resistance and pathogen evolution. DNA sequencing is now complete for some hosts and several pathogens. Microarrays make it possible to measure gene expression simultaneously for thousands of genes. These tools will contribute to plant disease epidemiology by providing information about which resistance or pathogenicity genes are present in individuals and populations, what genes other than those directly involved in resistance and virulence are important in epidemics, the role of the phenotypic status of hosts and pathogens, and the role of the status of the environmental metagenome. Conversely, models of group dynamics supplied by population biology and ecology may be used to interpret gene expression within individual organisms and in populations of organisms. Genomic tools have great potential for improving understanding of resistance gene evolution and the durability of resistance. For example, DNA sequence analysis can be used to evaluate whether an arms race model of co-evolution is supported. Finally, new genomic tools will make it possible to consider the landscape ecology of epidemics in terms of host resistance both as determined by genotype and as expressed in host phenotypes in response to the biotic and abiotic environment. Host phenotype mixtures can be modeled and evaluated, with epidemiological predictions based on phenotypic characteristics such as physiological age and status in terms of induced systemic resistance or systemic acquired resistance.     

Selection on Erysiphe graminis in pure and mixed stands of barley

The response of populations of Erysiphe graminis f. sp. hordei to selection by pure and mixed stands of three spring barley cultivars was studied in two field trials. The range of virulence of the pathogen genotypes selected in mixed host stands was dependent on the relative fitness of each genotype over all hosts. Unnecessary virulences were rapidly selected against on some hosts, but were less deleterious or favoured on others. In general there was selection for widely adapted pathogen genotypes in mixed host populations but this selection for flexibility limited the abilitytions in the absolute size of the pathogen population in host mixtures reduced the absolute frequencies of pathogen genotypes with combined virulences in comparison with those in pure stands. It is argued that host mixtures are therefore unlikely to favour rapid pathogen evolution towards races which are both widely adapted and highly virulent on all component cultivars which they can infect his definition docs not conform with conventional usage in population genetics.     

Host diversity can reduce potato late blight severity for focal and general patterns of primary inoculum

ABSTRACT The use of host diversity as a tool for management of potato late blight has not been viewed as promising in the past. But the increasing importance of late blight internationally has brought new consideration to all potential management tools. We studied the effect of host diversity on epidemics of potato late blight in Oregon, where there was little outside inoculum. The experimental system consisted of susceptible potato cv. Red LaSoda and a highly resistant breeding selection, inoculated with local isolates of US-8 Phytophthora infestans. Potatoes were grown in single-genotype plots and also in a mixture of 10 susceptible and 26 resistant potato plants. Half of the plots received inoculation evenly throughout the plot (general inoculation) and half received an equal quantity of inoculum in only one corner of the plot (focal inoculation). The area under the disease progress curve (AUDPC) was greater in single genotype stands of susceptible cv. Red LaSoda inoculated throughout the plot than with stands inoculated in one focus. The host-diversity effect on foliar late blight was significant in both years of the investigation; the AUDPC was reduced by an average of 37% in 1997 and 36% in 1998, compared with the mean disease level for the potato genotypes grown separately. Though the evidence for influence of inoculum pattern on host-diversity effects was weak (P = 0.15), in both years there was a trend toward greater host-diversity effects for general inoculation. Statistical significance of host-diversity effects on tuber yield and blight were found only in one of the two years. In that year, tuber yield from both the resistant and susceptible cultivar was increased in mixtures compared with single genotype stands and tuber blight was decreased in mixtures for susceptible cv. Red LaSoda.     

Phenotypic and molecular genetic characterization indicate no major race‐specific interactions between Xanthomonas translucens pv. graminis and Lolium multiflorum

Bacterial wilt of forage grasses, caused by the pathogen Xanthomonas translucens pv. graminis (Xtg), is a major disease of forage grasses such as Italian ryegrass (Lolium multiflorum). The plant genotype‐bacterial isolate interaction was analysed to elucidate the existence of race‐specific responses and to assist the identification of plant disease resistance genes. In a greenhouse experiment, 62 selected plant genotypes were artificially inoculated with six different bacterial isolates. Significant differences in resistance were observed among L. multiflorum genotypes (P < 0·001) and in virulence (intensity of disease symptoms) among Xtg isolates (P < 0·001) using the area under the disease progress curve (AUDPC). No significant genotype‐isolate interaction (P > 0·05) could be observed using linear regression modelling. However, additive main effects and multiplicative interaction effects (ammi ) analysis revealed five genotypes which did not cluster close to the origin of the biplot, indicating specific interactions between these genotypes and some bacterial isolates. Simple sequence repeat (SSR) markers were used to identify marker‐resistance associations using the same plant genotypes and bacterial isolates. The SSR marker NFA027 located on linkage group (LG) 5 was significantly associated with bacterial wilt resistance across all six bacterial isolates and explained up to 37·4% of the total variance of AUDPC values. Neither the inoculation experiment nor the SSR analyses revealed major host genotype‐pathogen isolate interactions, thus suggesting that Xtg resistance, observed so far, is effective across a broad range of different bacterial isolates and plant genotypes.     

The effects of host diversity and other management components on epidemics of potato late blight in the humid highland tropics

ABSTRACT A field study at three highland sites near Quito, Ecuador, was conducted to determine whether host-diversity effects on potato late blight would be as important as recently found in studies conducted in temperate areas. We compared three potato mixtures and use of mixtures in combination with different planting densities and two fungicide regimes. Treatment comparisons were made by absolute and relative measures of host-diversity effects and incorporating a truncated area under the disease progress curve as a means of standardizing comparisons across sites. Potato-faba intercrops consisting of only 10% potato provided an estimate of the effects of dilution of susceptible host tissue. Host-diversity effects were very different across study sites, with a large host-diversity effect for reduced disease only at the site most distant from commercial potato production. Planting density had little influence on host-diversity effects or on late blight in single-genotype stands. Fungicide use in combination with potato mixtures enhanced a host-diversity effect for reduced late blight. Potato-faba intercrops produced only a small decrease in potato late blight. Effects of host diversity on yield were variable, with the greatest increase in yield for mixtures treated with fungicides at the site most distant from commercial potato production. The effects of host diversity on late blight severity may be less consistent in the tropical highlands than in the temperate zone, but can contribute to integrated disease management.     

Effect of resistance variation in a natural plant host–pathogen metapopulation on disease dynamics

Existing theory suggests that increasing the diversity of resistance and virulence types in host–pathogen interactions will result in qualitative shifts in spatial and temporal dynamics, and greater among-population asynchrony in disease dynamics and prevalence. Here, data are presented from a biologically realistic metapopulation model of gene-for-gene interactions that indicate that population level variation in resistance diversity will be negatively associated with disease prevalence (fraction of individuals infected). The model also predicts that disease incidence (presence/absence) will be positively related to total resistance diversity across the metapopulation, because high resistance diversity also selects for more virulent pathogens. These results are then contrasted with empirical data from a natural host–pathogen system. While the argument that high resistance diversity should generally lead to lower disease levels has been applied extensively in agricultural situations, the connection between genetic diversity, resistance and disease dynamics has never been demonstrated in natural systems. Here, through analysis of multiyear data on disease prevalence in the context of knowledge of resistance variation among host populations in a natural plant host–pathogen metapopulation, the first evidence is provided that observed levels of asynchrony in disease dynamics may indeed be related to resistance structure.     

Evidence for selection pressure from resistant potato genotypes but not from fungicide application within a clonal Phytophthora infestans population

Insight into pathogen population dynamics provides a key input for effective disease management of the potato late blight pathogen Phytophthora infestans. Phytophthora infestans populations vary from genetically complex to more simple with a few clonal lineages. The presence or absence of certain strains of P. infestans may impact the efficacy of fungicides or host resistance. Current evidence indicates that genetically, the Irish populations of P. infestans are relatively simple with a few clonal lineages. In this study, P. infestans populations were genetically characterized based on samples collected at the national centre for potato breeding during the period 2012–16. The dominance of clonal lineages within this P. infestans population was confirmed and the potential selection pressure of fungicide treatment (2013–15) and host resistance (2016) on this clonal P. infestans population was then investigated. It was found that fungicide products did not notably affect the genetic structure of sampled populations relative to samples from untreated control plants. In contrast, samples taken from several resistant potato genotypes were found to be more often of the EU_13_A2 lineage than those taken from control King Edward plants or potato genotypes with low resistance ratings. Resistant potato varieties Sarpo Mira and Bionica, containing characterized R genes, were found to strongly select for EU_13_A2 strains.     

Aggressiveness and its role in the adaptation of plant pathogens

Aggressiveness, the quantitative component of pathogenicity, and its role in the adaptation of plant pathogens are still insufficiently investigated. Using mainly examples of biotrophic and necrotrophic fungal pathogens of cereals and Phytophthora infestans on potato, the empirical knowledge on the nature of aggressiveness components and their evolution in response to host and environment is reviewed. Means of measuring aggressiveness components are considered, as well as the sources of environmental variance in these traits. The adaptive potential of aggressiveness components is evaluated by reviewing evidence for their heritability, as well as for constraints on their evolution, including differential interactions between host and pathogen genotypes and trade-offs between components of pathogenicity. Adaptations of pathogen aggressiveness components to host and environment are analysed, showing that: (i) selection for aggressiveness in pathogen populations can be mediated by climatic parameters; (ii) global population changes or remarkable population structures may be explained by variation in aggressiveness; and (iii) selection for quantitative traits can influence pathogen evolution in agricultural pathosystems and can result in differential adaptation to host cultivars, sometimes leading to erosion of quantitative resistance. Possible links with concepts in evolutionary ecology are suggested.     

Resistance to Leaf Scald Disease Is Associated with Limited Colonization of Sugarcane and Wild Relatives by Xanthomonas albilineans

ABSTRACT A streptomycin- and rifampicin-resistant mutant of Xanthomonas al-bilineans was used to study symptom expression of leaf scald disease (LSD) and colonization of sugarcane (Saccharum spp.) and its wild relatives by this bacterial pathogen. A total of 40 sugarcane cultivars and 15 clones from the Saccharum complex that differed in resistance to LSD were inoculated by a decapitation technique in both field and greenhouse experiments. In the plant crop, disease severity varied between 0 for the most resistant genotypes and 100 for the most susceptible ones. Resistance to LSD was characterized by limited colonization of the host plant by X. albilineans. Although almost all genotypes were colonized by the pathogen, the greatest bacterial population densities were found in the susceptible cultivars. There was a high correlation between disease severity and pathogen population in the apex. Several genotypes exhibited no or slight symptoms even though they were highly colonized in the upper and/or basal nodes of stalks. Two mechanisms, therefore, may play an important role in resistance to LSD: resistance to colonization of the apex, which is characterized by absence of symptoms, and resistance to colonization of the upper and lower parts of the stalk. In contrast, disease severity and pathogen population densities in the first ratoon crop in the field were nil or very low in the stalks, except for the highly susceptible cv. CP68-1026. Sugarcane ratoons, therefore, may recover from the disease after plant cane infection. Nevertheless, because low levels of the pathogen were still detected in some stalks, it is possible that LSD could develop from latent infections if favorable environmental conditions occur.     

Pathogenicity and infectivity of Phytophthora ramorum vary depending on host species,infected plant part,inoculum potential,pathogen genotype,and temperature

A total of 25 ornamental plant species representing 10 families were inoculated using three genotypes, each representing one of the genetic lineages NA1, NA2, and EU1 of the pathogen Phytophthora ramorum. Leaves were inoculated using suspensions with two zoospore concentrations and exposure at three temperatures, while stems were inoculated using agar plugs colonized by mycelia. Susceptibility was determined by measuring either the success of pathogen reisolation or lesion length caused by the pathogen. Infectivity was determined by counting sporangia in washes of inoculated leaves or stems. Results from all three pathogen genotypes combined were used to rank each of the 25 plant species for susceptibility and infectivity, while pooled results per genotype from all 25 hosts combined were employed for a preliminary comparison of pathogenicity and infectivity among genotypes. Statistical analyses showed that leaf results were affected by the concentration of zoospores, temperature, plant host, pathogen genotype, and by the interaction between host and pathogen genotype. Stem results were mostly affected by host and by the interaction between host and pathogen genotype. Hosts ranked differently when looking at the various parameters, and differences in rankings were also significant when comparing stem and leaf results. Differences were identified among the 25 hosts and the three pathogen genotypes for all parameters: results can be used for decision-making regarding regulations or selection of plants to be grown where infestations by P. ramorum are an issue.     

Measures of durability of resistance

ABSTRACT Conventional models for the durability of resistant cultivars focus on the dynamics of the frequency of resistance genes. This leads to a definition of the durability of resistance as the time from introduction of the cultivar to the time when the frequency of the virulence gene reaches a preset threshold. It is questionable whether this is the most appropriate way to measure durability. Here we use a simple epidemiological model to link population dynamics and population genetics to compare three measures of durability: (i) the expected time until invasion of the virulent genotype, by mutation or immigration, and subsequent establishment of a population (T(invasion)); (ii) the virulence frequency related measure of the time for the virulent genotype to take-over the pathogen population ( T(take-over)); and (iii) the additional yield, measured by the additional number of uninfected host growth days (T(additional)). Specifically, we show how the measures of durability are affected by deployment and epidemiological parameters. We use a combination of numerical solution and analytical approximation of a model for the population dynamics of avirulent and virulent genotypes of a pathogen growing in dynamically changing populations of resistant and susceptible cultivars. The three measures of durability are compared. Some consequences of the results for durable resistance in multilines and mixtures and the regional deployment of resistant cultivars are discussed.     

Modelling the interactions between phenology and insecticide resistance genes in the codling moth Cydia pomonella

In the codling moth Cydia pomonella (L), insecticide resistance genes have been associated with pleiotropic effects affecting phenology. In this paper, we investigated whether an increase in the frequency of insecticide resistance in field populations of C pomonella was likely to entail significant divergences in the temporal occurrence of both susceptible and insecticide-resistant individuals. For this purpose, we built a phenological model that provided suitable predictions of the distinct and diverging seasonal evolutions of populations of a susceptible and two insecticide-resistant (at two and three loci) homozygous genotypes of C pomonella. Model simulations for each genotype were further compared with pheromone trap catches recorded in a field insecticide-treated population over an 8-year period (from 1992 to 2000), which reflected the progressive annual increase in the frequency of resistance in southeastern France. We found a significant delay in field adult emergence relative to those predicted by the homozygous susceptible model, and the magnitude of such a delay was positively correlated with increasing frequencies of insecticide resistance in the sampled field population of C pomonella. Adult emergence predicted in the theoretical population that was homozygous for resistance at two loci converged with those recorded in the field during the investigated 8-year period. This suggested that the pleiotropic effects of resistance were likely to result in a significant phenological segregation of insecticide-resistant alleles in the field. The results of this study emphasized the potential for pest populations exposed to chemical selection to evolve qualitatively with respect to phenology. This may raise critical questions regarding the use of phenological modelling as a forecasting tool for appropriate resistance management strategies that would take into account the diverging seasonal evolutions of both insecticide resistance and susceptibility.     

Effects of Wheat Cultivar Mixtures on Epidemic Progression of Septoria Tritici Blotch and Pathogenicity of Mycosphaerella graminicola

ABSTRACT The effects of host genotype mixtures on disease progression and pathogen evolution are not well understood in pathosystems that vary quantitatively for resistance and pathogenicity. We used four mixtures of moderately resistant and susceptible winter wheat cultivars naturally inoculated with Mycosphaerella graminicola to investigate impacts on disease progression in the field, and effects on pathogenicity as assayed by testing isolate populations sampled from the field on greenhousegrown seedlings. Over 3 years, there was a correspondence between the mixtures' disease response and the pathogenicity of isolates sampled from them. In 1998, with a severe epidemic, mixtures were 9.4% less diseased than were their component pure stands (P = 0.0045), and pathogen populations from mixtures caused 27% less disease (P = 0.085) in greenhouse assays than did populations from component pure stands. In 1999, the epidemic was mild, mixtures did not reduce disease severity (P = 0.39), and pathogen populations from mixtures and pure stands did not differ in pathogenicity (P = 0.42). In 2000, epidemic intensity was intermediate, mixture plots were 15.2% more diseased than the mean of component pure stands (P = 0.053), and populations from two of four mixtures were 152 and 156% more pathogenic than the mean of populations from component pure stands (P = 0.043 and 0.059, respectively). Mixture yields were on average 2.4 and 6.2% higher than mean component pure-stand yields in 1999 and 2000, respectively, but the differences were not statistically significant. The ability of mixtures challenged with M. graminicola to suppress disease appears to be inconsistent. In this system, host genotype mixtures evidently do not consistently confer either fitness benefits or liabilities on pathogen populations.     

Diversity and Complexity of Erysiphe graminis f.sp. hordei Collected from Barley Cultivar Mixtures or Barley Plots Treated with a Resistance Elicitor

Powdery mildew populations were analysed to determine the effects of a resistance elicitor and cultivar mixtures on genetic complexity and diversity. Isolations were made from a range of spring barley monocultures and mixtures in a field trial, and characterised for virulence and RAPD profile. In a second trial, isolates were taken from a single mixture from untreated and resistance elicitor-treated areas and from the components of the mixture in monoculture. The mildew population was not only highly heterogeneous for virulence characteristics, but also proved heterogeneous within pathotypes for molecular markers, indicating the major impact of sexual recombination on population structure and the lack of clonal dominance. Various diversity measurements were compared and the value of dissimilarity measurement for revealing genetic distance within a population was highlighted. There was a trend towards increasing complexity as the season progressed, but there was no consistent relationship between cultivar or mixture, disease control treatment, fertiliser treatment, replicate or position in trial, and pathogen genotype. Whilst the resistance elicitor did reduce mildew by 78% in the first trial, and there was no interaction with fertiliser level in its expression, control was substantially less in the second trial. There were no differences between mildew isolates from elicitor and control treatments. It was felt that more effective and consistent resistance elicitors need to be developed before it can be stated that they are unlikely to be eroded by selecting resistant or adapted mildew genotypes.     

Seasonally induced chaotic dynamics and their implications in models of plant disease

Two models of plant disease in crops regulated by a virus disease or by a hyperparasite are introduced. If the host crop is annually harvested or dies back, the pathogen population at the end of each year may fluctuate indefinitely and irregularly in a way which depends very precisely and for ever on the initial populations. This means that even in an environment which is identical every year, the pathogen populations in each year vary enormously and erratically. However, the combinations of pathogen and virus incidence or hyperparasite infection that can occur exhibit very well defined patterns, even if parameters in the model exhibit substantial random annual variation. It is important that pathologists should be aware that population fluctuations may not be caused by environmental fluctuations and may be, in principle, unpredictable by a deterministic model.     

Evidence of early defence to Ascochyta lentis within the recently identified Lens orientalis resistance source ILWL180

In plant–pathogen interactions, strong structural and biochemical barriers may induce a cascade of reactions in planta, leading to host resistance. The kinetic speed and amplitudes of these defence mechanisms may discriminate resistance from susceptibility to necrotrophic fungi. The infection processes of two Ascochyta lentis isolates (FT13037 and F13082) on the recently identified ascochyta blight (AB)‐resistant Lens orientalis genotype ILWL180 and two cultivated genotypes, ILL7537 (resistant) and ILL6002 (susceptible), were assessed. Using histopathological methods, significant differences in early behaviour of the isolates and the subsequent differential defence responses of the hosts were revealed. Irrespective of virulence, both isolates had significantly lower germination, shorter germ tubes and delayed appressorium formation on the resistant genotypes (ILWL180 and ILL7537) compared to the susceptible genotype (ILL6002); furthermore, these were more pronounced on genotype ILWL180 than on genotype ILL7537. Subsequently, host perception of pathogen entry led to the faster accumulation and notably higher amounts of reactive oxygen species and phenolic compounds at the penetration sites of the resistance genotypes ILWL180 and ILL7537. In contrast, genotype ILL6002 responded slowly to the A. lentis infection and reaffirmed previous gross disease symptomology reports as highly susceptible. Interestingly, quantification of H₂O₂ was markedly higher in ILWL180 particularly at 12 h post‐inoculation compared to ILL7537, potentially indicative of its superior resistance capability. Faster recognition of A. lentis is likely to be a major contribution to the superior resistance observed in genotype ILWL180 to the highly aggressive isolates of A. lentis assessed.     

Virulence Genes in Heterodera glycines: Allele Frequencies and Ror Gene Groups Among Field Isolates and Inbred Lines

ABSTRACT Genetic variation in field populations of Heterodera glycines is a key issue for both resistance gene deployment and basic understanding of virulence-gene flow in populations. In this study, we examined phenotypically defined genes for virulence under selection from host resistance. We separated the most common H. glycines genotypes in the United States into two virulence groups, based on their reproductive abilities on the resistant soybean plant introduction (PI) 88788. These groups correspond to previously identified virulence genes in the nematode, as follows: the dominant gene in H. glycines to PI88788, and the recessive genes to PI90763 and Pickett/Peking. Virulence allele frequencies and virulence genotype frequencies of selected field isolates were investigated by testing the host range of single-female-derived lines, which were developed through single-female inoculation on the standard susceptible soybean 'Lee 68'. By comparing virulence genotype frequencies between the original field isolates and their single-female-derived lines, we were able to determine allele frequencies in the field populations. The results suggest that tremendous variation in H. glycines virulence genes exists among field populations. Potential mechanisms of selection which could cause virulence genotype frequency increases are discussed as related to population genetics equilibrium theory.