The complex relationship between disease resistance and yield in crops.

In plants, growth and defence are controlled by many molecular pathways that are antagonistic to one another. This results in a 'growth-defence trade-off', where plants temporarily reduce growth in response to pests or diseases. Due to this antagonism, genetic variants that improve resistance often reduce growth and vice versa. Therefore, in natural populations, the most disease resistant individuals are often the slowest growing. In crops, slow growth may translate into a yield penalty, but resistance is essential for protecting yield in the presence of disease. Therefore, plant breeders must balance these traits to ensure optimal yield potential and yield stability. In crops, both qualitative and quantitative disease resistance are often linked with genetic variants that cause yield penalties, but this is not always the case. Furthermore, both crop yield and disease resistance are complex traits influenced by many aspects of the plant's physiology, morphology and environment, and the relationship between the molecular growth-defence trade-off and disease resistance-yield antagonism is not well-understood. In this article, we highlight research from the last 2 years on the molecular mechanistic basis of the antagonism between defence and growth. We then discuss the interaction between disease resistance and crop yield from a breeding perspective, outlining the complexity and nuances of this relationship and where research can aid practical methods for simultaneous improvement of yield potential and disease resistance.

Genome-wide identification of Sclerotinia sclerotiorum small RNAs and their endogenous targets.

BACKGROUND: Several phytopathogens produce small non-coding RNAs of approximately 18-30 nucleotides (nt) which post-transcriptionally regulate gene expression. Commonly called small RNAs (sRNAs), these small molecules were also reported to be present in the necrotrophic pathogen Sclerotinia sclerotiorum. S. sclerotiorum causes diseases in more than 400 plant species, including the important oilseed crop Brassica napus. sRNAs can further be classified as microRNAs (miRNAs) and short interfering RNAs (siRNAs). Certain miRNAs can activate loci that produce further sRNAs; these secondary sRNA-producing loci are called 'phased siRNA' (PHAS) loci and have only been described in plants. To date, very few studies have characterized sRNAs and their endogenous targets in S. sclerotiorum. RESULTS: We used Illumina sequencing to characterize sRNAs from fungal mycelial mats of S. sclerotiorum spread over B. napus leaves. In total, eight sRNA libraries were prepared from in vitro, 12 h post-inoculation (HPI), and 24 HPI mycelial mat samples. Cluster analysis identified 354 abundant sRNA clusters with reads of more than 100 Reads Per Million (RPM). Differential expression analysis revealed upregulation of 34 and 57 loci at 12 and 24 HPI, respectively, in comparison to in vitro samples. Among these, 25 loci were commonly upregulated. Altogether, 343 endogenous targets were identified from the major RNAs of 25 loci. Almost 88% of these targets were annotated as repeat element genes, while the remaining targets were non-repeat element genes. Fungal degradome reads confirmed cleavage of two transposable elements by one upregulated sRNA. Altogether, 24 milRNA loci were predicted with both mature and milRNA* (star) sequences; these are both criteria associated previously with experimentally verified miRNAs. Degradome sequencing data confirmed the cleavage of 14 targets. These targets were related to repeat element genes, phosphate acetyltransferases, RNA-binding factor, and exchange factor. A PHAS gene prediction tool identified 26 possible phased interfering loci with 147 phasiRNAs from the S. sclerotiorum genome, suggesting this pathogen might produce sRNAs that function similarly to miRNAs in higher eukaryotes. CONCLUSIONS: Our results provide new insights into sRNA populations and add a new resource for the study of sRNAs in S. sclerotiorum.

Association Mapping Combined with Whole Genome Sequencing Data Reveals Candidate Causal Variants for Sclerotinia Stem Rot Resistance in Brassica napus.

Canola (Brassica napus) yield can be significantly reduced by the disease sclerotinia stem rot (SSR), which is caused by Sclerotinia sclerotiorum, a necrotrophic fungal pathogen with an unusually large host range. Breeding cultivars that are physiologically resistant to SSR is desirable to enhance crop productivity. However, the development of resistant varieties has proved challenging due to the highly polygenic nature of S. sclerotiorum resistance. Here, we identified regions of the B. napus genome associated with SSR resistance using data from a previous study by association mapping. We then validated their contribution to resistance in a follow-up screen. This follow-up screen also confirmed high levels of SSR resistance in several genotypes from the previous study. Using publicly available whole genome sequencing data for a panel of 83 B. napus genotypes, we identified nonsynonymous polymorphisms linked to the SSR resistance loci. A qPCR analysis showed that two of the genes containing these polymorphisms were transcriptionally responsive to S. sclerotiorum infection. In addition, we provide evidence that homologues of three of the candidate genes contribute to resistance in the model Brassicaceae species Arabidopsis thaliana. The identification of resistant germplasm and candidate genomic loci associated with resistance are important findings that can be exploited by breeders to improve the genetic resistance of canola varieties.

Impacts of fludioxonil resistance on global gene expression in the necrotrophic fungal plant pathogen Sclerotinia sclerotiorum.

BACKGROUND: The fungicide fludioxonil over-stimulates the fungal response to osmotic stress, leading to over-accumulation of glycerol and hyphal swelling and bursting. Fludioxonil-resistant fungal strains that are null-mutants for osmotic stress response genes are easily generated through continual sub-culturing on sub-lethal fungicide doses. Using this approach combined with RNA sequencing, we aimed to characterise the effects of mutations in osmotic stress response genes on the transcriptional profile of the important agricultural pathogen Sclerotinia sclerotiorum under standard laboratory conditions. Our objective was to understand the impact of disruption of the osmotic stress response on the global transcriptional regulatory network in an important agricultural pathogen. RESULTS: We generated two fludioxonil-resistant S. sclerotiorum strains, which exhibited growth defects and hypersensitivity to osmotic stressors. Both had missense mutations in the homologue of the Neurospora crassa osmosensing two component histidine kinase gene OS1, and one had a disruptive in-frame deletion in a non-associated gene. RNA sequencing showed that both strains together differentially expressed 269 genes relative to the parent during growth in liquid broth. Of these, 185 (69%) were differentially expressed in both strains in the same direction, indicating similar effects of the different point mutations in OS1 on the transcriptome. Among these genes were numerous transmembrane transporters and secondary metabolite biosynthetic genes. CONCLUSIONS: Our study is an initial investigation into the kinds of processes regulated through the osmotic stress pathway in S. sclerotiorum. It highlights a possible link between secondary metabolism and osmotic stress signalling, which could be followed up in future studies.

Analysis of differentially expressed Sclerotinia sclerotiorum genes during the interaction with moderately resistant and highly susceptible chickpea lines.

BACKGROUND: Sclerotinia sclerotiorum, the cause of Sclerotinia stem rot (SSR), is a host generalist necrotrophic fungus that can cause major yield losses in chickpea (Cicer arietinum) production. This study used RNA sequencing to conduct a time course transcriptional analysis of S. sclerotiorum gene expression during chickpea infection. It explores pathogenicity and developmental factors employed by S. sclerotiorum during interaction with chickpea. RESULTS: During infection of moderately resistant (PBA HatTrick) and highly susceptible chickpea (Kyabra) lines, 9491 and 10,487 S. sclerotiorum genes, respectively, were significantly differentially expressed relative to in vitro. Analysis of the upregulated genes revealed enrichment of Gene Ontology biological processes, such as oxidation-reduction process, metabolic process, carbohydrate metabolic process, response to stimulus, and signal transduction. Several gene functional categories were upregulated in planta, including carbohydrate-active enzymes, secondary metabolite biosynthesis clusters, transcription factors and candidate secreted effectors. Differences in expression of four S. sclerotiorum genes on varieties with different levels of susceptibility were also observed. CONCLUSION: These findings provide a framework for a better understanding of S. sclerotiorum interactions with hosts of varying susceptibility levels. Here, we report for the first time on the S. sclerotiorum transcriptome during chickpea infection, which could be important for further studies on this pathogen's molecular biology.

fIdentification of B. napus small RNAs responsive to infection by a necrotrophic pathogen.

BACKGROUND: Small RNAs are short non-coding RNAs that are key gene regulators controlling various biological processes in eukaryotes. Plants may regulate discrete sets of sRNAs in response to pathogen attack. Sclerotinia sclerotiorum is an economically important pathogen affecting hundreds of plant species, including the economically important oilseed B. napus. However, there are limited studies on how regulation of sRNAs occurs in the S. sclerotiorum and B. napus pathosystem. RESULTS: We identified different classes of sRNAs from B. napus using high throughput sequencing of replicated mock and infected samples at 24 h post-inoculation (HPI). Overall, 3999 sRNA loci were highly expressed, of which 730 were significantly upregulated during infection. These 730 up-regulated sRNAs targeted 64 genes, including disease resistance proteins and transcriptional regulators. A total of 73 conserved miRNA families were identified in our dataset. Degradome sequencing identified 2124 cleaved mRNA products from these miRNAs from combined mock and infected samples. Among these, 50 genes were specific to infection. Altogether, 20 conserved miRNAs were differentially expressed and 8 transcripts were cleaved by the differentially expressed miRNAs miR159, miR5139, and miR390, suggesting they may have a role in the S. sclerotiorum response. A miR1885-triggered disease resistance gene-derived secondary sRNA locus was also identified and verified with degradome sequencing. We also found further evidence for silencing of a plant immunity related ethylene response factor gene by a novel sRNA using 5'-RACE and RT-qPCR. CONCLUSIONS: The findings in this study expand the framework for understanding the molecular mechanisms of the S. sclerotiorum and B. napus pathosystem at the sRNA level.

Identification of Sources of Sclerotinia sclerotiorum Resistance in a Collection of Wild Cicer Germplasm.

Sclerotinia sclerotiorum is an important fungal pathogen of chickpea (Cicer arietinum L.), and it can cause yield losses up to 100%. The wild progenitors are much more diverse than domesticated chickpea, and this study describes how this relates to S. sclerotiorum resistance. Initially, the pathogenicity of nine Australian S. sclerotiorum isolates was examined on three Cicer lines to develop a robust phenotyping assay, and significant differences in isolate aggressiveness were identified with six isolates being classed as highly aggressive and three as moderately aggressive. We identified two S. sclerotiorum isolates, CU8.20 and CU10.12, to be highly aggressive and moderately aggressive, respectively. A subsequent phenotyping assay was conducted using the two isolates to evaluate 86 wild Cicer accessions (Cicer reticulatum and Cicer echinospermum) and two C. arietinum varieties for resistance to S. sclerotiorum. A subset of 12 genotypes was further evaluated, and subsequently, two wild Cicer accessions with consistently high levels of resistance to S. sclerotiorum were examined using the initially characterized nine isolates. Wild Cicer accessions Karab_084 and Deste_063 demonstrated consistent partial resistance to S. sclerotiorum. There were significant differences in responses to S. sclerotiorum across wild Cicer collection sites. The Cermik, Karabahce, and Destek sites' responses to the aggressive isolate CU8.20 ranged from resistant to susceptible, highlighting an interaction between isolate genotype and chickpea collection site for sclerotinia stem rot resistance. This is the first evidence of partial stem resistance identified in wild Cicer germplasm, which can be adopted in chickpea breeding programs to enhance S. sclerotiorum resistance in future chickpea varieties.

A detailed in silico analysis of secondary metabolite biosynthesis clusters in the genome of the broad host range plant pathogenic fungus Sclerotinia sclerotiorum.

BACKGROUND: The broad host range pathogen Sclerotinia sclerotiorum infects over 400 plant species and causes substantial yield losses in crops worldwide. Secondary metabolites are known to play important roles in the virulence of plant pathogens, but little is known about the secondary metabolite repertoire of S. sclerotiorum. In this study, we predicted secondary metabolite biosynthetic gene clusters in the genome of S. sclerotiorum and analysed their expression during infection of Brassica napus using an existing transcriptome data set. We also investigated their sequence diversity among a panel of 25 previously published S. sclerotiorum isolate genomes. RESULTS: We identified 80 putative secondary metabolite clusters. Over half of the clusters contained at least three transcriptionally coregulated genes. Comparative genomics revealed clusters homologous to clusters in the closely related plant pathogen Botrytis cinerea for production of carotenoids, hydroxamate siderophores, DHN melanin and botcinic acid. We also identified putative phytotoxin clusters that can potentially produce the polyketide sclerin and an epipolythiodioxopiperazine. Secondary metabolite clusters were enriched in subtelomeric genomic regions, and those containing paralogues showed a particularly strong association with repeats. The positional bias we identified was borne out by intraspecific comparisons that revealed putative secondary metabolite genes suffered more presence / absence polymorphisms and exhibited a significantly higher sequence diversity than other genes. CONCLUSIONS: These data suggest that S. sclerotiorum produces numerous secondary metabolites during plant infection and that their gene clusters undergo enhanced rates of mutation, duplication and recombination in subtelomeric regions. The microevolutionary regimes leading to S. sclerotiorum secondary metabolite diversity have yet to be elucidated. Several potential phytotoxins documented in this study provide the basis for future functional analyses.

Analysis of cytochrome b(5) reductase-mediated metabolism in the phytopathogenic fungus Zymoseptoria tritici reveals novel functionalities implicated in virulence.

Septoria tritici blotch (STB) caused by the Ascomycete fungus Zymoseptoria tritici is one of the most economically damaging diseases of wheat worldwide. Z. tritici is currently a major target for agricultural fungicides, especially in temperate regions where it is most prevalent. Many fungicides target electron transfer enzymes because these are often important for cell function. Therefore characterisation of genes encoding such enzymes may be important for the development of novel disease intervention strategies. Microsomal cytochrome b5 reductases (CBRs) are an important family of electron transfer proteins which in eukaryotes are involved in the biosynthesis of fatty acids and complex lipids including sphingolipids and sterols. Unlike the model yeast Saccharomyces cerevisiae which possesses only one microsomal CBR, the fully sequenced genome of Z. tritici bears three possible microsomal CBRs. RNA sequencing analysis revealed that ZtCBR1 is the most highly expressed of these genes under all in vitro and in planta conditions tested, therefore ΔZtCBR1 mutant strains were generated through targeted gene disruption. These strains exhibited delayed disease symptoms on wheat leaves and severely limited asexual sporulation. ΔZtCBR1 strains also exhibited aberrant spore morphology and hyphal growth in vitro. These defects coincided with alterations in fatty acid, sphingolipid and sterol biosynthesis observed through GC-MS and HPLC analyses. Data is presented which suggests that Z. tritici may use ZtCBR1 as an additional electron donor for key steps in ergosterol biosynthesis, one of which is targeted by azole fungicides. Our study reports the first functional characterisation of CBR gene family members in a plant pathogenic filamentous fungus. This also represents the first direct observation of CBR functional ablation impacting upon fungal sterol biosynthesis.

Till death do us pair: Co-evolution of plant-necrotroph interactions.

Plants use programmed cell death as a potent defense response against biotrophic pathogens that require living host cells to thrive. However, cell death can promote infection by necrotrophic pathogens. This discrepancy creates specific co-evolutionary dynamics in the interaction between plants and necrotrophs. Necrotrophic pathogens produce diverse cell death-inducing effectors that act redundantly on several plant targets and sometimes suppress plant immune responses as an additional function. Plants use surface receptors that recognize necrotrophic effectors to increase quantitative disease resistance, some of which evolved independently in several plant lineages. Co-evolution has shaped molecular mechanisms involved in plant-necrotroph interactions into robust systems, relying on degenerate and multifunctional modules, general-purpose components, and compartmentalized functioning.

Surface frustration re-patterning underlies the structural landscape and evolvability of fungal orphan candidate effectors.

Pathogens secrete effector proteins to subvert host physiology and cause disease. Effectors are engaged in a molecular arms race with the host resulting in conflicting evolutionary constraints to manipulate host cells without triggering immune responses. The molecular mechanisms allowing effectors to be at the same time robust and evolvable remain largely enigmatic. Here, we show that 62 conserved structure-related families encompass the majority of fungal orphan effector candidates in the Pezizomycotina subphylum. These effectors diversified through changes in patterns of thermodynamic frustration at surface residues. The underlying mutations tended to increase the robustness of the overall effector protein structure while switching potential binding interfaces. This mechanism could explain how conserved effector families maintained biological activity over long evolutionary timespans in different host environments and provides a model for the emergence of sequence-unrelated effector families with conserved structures.

The broad host range pathogen Sclerotinia sclerotiorum produces multiple effector proteins that induce host cell death intracellularly.

Sclerotinia sclerotiorum is a broad host range necrotrophic fungal pathogen, which causes disease on many economically important crop species. S. sclerotiorum has been shown to secrete small effector proteins to kill host cells and acquire nutrients. We set out to discover novel necrosis-inducing effectors and characterize their activity using transient expression in Nicotiana benthamiana leaves. Five intracellular necrosis-inducing effectors were identified with differing host subcellular localization patterns, which were named intracellular necrosis-inducing effector 1-5 (SsINE1-5). We show for the first time a broad host range pathogen effector, SsINE1, that uses an RxLR-like motif to enter host cells. Furthermore, we provide preliminary evidence that SsINE5 induces necrosis via an NLR protein. All five of the identified effectors are highly conserved in globally sourced S. sclerotiorum isolates. Taken together, these results advance our understanding of the virulence mechanisms employed by S. sclerotiorum and reveal potential avenues for enhancing genetic resistance to this damaging fungal pathogen.

Diversity of fatty acid biosynthesis genes across the soybean pangenome.

Soybean (Glycine max) is a major crop that contributes more than half of global oilseed production. Much research has been directed towards improvement of the fatty acid profile of soybean seeds through marker assisted breeding. Recently published soybean pangenomes, based on thousands of soybean lines, provide an opportunity to identify new alleles that may be involved in fatty acid biosynthesis. In this study, we identify fatty acid biosynthesis genes in soybean pangenomes based on sequence identity with known genes and examine their sequence diversity across diverse soybean collections. We find three possible instances of a gene missing in wild soybean, including FAD8 and FAD2-2D, which may be involved in oleic and linoleic acid desaturation, respectively, although we recommend follow-up research to verify the absence of these genes. More than half of the 53 fatty acid biosynthesis genes identified contained missense variants, including one linked with a previously identified QTL for oil quality. These variants were present in multiple studies based on either short read mappings or alignment of reference grade genomes. Missense variants were found in previously characterized genes including FAD2-1A and FAD2-1B, both of which are involved in desaturation of oleic acid, as well as uncharacterized candidate fatty acid biosynthesis genes. We find that the frequency of missense alleles in fatty acid biosynthesis genes has been reduced significantly more than the global average frequency of missense mutations during domestication, and missense variation in some genes is near absent in modern cultivars. This could be due to the selection for fatty acid profiles in seed, though future work should be conducted towards understanding the phenotypic impacts of these variants.

The evolutionary and molecular features of the broad-host-range plant pathogen Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a pathogenic fungus that infects hundreds of plant species, including many of the world's most important crops. Key features of S. sclerotiorum include its extraordinary host range, preference for dicotyledonous plants, relatively slow evolution, and production of protein effectors that are active in multiple host species. Plant resistance to this pathogen is highly complex, typically involving numerous polymorphisms with infinitesimally small effects, which makes resistance breeding a major challenge. Due to its economic significance, S. sclerotiorum has been subjected to a large amount of molecular and evolutionary research. In this updated pathogen profile, we review the evolutionary and molecular features of S. sclerotiorum and discuss avenues for future research into this important species.

Identification of Sclerotinia stem rot resistance quantitative trait loci in a chickpea (Cicer arietinum) recombinant inbred line population.

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum , is one of the most economically devastating diseases in chickpea (Cicer arietinum L.). No complete resistance is available in chickpea to this disease, and the inheritance of partial resistance is not understood. Two hundred F7 recombinant inbred lines (RILs) derived from a cross between a partially resistant variety PBA HatTrick, and a highly susceptible variety Kyabra were characterised for their responses to SSR inoculation. Quantitative trait locus (QTL) analysis was conducted for the area under the disease progress curve (AUDPC) after RIL infection with S. sclerotiorum . Four QTLs on chromosomes, Ca4 (qSSR4-1, qSSR4-2), Ca6 (qSSR6-1) and Ca7 (qSSR7-1), individually accounted for between 4.2 and 15.8% of the total estimated phenotypic variation for the response to SSR inoculation. Candidate genes located in these QTL regions are predicted to be involved in a wide range of processes, including phenylpropanoid biosynthesis, plant-pathogen interaction, and plant hormone signal transduction. This is the first study investigating the inheritance of resistance to S. sclerotiorum in chickpea. Markers associated with the identified QTLs could be employed for marker-assisted selection in chickpea breeding.

Small RNA Analyses of a Ceratobasidium Isolate Infected with Three Endornaviruses.

Isolates of three endornavirus species were identified co-infecting an unidentified species of Ceratobasidium, itself identified as a symbiont from within the roots of a wild plant of the terrestrial orchid Pterostylis vittata in Western Australia. Isogenic lines of the fungal isolate lacking all three mycoviruses were derived from the virus-infected isolate. To observe how presence of endornaviruses influenced gene expression in the fungal host, we sequenced fungus-derived small RNA species from the virus-infected and virus-free isogenic lines and compared them. The presence of mycoviruses influenced expression of small RNAs. Of the 3272 fungus-derived small RNA species identified, the expression of 9.1% (300 of 3272) of them were up-regulated, and 0.6% (18 of 3272) were down-regulated in the presence of the viruses. Fourteen novel micro-RNA-like RNAs (Cer-milRNAs) were predicted. Gene target prediction of the differentially expressed Cer-milRNAs was quite ambiguous; however, fungal genes involved in transcriptional regulation, catalysis, molecular binding, and metabolic activities such as gene expression, DNA metabolic processes and regulation activities were differentially expressed in the presence of the mycoviruses.

Positive Selection of Transcription Factors Is a Prominent Feature of the Evolution of a Plant Pathogenic Genus Originating in the Miocene.

Tests based on the dN/dS statistic are used to identify positive selection of nonsynonymous polymorphisms. Using these tests on alignments of all orthologs from related species can provide insights into which gene categories have been most frequently positively selected. However, longer alignments have more power to detect positive selection, creating a detection bias that could create misleading results from functional enrichment tests. Most studies of positive selection in plant pathogens focus on genes with specific virulence functions, with little emphasis on broader molecular processes. Furthermore, no studies in plant pathogens have accounted for detection bias due to alignment length when performing functional enrichment tests. To address these research gaps, we analyze 12 genomes of the phytopathogenic fungal genus Botrytis, including two sequenced in this study. To establish a temporal context, we estimated fossil-calibrated divergence times for the genus. We find that Botrytis likely originated 16-18 Ma in the Miocene and underwent continuous radiation ending in the Pliocene. An untargeted scan of Botrytis single-copy orthologs for positive selection with three different statistical tests uncovered evidence for positive selection among proteases, signaling proteins, CAZymes, and secreted proteins. There was also a strong overrepresentation of transcription factors among positively selected genes. This overrepresentation was still apparent after two complementary controls for detection bias due to sequence length. Positively selected sites were depleted within DNA-binding domains, suggesting changes in transcriptional responses to internal and external cues or protein-protein interactions have undergone positive selection more frequently than changes in promoter fidelity.

Modeling first order additive × additive epistasis improves accuracy of genomic prediction for sclerotinia stem rot resistance in canola.

The fungus Sclerotinia sclerotiorum infects hundreds of plant species including many crops. Resistance to this pathogen in canola (Brassica napus L. subsp. napus) is controlled by numerous quantitative trait loci (QTL). For such polygenic traits, genomic prediction may be useful for breeding as it can capture many QTL at once while also considering nonadditive genetic effects. Here, we test application of common regression models to genomic prediction of S. sclerotiorum resistance in canola in a diverse panel of 218 plants genotyped at 24,634 loci. Disease resistance was scored by infection with an aggressive isolate and monitoring over 3 wk. We found that including first-order additive × additive epistasis in linear mixed models (LMMs) improved accuracy of breeding value estimation between 3 and 40%, depending on method of assessment, and correlation between phenotypes and predicted total genetic values by 14%. Bayesian models performed similarly to or worse than genomic relationship matrix-based models for estimating breeding values or overall phenotypes from genetic values. Bayesian ridge regression, which is most similar to the genomic relationship matrix-based approach in the amount of shrinkage it applies to marker effects, was the most accurate of this family of models. This confirms several studies indicating the highly polygenic nature of sclerotinia stem rot resistance. Overall, our results highlight the use of simple epistasis terms for prediction of breeding values and total genetic values for a complex disease resistance phenotype in canola.

Bioinformatic Detection of Positive Selection Pressure in Plant Pathogens: The Neutral Theory of Molecular Sequence Evolution in Action.

The genomes of plant pathogenic fungi and oomycetes are often exposed to strong positive selection pressure. During speciation, shifts in host range and preference can lead to major adaptive changes. Furthermore, evolution of total host resistance to most isolates can force rapid evolutionary changes in host-specific pathogens. Crop pathogens are subjected to particularly intense selective pressures from monocultures and fungicides. Detection of the footprints of positive selection in plant pathogen genomes is a worthwhile endeavor as it aids understanding of the fundamental biology of these important organisms. There are two main classes of test for detection of positively selected alleles. Tests based on the ratio of non-synonymous to synonymous substitutions per site detect the footprints of multiple fixation events between divergent lineages. Thus, they are well-suited to the study of ancient adaptation events spanning speciations. On the other hand, tests that scan genomes for local fluctuations in allelic diversity within populations are suitable for detection of recent positive selection in populations. In this review, I briefly describe some of the more widely used tests of positive selection and the theory underlying them. I then discuss various examples of their application to plant pathogen genomes, emphasizing the types of genes that are associated with signatures of positive selection. I conclude with a discussion of the practicality of such tests for identification of pathogen genes of interest and the important features of pathogen ecology that must be taken into account for accurate interpretation.