EVOLUTION AND SPECIATION OF PLANT-PATHOGENIC FUNGI

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0180234
• Project Start Date: Feb 1, 2009
• Project End Date: Jan 31, 2014
• Program Code: [(N/A)]- (N/A)

Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001

Performing Department
Plant Pathology

Non Technical Summary
Speciation mechanisms are poorly understood in almost all biological systems and particularly in plant-pathogenic fungi. Plant pathologists have long recognized the significance of host specificity as a taxonomic character to identify and classify pathogens, but the evolution of host specificity and its role in the speciation process is poorly understood. Ascochyta spp. causing diseases of cool season food legumes and A. alternata on citrus represent ideal model systems with which to study the evolution of pathogenicity, genetics of host specificity and mechanisms of speciation. These pathosystems allow comparison of predominantly sexually (Ascochyta spp.) and asexually (Alternaria spp.) reproducing pathogens and provide insight into speciation processes in fungi with diverse mating systems. The study of speciation and the genetics of host specificity will contribute to our basic knowledge of the evolution of plant pathogenesis, the emergence of disease and the design of more effective and environmentally sound methods of disease control. In addition to improving our understanding of speciation processes in fungi, systematic study of plant-pathogenic fungi at or near the population/speciation interface is important from a taxonomic perspective and has important economic ramifications in terms of regulatory constraints on the movement of pathogens, quarantines, and biosecurity.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	0999	1080	20%
212	1419	1080	80%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1419 - Leguminous vegetables, general/other; 0999 - Citrus, general/other;

Field Of Science
1080 - Genetics;

Keywords

ascochyta, alternaria, cool season food legumes, citrus, host

specificity, fungal speciation, fungal evolution, horizontal gene

transfer, fungal systematics

Goals / Objectives
The objectives of this project are to use the techniques of molecular biology to address questions about the genetics, systematics, evolution and ecology of plant pathogenic fungi. Specific objectives include 1) the development of genomic resources for continued systematic study of Ascochyta and Alternaria, 2) the estimation of robust, multigene phylogenies of Ascochyta and Alternaria species and their host plants to test evolutionary hypotheses, 3) to quantitatively estimate of species boundaries based on transitions in branching rates at the population/species boundary and the cessation of gene flow between sympatric hosts colonized by host-specific and generalist Ascochyta and Alternaria fungi, and 4) determine the genetic control of host specificity and the role that host specificity has played in pathogen evolution and speciation.

Project Methods
Genomic regions for population-level systematics will be developed using a combination of approaches. Ten microsatellite loci will be developed for closely related Ascochyta spp. using standard enrichment and cloning techniques. For Alternaria spp., a whole genome sequencing approach will be pursued. First, we will generate 6X coverage for a single representative species, and then 1X coverage will be generated for each of 6 additional, genetically distinct taxa within each group of taxa and aligned with the primary representative for the group. Homologous, variable regions will be identified among the 6 taxa in each species group and primers specific for each region will be developed for subsequent locus-specific sequencing efforts. Phylogenies for Ascochyta spp. and Alternaria spp. will be estimated using standard maximum likelihood approaches in both frequentist and Bayesian statistical frameworks. Robust multilocus phylogenies will be used to model the boundary of population-level processes (coalescence) and species-level population processes (divergence) to quantitatively define the limits of species. Maximum likelihood transitions of branching rates will provide a criterion for the delimitation of species and allow comparison with species defined based on population genetic criteria (ie. gene flow). Population-scale samples from sympatric hosts will be employed to estimate divergence times and gene flow using divergence population genetics approaches. Methods that capture the genealogical process using coalescent simulations will be used to estimate critical population parameters and direction(s) of gene flow since divergence. Host specificity of Ascochyta spp. will be tested with host inoculations of selected isolates on selected legume hosts. Intrinsic, genetic mating barriers in Ascochyta will be determined by performing crosses of host-specific forms and assaying reproductive fitness through pseudothecial production. Postzygotic mating barriers will be estimated by performing genetic crosses of selected isolates and measuring the parasitic fitness of Ascochyta spp. hybrids in vitro and on plants. Long-term field plots will be established with sympatric legume hosts and used to test for gene flow among host-specific hosts using microsatellite markers.

Progress 02/01/09 to 01/31/14

Outputs
Target Audience: Scientific community Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Project has provided training and professional development for one female post-doctoral scientist How have the results been disseminated to communities of interest? Results have been disseminated via publication in scientific journals, presentations at conferences, and through invited presentations at academic institutions What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Quantitative species recognition in asexual fungi Using a worldwide sample of Alternaria alternata, causal agent of citrus brown spot, the evolutionary histories of five nuclear loci were estimated using coalescent analyses. Species boundaries were estimated using several approaches that incorporate uncertainty in gene genealogies when lineage sorting and non-reciprocal monophyly of gene trees is common. Coalescent analyses revealed three phylogenetic lineages strongly influenced by incomplete lineage sorting and recombination. Divergence of two lineages from the other was strongly supported at all loci. A consensus of species tree estimation methods supported two species of Alternaria causing citrus brown spot worldwide. Based on substitution rates at the endo-polygalacturonase locus, the main divergence event was estimated to have occurred between 1- 12 my before present, predating the human-mediated movement of citrus and associated pathogens out of SE Asia. The number of Alternaria species identified previously as causing brown spot of citrus worldwide using morphological criteria has been overestimated and little support was found for most of these morphospecies using quantitative species recognition approaches. Our research showed that multilocus phylogenetic methods that allow for recombination and incomplete lineage sorting can be useful for the quantitative delimitation of asexual species that are morphologically indistinguishable. Mating system of asexual plant-pathogenic fungi The mode of reproduction of an Alternaria alternata population causing citrus brown spot in central Florida was estimated. Mating type of each isolate was determined, and isolates were sequenced at six unlinked loci. Three genetically distinct sub-populations were identified using network and Bayesian population structure analyses. Results demonstrate that most sub-populations of A. alternata associated with citrus are clonal but some have the ability to extensively recombine through a cryptic sexual cycle and/or parasexual cycle. Although isolates were sampled in close physical proximity, we were able to reject a random mating model using multilocus gametic disequilibrium tests for two sub-populations suggesting that these sub-populations were predominantly asexual. However, three recombination events were identified in these populations, and localized to individuals of opposite mating type, possibly indicating meiotic recombination. In contrast, in the third sub-population, where only one mating type was present, extensive reticulation was evident in network analyses and multilocus gametic disequilibrium tests were consistent with recombination. Recombination among isolates of the same mating type suggests that a non-meiotic mechanism of recombination such as the parasexual cycle may be operating in this sub-population. The level of gene flow detected among sub-populations does not appear to be sufficient to prevent differentiation, and perhaps future speciation, of these sub-populations. Emergence of eucalyptus rust in Brazil We employed microsatellite markers to genotype P. psidii samples from eucalypts and guava plus five additional myrtaceous hosts across a wide geographic range of southeastern Brazil and Uruguay in order to infer the origin of the pathogen on eucalypts. Multiple analyses revealed two major genetic clusters among the sampled isolates, one associated with guava and another associated with eucalypts and three additional hosts. Multilocus genotypes infecting guava differed by multiple mutational steps at eight loci compared to those infecting eucalypts. Approximate Bayesian computation revealed that evolutionary scenarios involving a coalescence event between guava- and eucalypt-associated pathogen populations within the past 1000 years are highly unlikely. None of the analyses supported the hypothesis that eucalypt-infecting P. psidii in Brazil originated via host jump from guava since the introduction of eucalypts to Brazil approximately 185 years ago.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Ozkilinc, H., K.A. Thomas, M. Abang and T.L. Peever. 2014. Reproductive mode of Didymella fabae in Syria. Plant Pathology 63: 000-000 (in press).
• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Kim, W., C.-H. Park, J.-J. Park, H. Akamatsu, T.L. Peever, M. Xian, D.R. Gang, G. Vandermark and W. Chen. 2014. Functional analyses of the diels-alderase gene sol5 of Ascochyta rabiei and Alternaria solani indicate that the phytotoxin solanapyrones are nonessential for pathogenicity. Molecular Plant-Microbe Interactions 27: 000-000 (in press).
• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Carris, L.M. and T.L. Peever. 2014. Anamorphic states of Disciotis, Gyromitra and Morchella identified in the late fall and early winter in the inland Pacific Northwestern U.S.A. Mycologia 104: (in press).
• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Huang, F., Y. Fu, D. Nie, J.E. Stewart, T.L. Peever and H. Li. 2014. Identification of a novel phylogenetic lineage of Alternaria alternata causing citrus brown spot in China. Fungal Biology 118: 000-000 (in press).
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Chilvers, M.I., S. Jones, J. Meleca, T.L. Peever, S. Pethybridge, and F. Hay. 2014. Characterization of mating type genes supports the hypothesis that Stagonosporopsis chrysanthemi is homothallic and provides evidence that Stagonosporopsis tanaceti is heterothallic. Current Genetics 60: 295-302.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Inami, K., T. Kashiwa, M. Kawabe, A. Onokubo-Okabe, N. Ishikawa, E. Rodriguez Perez, T. Hozumi, L. Aragon Caballero, F. Caceres de Baldarrago, M. Jimenez Roco, K.A. Madadi, T.L. Peever, T. Teraoka, M. Kodama and T. Arie. 2014. The tomato wilt fungus Fusarium oxysporum f. sp. lycopersici shares common ancestors with nonpathogenic F. oxysporum isolated from wild tomatoes in the Peruvian Andes. Microbes and Environments 29: 200-210.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Stewart, J.E, L.M. Timmer, C.B. Lawrence, B.M. Pryor and T.L. Peever. 2014. Discord between morphological and phylogenetic species boundaries: Incomplete lineage sorting and recombination results in fuzzy species boundaries in an asexual fungal pathogen. BMC Evolutionary Biology 14: 38 (3 March 2014).
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Njambere, E.N., T.L. Peever, G. Vandermark and W. Chen. 2014. Genotypic variation and population structure of Sclerotinia trifoliorum infecting chickpea in California. Plant Pathology 63: 994-1004.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Scientific community Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? 1 female domestic PhD student trained and graduated, 1 female domestic PhD student supervised, 1 female foreign (Iran) visiting scientist trained How have the results been disseminated to communities of interest? Disseminated to the scientific community via journal publications What do you plan to do during the next reporting period to accomplish the goals? Speciation genes will be localized in the genome and identified using high-throughput, map-based cloning. Cultivated pea (Pisum sativum) will be inoculated with 100 haploid ascospore progeny from a cross of Ascochyta spp. differing in host response to score disease phenotype. Differential host specificities between the parents of several Ascochyta crosses has been demonstrated in my lab. Disease phenotype will be scored using standard methods. Loci controlling disease reaction will be identified by map-based cloning with barcoded, multiplexed, Illumina Hi Seq sequencing of progeny with paired-end reads at 5X coverage per progeny genome. Loci contributing significantly to QTLs or qualitative loci controlling virulence on each host will be localized in the genome, cloned and characterized using standard QTL mapping approaches). The availability of many crosses between parents of varying divergence will allow the study of speciation mechanisms at different evolutionary scales which, to our knowledge, has never been attempted. Complementary to the map-based cloning approach described above, we will attempt to identify putative speciation genes by comparing closely related Ascochyta spp. genome sequences and identifying genetic loci under strong selection. It has been predicted that genes under strong positive selection may be important in the early events of speciation and in the adaptation of pathogens to cultivated plants. Genome comparisons will employ sequences of parental isolates of all the crosses performed among isolates in the A. pisi/A. lentis clade. Non-synonymous to synonymous substitution rate ratios (dN/dS) will be compared with nested codon models to infer selection on individual genes across the genomes using codon-based likelihood analyses implemented in PAML. The cross between A. pisi infecting cultivated pea and A. fabae infecting faba bean described above was made on senescent chickpea stems, a non-host for most of these fungi. This apparent lack of host preference for mating raises the possibility that these taxa are not necessarily subject to assortative mating between host-adapted forms in nature. To address this hypothesis, an artificial “hybrid zone” will be used to estimate gene flow between fungi colonizing these hosts. Geographically separated field sites will each be planted with mixtures of two different host plants and inoculated with both mating types of Ascochyta fungi specific for each host in the mixture. Each inoculated fungus will have a unique multilocus microsatellite genotype to estimate gene flow and specific markers for each host will be developed from sequences of the chloroplast matK gene. Infected plants of each host species will be sampled twice per growing season and the pathogen and host characterized genetically. Over-wintered infected debris will be harvested in early spring and assessed for the presence of sexual fruiting bodies and genotyped. Ascospores will be collected to confirm identity of the fungi colonizing each piece of plant debris and to detect outcrossing between host-specific forms. If each fungus is host-specific throughout its entire life cycle, we expect to see little or no gene flow between host-adapted fungi in both the pathogenic phase and sexual phase and all sexual fruiting bodies will result from fertilization by one host-adapted form or the other with no evidence of hybridization.

Impacts
What was accomplished under these goals? Using a worldwide sample of Alternaria alternata, the causal agent of citrus brown spot, the evolutionary histories of five nuclear loci including an endo-polygalacturonase gene, two anonymous loci, and two microsatellite flanking regions were estimated using the coalescent. Species boundaries were estimated using several approaches including those that incorporate uncertainty in gene genealogies when lineage sorting and non-reciprocal monophyly of gene trees is common. Coalescent analyses of five genomic regions revealed three phylogenetic lineages strongly influenced by incomplete lineage sorting and recombination. Divergence of the citrus 2 lineage from the citrus 1 and citrus 3 lineages was strongly supported at all loci. A consensus of species tree estimation methods supported two species of Alternaria causing citrus brown spot worldwide. Based on substitution rates at the endo-polygalacturonase locus, divergence of the citrus 2 lineage from citrus 1 and 3 was estimated to have occurred between 1-12 my before present, predating the human-mediated movement of citrus and associated pathogens out of SE Asia. The number of Alternaria species identified as causing brown spot of citrus worldwide using morphological criteria has been overestimated. Little support was found for most of these morphospecies using quantitative species recognition approaches. Correct and accurate species delimitation of plant-pathogenic fungi is critical for understanding the evolution of pathogenicity, introductions of pathogens to new areas, and for regulating the movement of pathogens including the enforcement of quarantines. This research shows that multilocus phylogenetic methods that allow for recombination and incomplete lineage sorting can be useful for the quantitative delimitation of asexual species that are morphologically indistinguishable. Two phylogenetic species of Alternaria were identified as causing citrus brown spot worldwide. Further research is needed to determine how these species were introduced worldwide, how they may differ phenotypically and how these species are maintained. The mode of reproduction of an Alternaria alternata population causing citrus brown spot in central Florida was estimated. Mating type of each isolate was determined, and isolates were sequenced at six putatively unlinked loci. Three genetically distinct sub-populations (citrus1, citrus2, citrus3) were identified using network and Bayesian population structure analyses. Results demonstrate that most sub-populations of A. alternata associated with citrus are clonal but some have the ability to extensively recombine through a cryptic sexual cycle and/or parasexual cycle. Although isolates were sampled in close physical proximity (~2500 m2 area), we were able to reject a random mating model using multilocus gametic disequilibrium tests for two sub-populations, citrus 1 and citrus3, suggesting that these sub-populations were predominantly asexual. However, three recombination events were identified in these populations, and localized to individuals of opposite mating type, possibly indicating meiotic recombination. In contrast, in the third sub-population, citrus2, where only one mating type was present, extensive reticulation was evident in network analyses and multilocus gametic disequilibrium tests were consistent with recombination. Recombination among isolates of the same mating type suggests that a non-meiotic mechanism of recombination such as the parasexual cycle may be operating in this sub-population. The level of gene flow detected among sub-populations does not appear to be sufficient to prevent differentiation, and perhaps future speciation, of these A. alternata sub-populations. We employed 10 microsatellite markers to genotype 148 P. psidii samples from eucalypts and guava plus five additional myrtaceous hosts across a wide geographic range of southeastern Brazil and Uruguay in order to infer the origin of the pathogen on eucalypts. Principal coordinates analysis, a Bayesian clustering analysis, and a minimum spanning network revealed two major genetic clusters among the sampled isolates, one associated with guava and another associated with eucalypts and three additional hosts. Multilocus genotypes infecting guava differed by multiple mutational steps at eight loci compared to those infecting eucalypts. Approximate Bayesian computation revealed that evolutionary scenarios involving a coalescence event between guava- and eucalypt-associated pathogen populations within the past 1000 years are highly unlikely. None of the analyses supported the hypothesis that eucalypt-infecting P. psidii in Brazil originated via host jump from guava since the introduction of eucalypts to Brazil approximately 185 years ago. The existence of host-associated biotypes of P. psidii in Brazil indicates that this diversity must be considered when assessing the invasive threat posed by this pathogen to myrtaceous hosts worldwide.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Gra�a, R.N., Ross-Davis, N.B. Klopfenstein, M.-S. Kim, T.L. Peever, P.G. Cannon, C.P. Aun, E.S.G. Mizubuti and A.C. Alfenas. 2013. Rust disease of eucalypts, caused by Puccinia psidii, did not originate via host jump from guava in Brazil. Molecular Ecology 22: 5963-6197.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Stewart, J.E., K.A. Thomas, C.B. Lawrence, H. Dang, B.M. Pryor, L.M. Timmer and T.L. Peever. 2013. Signatures of recombination in clonal lineages of the citrus brown spot pathogen, Alternaria alternata sensu lato. Phytopathology 103: 741-749.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Stewart, J.E., M. Andrew, X. Bao, M.I. Chilvers, L.M. Carris and T.L. Peever. 2013. Development and utility of sequence characterized amplified genomic regions for lower-level fungal systematics: Proof of principle using Alternaria, Ascochyta and Tilletia. Mycologia 105: 1077-1086.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Lawrence, D.P., P.B. Gannibal, T.L. Peever and B.M. Pryor. 2013. The Sections of Alternaria: Formalizing species-group concepts. Mycologia 105: 530-546.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Dung, J.K.S., T.L. Peever and D.A. Johnson. 2013. Genetic Differentiation of Verticillium dahliae from Potato and Mint. Phytopathology 103: 445-459.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Zhuang, X., K. McPhee, T.E. Coram, T.L. Peever and M.I. Chilvers. 2013. Development and characterization of 37 novel EST-SSR markers in Pisum sativum (Fabaceae). Applications in Plant Sciences 1:1200249 (http://www.bioone.org/doi/full/10.3732/apps.1200249)
• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Stewart, J.E, L.M. Timmer, C.B. Lawrence, B.M. Pryor and T.L. Peever. 2014. Discord between morphological and phylogenetic species boundaries: Incomplete lineage sorting and recombination results in fuzzy species boundaries in an asexual fungal pathogen. BMC Evolutionary Biology 14: 000-000 (in press).
• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Njambere, E.N., T.L. Peever, G. Vandermark and W. Chen. 2014. Genotypic variation and population structure of Sclerotinia trifoliorum infecting chickpea in California. Plant Pathology 63: 000-000 (in press).

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Genes controlling production of ACR-toxin in A. alternata infecting rough lemon (Citrus jambhiri) were localized to a conditionally dispensable 1.5-Mb chromosome carrying the ACR-toxin biosynthesis gene cluster (ACRT) in the genome of the rough lemon pathotype. A genomic BAC clone containing a portion of the ACRT cluster was sequenced which allowed identification of three open reading frames present only in the genomes of ACR-toxin producing isolates. The functional role of two of these open reading frames, ACRTS1 which encodes a putative hydroxylase, and ACRTS2 which encodes a polyketide synthase in toxin biosynthesis was studied by homologous recombination-mediated gene disruption and RNA silencing. At least three copies of the ACRTS1 gene in the genome were identified and disruption of two copies of this gene significantly reduced ACR-toxin production as well as pathogenicity. However, transcription of ACRTS1 and production of ACR-toxin were not completely eliminated due to remaining functional copies. RNA silencing was used to knock down the remaining ACRTS1 transcripts to levels undetectable by reverse transcription-polymerase chain reaction. The silenced transformants did not produce detectable ACR-toxin and were not pathogenic. Similarly, the combined use of homologous recombination-mediated gene disruption and RNA silencing allowed examination of the functional role of multiple paralogs of ACRTS2 in ACR-toxin production. These results indicate that ACRTS1 and ACRTS2 are essential genes in ACR-toxin biosynthesis in the rough lemon pathotype of A. alternata and are required for full virulence of this fungus. In order to further characterize the genomic organization of conditionally dispensable chromosomes (CDC) carrying important pathogenicity determinants in Alternaria plant pathogens, we sequenced the entire genome of the tomato pathotype of A. arborescens in collaboration with Tom Mitchell and colleagues at the Ohio State University. The sequence of the CDC (approximately 1 Mb) was subtracted from the non-CDC portion of the genome by comparison with another species known to not carry CDCs and a de-novo assembly of the CDC and its predicted gene content was performed. Identification of the CDC was validated by Southern hybridization of putative CDC and non-CDC genes to membranes generated from whole chromosome (CHEF) gels. Predicted genes were functionally annotated through BLAST and gene ontology terms were assigned, and conserved domains identified. Differences in nucleotide usage were found between CDC genes and those on the essential chromosome (EC), including GC3-content, codon usage bias, and repeat region load. Genes carrying PKS and NRPS domains were identified in clusters on the CDC and evidence supporting the origin of the CDC through horizontal transfer from an unrelated fungus. Evidence supporting the hypothesis that the CDC in A. arborescens was acquired through horizontal transfer, likely from an unrelated fungus was presented. We also identified several predicted CDC genes under positive selection that may serve as candidate virulence factors. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Evolutionary biologists, plant pathologists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The identification and characterization of genes controlling host-specific toxin production in the model plant-pathogenic fungus Alternaria alternata has furthered our understanding of the evolution of pathogenicity in plant-pathogenic fungi and has broad implications to many different pathosystems. This research was extended to the whole genome level in A. aborescens, which allowed the study of the evolution of pathogenicity at a much larger scale (i.e., the whole genome). This research is important because it allows us to understand how plant pathogenic fungi have evolved to become parasites of plants and explain the emergence of new diseases threatening US and global agro-ecosystems. Recent data from several different research labs has indicated that plant-pathogenic fungi are able to frequently acquire novel genetic material from other strains of the same species and/or other species, and even from bacteria. This process appears to be mediated through horizontal genetic transfer which is a non-Mendelian mode of creating genomic novelty that may be particular common in bacteria and fungi. This transfer of genetic material can include novel genes allowing the recipient strain to produce a host-specific toxin or other pathogenicity/virulence determinant. This acquisition then allows the recipient fungus to become a pathogen or for an existing pathogenic fungis to expand its host range. Both of these processes are highly significant to pathogen evolution and the emergence of disease. An improved understanding of the evolutionary processes controlling pathogen evolution will allow the design and deployment of novel disease control strategies that specifically target these pathogenicity determinants.

Publications

• Hu J., C. Chen, T. Peever, H. Dang, C. Lawrence, T. Mitchell 2012. Genomic characterization of the conditionally dispensable chromosome in Alternaria arborescens provides evidence for horizontal gene transfer. BMC Genomics 13:171.
• Peever T., W. Chen, Z. Abdo, W.J. Kaiser 2012. Genetics of virulence in Ascochyta rabiei. Plant Pathology. 61(4):754-760.
• Akamatsu H., M. Chilvers, W.J. Kaiser, T. Peever 2012. Karyotype polymorphisms in the phytopathogenic fungus Ascochyta rabiei and chromosomal rearrangement during meiosis. Fungal Biology. 116:1119-1133.
• Zhuang X., K. McPhee, T. Coram, T. Peever, M. Chilvers 2012. Rapid characterization of the host-pathogen pea-Sclerotinia sclerotiorum transcriptome with 454 pyrosequencing, and parsing of host-pathogen sequences for non-model organisms. BMC Genomics 13:668.
• Izumi Y., K. Ohtani, Y. Miyamoto, A. Masunaka, T. Fukumoto, K. Gomi, Y. Tada, K. Ichimura, T. Peever, K. Akimitsu 2012. A polyketide synthase gene, ACRTS2, is responsible for biosynthesis of host-selective ACR-toxin in the rough lemon pathotype of Alternaria alternata. Molecular Plant-microbe Interactions. 25(11):1419-29. doi: 10.1094/MPMI-06-12-0155-R.
• Izumi Y., E. Kamei, Y. Miyamoto, K. Ohtani, A. Masunaka, T. Fukumoto, K. Gomi, Y. Tada, K. Ichimura, T. Peever, K. Akimitsu 2012. Role of the pathotype-specific ACRTS1 gene encoding a hydroxylase involved in the biosynthesis of host-selective ACR-toxin in the rough lemon pathotype of Alternaria alternata. Phytopathology. 102:741-748.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: The molecular evolution of the Alternaria MAT1 locus was explored using nucleotide diversity, nonsynonymous vs. synonymous substitution (dn/ds) ratios and codon usage statistics. Likelihood ratio tests of site-branch models failed to detect positive selection on MAT1-1-1 or MAT1-2-1. Codon-site models demonstrated that both MAT1-1-1 and MAT1-2-1 are under purifying selection and significant differences in codon usage were observed between MAT1-1-1 and MAT1-2-1. Mean GC content at the third position (GC3) and effective codon usage (ENC) were significantly different between MAT1-1-1 and MAT1-2-1. In contrast, codon usage of Pleospora spp., a closely related Dothideomycete genus, was not significantly different between MAT1-1-1 and MAT1-2-1. The purifying selection and biased codon usage detected at the MAT1 locus in Alternaria spp. suggest a recent sexual past, cryptic sexual present and/or that MAT1 plays important cellular role(s) in addition to mating. Many attempts have been made to classify variation in virulence of the chickpea Ascochyta blight pathogen, Ascochyta rabiei, into discrete categories referred to as "pathogenic groups", "races" or "pathotypes". Results have been inconsistent and conflicting due to lack of control of the genetic backgrounds of host and pathogen as well as environmental variation in disease assays. In order to critically test the hypothesis that virulence variation in this pathosystem is a discrete character under simple genetic control, a genetic cross was made between a highly virulent isolate of A. rabiei from Syria and a less virulent isolate from the US. Two independent virulence assays conducted by inoculating susceptible and resistant chickpea cultivars under controlled conditions with 77 independent progeny isolates from this cross revealed a continuous distribution of disease phenotypes. Statistical tests of the progeny phenotype distribution did not support bimodality as would be predicted for the segregation of virulence under simple genetic control. ANOVA revealed highly significant pathogen genotype X host genotype interactions demonstrating the segregation of genes controlling specialization on the two cultivars tested. These interactions could be localized to two isolates that changed virulence rank on the cultivars. We conclude that variation in virulence to these two cultivars is under quantitative genetic control. If this conclusion applies to other cultivars, we speculate that the discrete categories of virulence variation identified in previous studies were likely due to incomplete sampling of host resistance or pathogen virulence variation and/or to selection for increased virulence in contemporary A. rabiei populations. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our research on the evolution of the mating locus in a putatively asexual fungus such as Alternaria has raised significant questions concerning the role of these loci in asexual fungi. If this locus is generally under strong negative selection, it may be functioning in some sort of cryptic sexual cycle or it is important in functions in addition to mating. Currently the latter hypothesis is favored based on another study in our laboratory which demonstrated that Alternaria populations on citrus are largely clonal as would be predicted for an asexual fungus. However, one population sampled was freely recombining. This recombination could be due to a cryptic sexual cycle occurring only in this population or to parasexual processes. The observation that the recombining population consisted of only one mating type favors the latter hypothesis. These results are significant because it calls into question our assumptions about fungal mating. Fungal mating is important because recombining populations of plant-pathogenic fungi may be able to adapt more quickly to specific disease control tactics employed in agroecosystems and/or adapt more quickly to new hosts which may allow the emergence of new pathogens. Our study of the genetics of virulence in Ascochyta rabiei is significant because it represents the first genetic analysis of virulence in this pathosystem and challenges the prevailing notion that qualitative differences in virulence exist among isolates in A. rabiei populations. These have been referred to as "races" or "pathotypes" in previous research. Our study questions this dogma and provides the first evidence that differences in virulence may be quantitative in nature. The pattern of inheritance of virulence is important from a practical perspective because it suggests that the interaction between A. rabiei and chickpea is fundamentally different from qualitative interactions and the study of these interactions should proceed in a fundamentally different manner.

Publications

• No publications reported this period

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: We estimated phylogenies of antibiotic synthesis genes and housekeeping genes in several soil-inhabiting bacteria and performed topological comparisons of these phylogenies. The goal was to study the evolution of the phenazine synthesis pathway and to compare this evolution with the rest of the bacterial genome. The topologies of 5 housekeeping genes and several genes within the phenazine gene cluster were each congruent indicating a common evolutionary history. Topology of the phenazine gene phzF was incongruent with the concatenated housekeeping gene phylogeny. This incongruence provides evidence for horizontal transmission of phzF consistent with the gain and loss of phenazine biosynthesis machinery by soil-inhabiting bacteria. We used the technique of pulsed-field gel electrophoresis (PFGE) to study the structure and organization of a large collection of chickpea pathogen (Ascochyta rabiei) isolates from worldwide populations and investigate intra-specific karyotype variation for this fungus. Thirteen to sixteen chromosomes were revealed of approximately 5 to 1 megabase. Overall karyotypes among 112 isolates from 21 countries were broadly similar although length polymorphisms were common. Within A. rabiei, a chromosome length polymorphism was detected for the largest chromosome with approximately one-third of isolates possessing a chromosome over 4.0 Mb in size. This size of chromosome was absent from the remainder of the isolates. PFGE analysis of progeny from an in vitro sexual cross between a Syrian and US isolate revealed one of 16 progeny with an rDNA-encoding chromosome larger than 4.0 Mb, even though a chromosome of this size was not present in either parent. Using quantitative PCR, no expansion of the rDNA cluster was detected in the progeny carrying the >4.0 Mb chromosome so the increase in chromosome size was not due to an expansion in number of rDNA repeats. We identified two genes controlling the biosynthesis of host-specific toxins in Alternaria alternata pathogenic on citrus. Sequence analysis of a genomic BAC clone identified two ORFs, designated ACTTS3, encoding a putative polyketide synthase (PKS) and ACTTS2 encoding a putative enoyl-reductase. As for previously identified genes controlling host-specific toxin biosynthesis, genomic Southern blots demonstrated that both ACTTS2 and ACTTS3 are present on a dispensable small chromosome (approx 1.7 Mb) in the genome of the tangerine pathotype of A. alternata. The presence of ACTTS2 and ACTTS3 in the genome is highly correlated to ACT-toxin production and pathogenicity, and targeted gene disruption of ACTTS2 and ACTTS3 led to a complete loss of ACT-toxin production and pathogenicity. These results indicate that both ACTTS3 and ACTTS2 are essential genes for ACT-toxin biosynthesis in the tangerine pathotype of A. alternata, that they are required for pathogenicity, and are responsible for the host specificity of this fungal pathogen. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Phylogenetic analyses of antibiotic synthesis genes and housekeeping genes in several distinct taxa of soil-inhabiting bacteria have given us important insights into the evolution of antibiotic genes potentially important in biological control of soilborne plant diseases. Topological comparisons of phenazine gene phylogenies has revealed incongruence between the phzF phylogeny and a phylogeny estimated from the rest of the genome. This incongruence is likely due to horizontal gene transfer. Our data suggests that horizontal gene transfer of the entire phenazine biosynthetic cluster has occurred numerous times among several major groups of soil-inhabiting bacteria and indicates that antibiotic production can be gained or lost in different environments. Electrophoretic karyotype analysis of the chickpea pathogen, Ascochyta rabiei, has indicated that the karyotype of this fungus is highly variable and labile. Our study is the largest of its kind in fungi and is the first to perform a population-scale karyotype analysis. It appears that at least some of the relatively large-scale genomic rearrangements we observed in A. rabiei populations may be associated with meiosis. One such rearrangement of the largest chromosome was experimentally demonstrated using in vitro crosses of the fungus. We further demonstrated that this change in size of the largest chromosome in the genome was not associated with an expansion of the nuclear ribosomal region. The significance of karyotype variation in A. rabiei is not yet understood but may be significant in terms of fungal speciation. The cloning and characterization of two additional genes controlling host-specific toxin production in Alternaria alternata causing brown spot of citrus and demonstration of their role in pathogenicity adds to our growing knowledge of the role of toxin production in brown spot disease. With the recent access to entire genome sequences of these and related species, we will shortly be able to study the evolution of the entire toxin cluster in these fungi which is located on a small, conditionally dispensable chromosome. This will facilitate a better understanding of the role of toxin genes in disease and the evolution of pathogenicity and host specificity in fungal plant pathogens.

Publications

• Mavrodi, D.V., T.Peever, O.Mavrodi, J.A.Parejko, J.M.Raiaijmakers, P.Lemanceau, S.Mazurier, L.Heide, W.Blankenfeldt, D.M.Weller, and L.S.Thomashow. 2010. Diversity and Evolution of the Phenazine Biosynthesis Pathway.. Applied and Environmental Microbiology 76(3):866-879.
• Miyamoto, Y., A.Masunaka, T.Tsuge, M.Yamamoto, K.Ohtani, T.Fukumoto, K.Gomi, T.Peever, Y.Tada, K.Ichimura, and K.Akimitsu. 2010. ACTTS3 encoding a polyketide synthase is essential for the biosynthesis of ACT-toxin and pathogenicity in the tangerine pathotype of Alternaria alternata. Molecular Plant-microbe Interactions : MPMI. 23:406-414.
• Frenkel, O., T.Peever, M.Chilvers, S.Abbo, D.Shtienberg, and A.Sherman. 2010. Ecological divergence of the fungal pathogen Didymella rabiei on sympatric wild and domesticated chickpea. Applied and Environmental Microbiology 76:30-39.
• Ajiro, N., Y.Miyamoto, A.Masunaka, T.Tsuge, M.Yamamoto, K.Ohtani, T.Fukumoto, K.Gomi, T.Peever, Y.Izumi, Y.Tada, and K.Akimitsu. 2010. Role of host-selective ACT-toxin synthesis gene ACTTS2 encoding an enoyl-reductase for the pathogenicity of the tangerine pathotype of Alternaria alternata. Phytopathology. 100:120-126.
• Akamatsu, H., M.Chilvers, J.Stewart, and T.Peever. 2010. Identification and function of a polyketide synthase gene responsible for 1,8-dihydroxynaphthalene-melanin pigment biosynthesis in Ascochyta rabiei.. Current Genetics. 56:349-360.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Genetic divergence was estimated between Ascochyta rabiei populations sampled from wild Cicer judaicum and cultivated Cicier arietinum growing sympatrically in Israel to address the hypothesis that the pathogen has adapted to spring-sown cultivated chickpea through evolution of an increased temperature optimum. Microsatellite markers showed strong genetic differentiation between pathogen populations from the two hosts that indicated a lack of gene flow between them. Isolates from domesticated chickpea demonstrated increased adaptation to higher temperatures when grown in vitro compared with isolates from the wild host, consistent with the temperature adaptation hypothesis. Temperature response among progeny from crosses of C. judaicum isolates x C. arietinum isolates was continuous, suggesting polygenic control of this trait. Inoculations of host plants with progeny and the parents of the crosses indicated that pathogenic fitness of the parental isolates was higher than the hybrid progeny from these crosses. Based on these results, we hypothesize that this pathogenic fitness cost is due to breakdown of coadapted gene complexes controlling pathogenic fitness on each host. This fitness cost may be responsible for maintenance of genetic differentiation between the two populations and represent the first step in speciation of these populations. The role of 1,8-dihydroxynaphthalene-melanin pigment (DHN-melanin) in the biology of Ascochyta rabiei was investigated by cloning and characterizing a polyketide synthase gene. Degenerate PCR primers were designed to isolate a partial ArPKS1 sequence from A. rabiei with high amino acid similarity to PKS genes involved in biosynthesis of DHN-melanin in other ascomycetous fungi. Site-directed mutagenesis of ArPKS1 in A. rabiei generated melanin-deficient pycnidial mutants. Pycnidiospores in ArPKS1-mutant pycnidia showed higher sensitivity to UV light exposure compared to pycnidiospores in melanized pycnidia of the wild-type progenitor isolate. Integration of an orthologous PKS1 gene from Bipolaris oryzae into the genome of the mutants complemented the dysfunctional ArPKS1 gene. Several of the sexual stages (teleomorphs) associated with ascomycete legume pathogens in the genus Ascochyta have been described including the Ascochyta pathogens of chickpea, faba bean and lentil. An exception is the teleomorph of A. pisi, a pathogen of pea (Pisum sativum), which has never been described. Based on our previous phylogenetic analyses of Ascochyta spp. sampled from legumes, we predicted that A. pisi would be connected to a Didymella teleomorph as is the case for all other Ascochyta pathogens of cool season food legumes. We induced the teleomorph of A. pisi in the laboratory using standard mating techniques that have been successful with other Ascochyta species. These pairings demonstrated that A. pisi has a heterothallic mating system similar to that reported previously for other Ascochyta spcies. Based on morphological and molecular characters, we placed the teleomorph within the genus Didymella as D. pisi. We also described a heterothallic mating system using a PCR-based mating type assay and in vitro crosses. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our research and the research of others have demonstrated that pathogenicity, host-specificity and speciation are interrelated phenomena in plant-pathogenic fungi. However, the mechanisms controlling pathogenicity at the species level are poorly understood in most host-pathogen systems and our understanding of the speciation processes of plant-pathogenic fungi is almost non-existent. Understanding these phenomena is important in terms of basic studies of pathogen evolution, such as host shifts and the emergence of new pathogens and also in terms of more applied questions such as pathogen identification. We have continued to explore the mechanisms of speciation in both asexual and sexual plant pathogenic fungi using two systems: the small-spored Alternaria spp. and Ascochyta spp. attacking cool season food legumes. These systems represent ideal systems with which to study the evolution of pathogenicity, genetics of host specificity and mechanisms of speciation. These pathosystems allow comparison of a predominantly asexually reproducing pathogen (Alternaria spp.) and a predominantly sexually reproducing pathogen (Ascochyta spp.) and provide insight into speciation processes in fungi with diverse mating systems. We have demonstrated that the first events in pathogen speciation in Ascochyta are likely to be the evolution of host specificity. Therefore, in plant-pathogenic fungi, and possibly other parasites, host specificity genes may represent speciation genes. The study of speciation, pathogenicity mechanisms and the genetics of host specificity will contribute to our basic knowledge of the evolution of plant pathogenesis, the emergence of disease and the design of more effective methods of disease control. In addition to providing the key to our understanding of speciation processes in fungi, phylogenetic study of fungi at or near the population/speciation interface is important from a taxonomic perspective and has important economic ramifications in terms of quarantines and biosecurity.

Publications

• Chilvers, M., J.D.Rogers, F.M.Dugan, J.Stewart, W.Chen, and T.Peever. 2009. Didymella pisi sp. nov., the teleomorph of Ascochyta pisi. Mycological Research. 113:391-400.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: In order to test the hypothesis that the mating locus (MAT) plays a role in pathogenicity in ascomycete fungi, gene disruption mutants were created for both MAT idiomorphs of A. brassicicola, a cabbage pathogen. A. brassicicola isolate 01-19a-s was shown to carry the MAT1-1 idiomorph. We successfully disrupted the respective genes in both strains as shown by PCR analysis. Transformants were purified by isolating single conidia and selecting on PDA medium containing hygromycin. Mutants and corresponding parental wildtype strains were inoculated onto 4-week old green cabbage leaves and disease was allowed to develop. The virulence of each isolate was estimated by measuring lesion diameter for wildtype and mutant isolates paired on the same leaf with 15 replicate leaves per treatment. We observed a 50% reduction in the average lesion diameter for the MAT1-1 mutants compared to wildtype and this was statistically significant (P < 0.01). The MAT1-1 mutant, but not the MAT1-2 mutant, was significantly reduced in virulence and therefore MAT1-1 appears to represent a virulence factor on cabbage. This preliminary data provides strong evidence that the observed reduction in virulence is due to specific mutation at the MAT locus and is the first report of MAT locus control of pathogenicity in an ascomycete. Future studies will involve verification of the specificity of these mutations and complementation of mutants with wildtype genes to verify phenotypes. We have made significant advancements in the species-level systematics of small-spored Alternaria spp. by performing phylogenetic analyses of 150 isolates of the alternata species-group spanning 25 morphospecies using sequence data from an endopolygalacturonase gene as well as three anonymous loci. Associations among phylogenetic lineage, morphological classifications, geography, and host were evaluated for use as practical taxonomic characters. No associations were found between host or geographic associations and phylogenetic lineage, indicating that these characters were not useful for cladistic classification within the alternata species-group. Thirty-four isolates that grouped morphologically with A. arborescens fell into discrete clades for all loci. Thus, in some cases there are both morphological and molecular characters that can separate taxa within this species-group via standard methods. However, strict congruence between morphology and phylogenetic lineage was not found among isolates that grouped morphologically with A. alternata or A. tenuissima. Among these taxa, phylogenetic analyses revealed five to seven well-supported clades depending on the locus. We found evidence for recombination within two of the anonymous loci as well as statistically significant incongruence among the phylogenies estimated from each genomic region. It is hypothesized that much of this incongruence is due to incomplete lineage sorting. In order to accommodate these stochastic processes into an accurate estimate of the species phylogeny, additional regions of the genome and probabilistic modeling of species trees and joint estimation of gene trees in a Bayesian framework are required. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Mechanisms of pathogenicity and speciation are poorly understood in most host-pathogen systems and our understanding of the speciation processes of plant-pathogenic fungi is almost non-existent. It has long been thought by plant pathologists that host specificity was important in speciation as the historical taxonomy of plant-pathogenic fungi reflects a host bias. However, the genetic control of host specificity at the species level in plant pathogenic fungi and how this may contribute to speciation of fungi is poorly understood. Small-spored Alternaria spp. and Ascochyta spp. attacking cool season food legumes represent ideal systems with which to study the evolution of pathogenicity, genetics of host specificity and mechanisms of speciation. These pathosystems allow comparison of a predominantly asexually reproducing pathogen (Alternaria spp.) and a predominantly sexually reproducing pathogen (Ascochyta spp.) and provide insight into speciation processes in fungi with diverse mating systems. The study of speciation, pathogenicity mechanisms and the genetics of host specificity will contribute to our basic knowledge of the evolution of plant pathogenesis, the emergence of disease and the design of more effective methods of disease control. In addition to providing the key to our understanding of speciation processes in fungi, phylogenetic study of fungi at or near the population/speciation interface is important from a taxonomic perspective and has important economic ramifications in terms of quarantines and biosecurity.

Publications

• Peres, N., S.McKenzie, T.Peever, and L.W.Timmer. 2008. Postbloom fruit drop of citrus and Key lime anthracnose are caused by distinct phylogenetic lineages of Colletotrichum acutatum. Phytopathology. 98:345-352.
• Andrew, M., T.Peever, and B.M.Pryor. 2008. An expanded multilocus phylogeny does not resolve species among the small-spored Alternaria species complex. Mycologia. 101:95-109.
• Kaiser, W.J., J.Viruega, T.Peever, and A.Trapero. 2008. An outbreak of Ascochyta blight of pea caused by Ascochyta pisi in Spain. Plant Disease. 92:1365.
• Miyamoto, Y., A.Masunaka, T.Tsuge, M.Yamamoto, K.Ohtani, T.Fukumoto, K.Gomi, T.Peever, and K.Akimitsu. 2008. Functional analysis of a multicopy host-selective ACT-toxin biosynthesis gene in the tangerine pathotype of Alternaria alternata usingRNA silencing. Molecular Plant-microbe Interactions : MPMI. 21:1591-1599.
• Akamatsu, H., M.Chilvers, and T.Peever. 2008. First report of spring black stem and leaf spot of Medicago sativa (alfalfa) in Washington State caused by Phoma medicaginia. Australasian Plant Disease Notes 92:833.
• Rhaiem, A., M.Cherif, T.Peever, P.S.Dyer, M.Harrabi, and R.Strange. 2008. Population structure and mating system of Ascochyta rabiei in Tunisia revealed with locus-specific microsatellites and a mating type marker. Plant Pathology. 57:540-551.

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: In collaboration with Dr. Martin Chilvers, I have completed development of a PCR assay to identify and differentiate three Ascochyta species associated with the "Ascochyta complex" of pea. It is difficult to separate these three fungi based on traditional methods and the contribution of each species to the disease complex is not known. The assay is based on selective PCR amplification of the intergenic spacer of the nuclear ribosomal gene cluster. The utility of the assay was recently demonstrated with a sample of diseased winter peas from Potlatch, ID. Two of the three Ascochyta species were associated with disease on these peas, Ascochyta pinodella and A. pinodes. Previous research has indicated that the former fungus colonizes stem bases and crowns and the latter species colonizes stems and leaves above the soil level. We found that the distribution of the two species was random from soil level to the tops of the plants calling into question previous data showing spatial separation of these species on pea plants. This assay will prove useful to plant pathologists and plant breeders interested in better characterizing the pathogens observed in pea resistance screening nurseries and we have received commercial interest in the assay. During the coming year, this qualitative PCR assay will be converted to a quantitative assay using real-time PCR. A quantitative assay will allow quantification of the pathogen in pea tissues without isolation of the pathogen in pure culture. In collaboration with Dr. Hajime Akamatsu, we have developed a set of codominant molecular markers for the pea pathogen Aphanomyces euteiches. Our initial objective was to clone and characterize microsatellite markers using standard enriched library techniques. We were unable to identify a significant number of microsatellites in several different libraries. Our results suggest that these types of repeats are rare in the A. euteiches genome and forced us to develop alternative types of markers such as single nucleotide polymorphisms. These markers should prove extremely useful to the A. euteiches research community by facilitating population studies of the pathogen and improving resistance screening approaches. Also in collaboration with Dr. Akamatsu, we have continued to study the biological control of the sexual stage of Ascochyta rabiei on chickpea using another antagonistic fungus, Aureobasidum pullulans. Dr. Akamatsu has completed a series of studies aimed at determining the exact timing of application of biocontrol agent relative to pathogen colonization. He has found that the biocontrol agent is effective in preventing sexual development of the pathogen when inoculated up to 4 weeks following colonization of the chickpea stubble by the pathogen. If inoculated more than 4 weeks following colonization of the debris by the pathogen, the biocontrol agent is ineffective. These data indicate that A. pullulans can be effective under field conditions if applied less than a month after harvest. Elimination of the sexual stage of A. rabiei will reduce levels of initial inoculum and result in reductions in fungicide use later in the season. PARTICIPANTS: Martin I. Chilvers, Hajime Akamatsu

Impacts
Our previous efforts to develop a robust phylogeny of Ascochyta species associated with legumes has enabled us to design several robust PCR assays that are able to detect and quantify the economically important species in several plant tissues. All of these fungi are seedborne and seed infection represents an important source of primary inoculum and route of introduction into new regions. Knowledge of seed infection levels can inform disease management strategies by allowing growers to avoid planting infected seed or treating it prior to planting. Detection of the pathogen on seed will allow growers to prevent the movement of the pathogen into new areas and to eliminate a potential source of initial inoculum. Similarly, our development of molecular markers for A. eutieiches will allow researchers to estimate the population structure of the pathogen in many areas where it is important and to implement improved resistance screening methods. The biocontrol fungus, Aureopbasidium pullulans, has demonstrated ability to completely suppress development of the sexual stage of Ascochyta rabiei and production of ascospores in laboratory and growth chamber studies. This suppression of the sexual stage could result in reduction in levels of initial inoculum of A. rabiei in the field if timing of application is optimized. Ascospores are considered an important source of primary inoculum in the US Pacific Northwest and reductions in the levels of this inoculum will reduce the need for fungicide applications later in the season, improving the profitability of growing chickpeas.

Publications

• Peever, T. 2007. The Role of Host Specificity in the Speciation of Ascochyta Pathogens of Cool Season Food Legumes. European Journal of Plant Pathology / European Foundation for Plant Pathology. 119:119-126.
• Rhaiem, A., M.Cherif, P.S.Dyer, T.Peever, and R.Strange. 2007. Distribution of mating types and genetic structure of Ascochyta rabiei populations in Tunisia. Phytopathologische Zeitschrift. Journal of Phytopathology. 155:596-605.
• Peever, T., M.Barve, L.J.Stone, and W.J.Kaiser. 2007. Evolutionary relationships among Ascochyta species infecting wild and cultivated hosts in the legume tribes Cicereae and Vicieae. Mycologia. 99:59-77.
• Akamatsu, H., N.Grunwald, M.Chilvers, L.Porter, and T.Peever. 2007. Development and characterization of codominant simple sequence repeat, single nucleotide polymorphism and sequence characterized amplified region mark. Journal of Microbiological Methods. 71:82-86.
• Chilvers, M., T.Peever, H.Akamatsu, W.Chen, and F.J.Muehlbauer. 2007. Didymella rabiei primary inoculum release from chickpea debris in relation to weather variables in the Pacific Northwest of the United States. Canadian Journal of Plant Pathology. Revue Canadienne De Phytopathologie. 29:365-371

Progress 01/01/06 to 12/31/06

Outputs
A large-scale, multilocus phylogenetic analysis of small-spored Alternaria species was completed by Marion Andrew, an MS student in my laboratory. Her analysis confirmed and extended our previous phylogenetic analyses focused on small-spored Alternaria spp. associated with citrus. From this research, we can conclude that isolates classified morphologically as A. alternata and A. tenuissima from a number of plant hosts and geographic locations cannot be differentiated phylogenetically. This indicates that the sporulation characters used to differentiate these taxa are not phylogenetically informative. In contrast, isolates classified morphologically as A. arborescens were phylogenetically distinct indicating that the sporulation characters controlling this phenotype were phylogenetically informative. In collaboration with a visiting Fulbright fellow we cloned and characterized the mating locus from Ascochyta lentis, a pathogen of lentil. Sequence data from this locus allowed us to design a PCR primer set that can be used in a mating type-specific PCR assay for A. lentis as well as three other closely related Ascochyta species infecting legumes. In collaboration with a visiting scientist from Israel, Omer Frenkel, we made genetic crosses between A. rabiei isolates isolated from wild chickpeas in Israel as well as between wild chickpea isolates and isolates from cultivated chickpea. Omer had previously detected a large difference in growth temperature phenotype between isolates from wild hosts (low temperature optimum) and isolates from cultivated hosts (higher optimum). We made two crosses between isolates with large difference in growth temperature phenotype and picked approximately 120 progeny from each cross for genetic analyses. Omer screened these progeny for variation in temperature optimum and developed genetic markers for the crosses. Our goal is to use these crosses to study the genetics of growth temperature optimum and to study the evolution of this character and its role in the evolution of A. rabiei on cultivated chickpea. During this year we published the first two of a series of disease first reports describing Ascochyta spp. isolated form wild legume hosts in Central Europe and West Asia. We performed host inoculations and sequenced three regions of the genome for several isolates of Ascochyta spp. from wild legume hosts in the Republic of Georgia. In collaboration with Norm Dart and Gary Chastagner, Washington State University Puyallup, we completed a population structure study which examined the distribution of genetic variation of Heterobasidion annosum at several spatial scales in a Pacific Northwest Christmas tree plantation. Norm's research demonstrated definitively that clonal spread among trees within a disease focus did not occur and that disease within each infected tree and root system within a disease focus was likely initiated by a different basidiospore. In collaboration with Martin Chilvers and Lindsey duToit, WSU, Mount Vernon, we completed a study in which a real-time PCR assay was developed to detect Botrytis spp. associated with neck rot of onion.

Impacts
Inclusion of DNA sequence data and BLAST search results in our disease reports of Ascochyta spp. associated with wild legumes is an attempt to establish a new standard for these sorts of publications. With the ever-growing databases of fungal DNA sequences, it is obvious that DNA sequence data will be used routinely for pathogen identification in the future. These disease first reports document the existence of a particular disease but are much more important in terms of adding to our phylogeny of Ascochyta spp. This phylogeny forms the basis for our studies of species-level host specificity and the role of this specificity in fungal speciation. The development of a mating type-specific PCR assay for A. lentis will prove useful to many researchers interested in these fungi. Our A. rabiei MAT-specific PCR assay is already in use by several research laboratories around the world. Our phylogenetic studies of Alternaria spp. have greatly clarified the evolutionary relationships among the small-spored taxa and have allowed us to better define species limits in this genus. Alternaria species are fungi of substantial medical interest and also plant pathogens of substantial quarantine interest and the taxonomy of this genus has been in disarray for many years. Our research will allow much better predictive association of evolutionarily meaningful names with the biology of the species (pathogenicity, habitat, reproduction, physiology) and will contribute to a revised taxonomic system for this important genus.

Publications

• Cherif, M., M.Chilvers, H.Akamatsu, and T.Peever. 2006. Cloning of the mating type locus from Ascochyta lentis (teleomorph: Didymella lentis) and development of a multiplex PCR mating assay for Ascochyta sp. Current Genetics. 50(3):203-215.
• Hernandez-Bello, M., M.Chilvers, H.Akamatsu, and T.Peever. 2006. Host specificity of Ascochyta species infecting legumes of the Viciae and Cicerae tribes and the pathogenicity of an interspecific hybrid. Phytopathology. 96:1148-56.
• Marin, J., H.Fernandez, N.Peres, M.Andrew, T.Peever, and L.W.Timmer. 2006. First Report of Alternaria Brown Spot of Citrus Caused by Alternaria alternata in Peru. Plant Disease. 90:686.

Progress 01/01/05 to 12/31/05

Outputs
Evolutionary relationships were inferred among a worldwide sample of Ascochyta spp. isolates from wild and cultivated legume hosts in the Viciae and Cicereae tribes based on phylogenetic analyses of DNA sequence data from four regions of the genome. Analysis of the combined dataset revealed that isolates sampled from cultivated pea (P. sativum), lentil (Lens culinaris), faba bean (Vicia faba) and chickpea (Cicer arietinum) in diverse geographic locations each had identical or similar sequences at each locus. Isolates from these hosts clustered in well-supported clades specific for each host suggesting a tight co-evolutionary history between pathogen and cultivated host. Isolates sampled from Vicia grandiflora (bigflower vetch) were polyphyletic suggesting that either this host is colonized by phylogenetically distinct lineages of Ascochyta or that the hosts are polyphyletic with distinct evolutionary lineages of the pathogen colonizing each host lineage. We have identified several closely related, host-specific Ascochyta taxa that were crossed in the laboratory and produced normal progeny. No statistically significant differences in AFLP marker segregation (P = 0.14) were detected among progeny from interspecific and intraspecific crosses but progeny from the former did not cause disease on either host plant. These data support the hypothesis that host specificity may function as both a prezygotic and postzygotic mating barrier and limit gene flow between taxa adapted to each host. Ascospores of A. rabiei were sampled at two locations during the spring of 2005 using trap plants deployed around small plots of overwintered, A. rabiei-infested chickpea debris. Simultaneous collection of environmental data has allowed us to relate pseudothecium development and ascospore dispersal to weather data. Dispersal of ascospores was tightly correlated to rainfall events and all dispersal events ended by the end of May. Nineteen sequence-characterized amplified regions (SCAR) were developed for Alternaria spp. by cloning and sequencing random PCR amplicons. To determine if any of the five regions represented genes, sequences were translated in six frames and BlastX searches performed to assess similarity to known proteins. None of the 5 regions had significant similarity to any known proteins in the databases. We have completed phylogenetic analyses of endoPG, and three anonymous regions of the genome for alternata species group isolates sampled from five distinct hosts and geographic regions. Isolates classified morphologically as A. tenuissima, A. alternata, A. limoniasperae and A. citriarbusti were all paraphyletic with lack of congruence between morphology and phylogenetic lineage. Exceptions included the arborescens and tangelonis morphospecies groups which are correlated to distinct clades in analyses of all four genomic regions. Approximately 18 percent (4 of 22) of isolates identified morphologically by us were identified differently by other workers (Serdani et al 2002). Additionally, thirty one percent of isolates examined in this study (10 of 32 isolates) did not sporulate sufficiently to allow morphological classification.

Impacts
Our phylogenetic, host specificity and speciation studies of Ascochyta spp. represents one of the few studies of the genetics of host specificity at this level of biological organization. Little is known about the mechanisms of fungal speciation and this research will allow us to determine how pathogens colonize new hosts and to make predictions about future host shifts of plant-pathogenic fungi. The evolution of emerging diseases of both plants and animals is of great theoretical and practical interest and we hope to contribute to this effort. Our epidemiological studies of ascospore dispersal in A. rabiei have led to the development of a weather-driven model of sexual stage development and ascospore dispersal which will allow growers to make more informed decisions concerning disease control measures and to better time fungicide applications. Alternaria species are fungi of substantial medical interest and also plant pathogens of substantial quarantine interest but the taxonomy of this genus is in disarray. There is a great need for predictive association of names with the biology of the species (pathogenicity, habitat, reproduction, physiology) and such a taxonomic system will allow identification by a large number of workers without specialized knowledge and equipment.

Publications

• Dugan, F.M., S.L. Lupien, M. Hernandez-Bello, T.L. Peever and W. Chen. 2005. Fungi resident in chickpea debris and their suppression of growth and reproduction of Didymella rabiei under laboratory conditions. Journal of Phytopathology 153: 431-439.
• Golmohammadi, M., M. Andrew, T.L. Peever, N.A. Peres, and L.W. Timmer. 2005. First report of brown spot of Minneola and Page tangelo and Fortune tangerine caused by Alternaria alternata in Iran. BSPP New Disease Reports 12: (http://www.bspp.org.uk/ndr/jan2006/2005-98.asp)
• Peever, T.L., L. Carpenter-Boggs, L.M. Carris, L.W. Timmer and A. Bhatia. 2005. Citrus black rot is caused by phylogenetically distinct lineages of Alternaria alternata. Phytopathology 95: 512-518

Progress 01/01/04 to 12/31/04

Outputs
Ascospores of A. rabiei were sampled at two locations during the spring of 2004 using trap plants deployed around small plots of overwintered, A. rabiei-infested chickpea debris. Simultaneous collection of environmental data allowed us to relate pseudothecium development and ascospore dispersal to weather data. Dispersal of ascospores was tightly correlated to rainfall events and all dispersal events ended by the end of May. An extensive sample of isolates of Ascochyta rabiei was collected from ascochyta blight-infected chickpea in the Central Valley of California from 1996 to 2003. Isolates were screened for genetic variation at 6 microsatellite loci and the mating type locus. All isolates sampled prior to 2000 belonged to 2 genotypes and all were a single mating type. The alternate mating type was detected at low frequency at one location in 2000 and increased in frequency between 2000 and 2003. The introduction of the other mating type was accompanied by an increase in diversity at the microsatellite loci which was likely the result of new introductions of the pathogen on seed imported for agronomic evaluation. Despite the introduction of many new microsatellite alleles and the other mating type, no evidence for genetic recombination through the sexual stage was found in this population. Ascochyta spp. were sampled from various legume hosts worldwide and we PCR-amplified and sequenced 4 regions of the genome in order to infer a phylogeny among these taxa. Isolates representing a broad geographic sample from each host had identical sequences for each genomic region and formed a monophyletic group. This indicates that host specificity is a strong isolating mechanism in this group of fungi and that isolates causing disease on each host are evolving independently. Through these phylogenetic analyses, we have identified several closely related, host-specific taxa that were crossed in the laboratory and produced normal progeny. Progeny from these crosses are being used to determine the genetics of species-level host specificity. This project is related to the phylogenetic study above and we are trying to better understand the evolutionary history of Ascochyta spp. on legumes and the role of host specificity controlling speciation in this group. We screened the progeny of several inter-specific crosses for pathogenicity in the growth chamber and started to develop AFLP markers segregating in these crosses as well as intra-specific crosses. Our results support the hypothesis that host specificity may function as a pre-zygotic mating barrier and limit gene flow between taxa adapted to each host. The hypothesis that small-spored Alternaria taxa from citrus and non-citrus hosts are able to cause black rot was tested by performing inoculation studies of citrus fruit. We experimentally demonstrated that all small-, catenulate-spored taxa regardless of host of origin are able to cause this disease and that black rot of citrus is caused by isolates from several monophyletic lineages. This manuscript was submitted to Phytopathology during 2004 and has been accepted for publication.

Impacts
Epidemiological studies of ascospore dispersal in Ascochyta rabiei will allow the development of a weather-driven model of sexual stage development and ascospore dispersal. This model will be used by growers to improve timing of fungicide applications and will result in fewer fungicide applications saving growers money and reducing environmental impacts. Determination of the population structure of A. rabiei in the US Pacific Northwest and California and worldwide has allowed us to pinpoint likely routes of introductions of plant-pathogenic fungi into the US. This information will be used to strengthen quarantine regulations and improve early detection of this and other pathogens. Our study of the mating system transition of A. rabiei in California will allow us to make predictions about fungal dispersal as the fungus shifts from a clonal mode of reproduction through splash-dispersed conidia to a mixed mating system involving the production of both conidia and airborne ascospores. Phylogenetic analyses and host specificity studies with Ascochyta spp. have revealed mechanisms of host specificity and fungal speciation which will allow predictions to be made about the frequency with which pathogens adapt to new hosts and the mechanisms involved in host shifts in pathogens. Phylogenetic analysis of citrus black rot isolates have revealed that several species of Alternaria can cause black rot of citrus. This information has forced us to re-examine of the potential role of saprophytic and endophytic Alternaria spp. associated with citrus as black rot pathogens.

Publications

• Peever, T.L., G. Su, L. Carpenter-Boggs, and L.W. Timmer. 2004. Molecular systematics of citrus-associated Alternaria species. Mycologia 96:119-134.
• Peever, T.L., S.S. Salimath, G. Su, W.J. Kaiser and F.J. Muehlbauer. 2004. Historical and contemporary multilocus population structure of Ascochyta rabiei (teleomorph: Didymella rabiei) in the US Pacific Northwest. Molecular Ecology 13:219-309..
• Peres, N. A. R., N.L. Souza, T.L. Peever, and L.W. Timmer, 2004. Benomyl sensitivity of isolates of Colletotrichum acutatum and C. gloeosporioides from citrus. Plant Disease 88:125-130.
• Richardson, B.A., N.B. Klopfenstein and T.L. Peever. 2005. Assessing forest-pathogen interactions at the population level. Pages 21-30 in: Integrating Scales in Forest Pathology, J.E. Lundquist, R.C. Hamelin and C. Aquirre-Bravo eds., American Phytopathological Society Press, St. Paul, MN, USA.
• Timmer, L.W., T.L. Peever, Z. Solel and K. Akimitsu. 2004. Alternaria diseases of citrus - Novel pathosystems. Phytopathologia Mediterranea 42: 99-112.
• Dart, N., T.L. Peever and G.C. Chastagner. 2004. Population Biology of Heterobasidion parviporum (H. annosum S-Strain) infecting Christmas Trees in the Pacific Northwest. Phytopathology 94: (Suppl.) S24.
• Dugan, F.D., T.L. Peever and W. Chen. 2004. Fungi resident on chickpea debris and competitive interactions with Ascochyta rabiei. Phytopathology 94: (Suppl.) S27.
• Hernandez-Bello, M.A. and T.L. Peever. 2004. Genetics of host specificity of Ascochyta species infecting legumes. Phytopathology 94: (Suppl.) S41.
• Hoff, J.A., N.B. Klopfenstein, J.R. Tonn, P.J. Zambino, P.F. Hessburg, J.D. Rogers, T.L. Peever and L.M. Carris. 2004. Fungal diversity in woody roots of Douglas-fir (Pseudotsuga menziesii) and ponderosa pine (Pinus ponderosa). Forest Pathology 34:1-17.
• Peever, T.L. 2004. Inferring patterns of migration and gene flow in introduced populations of plant pathogens using the chickpea pathogen Ascochyta rabiei as a model. Phytopathology 94: (Suppl.) S127.

Progress 01/01/03 to 12/31/03

Outputs
During 2003, we pursued three main lines of research involving Ascochyta species associated with legumes. The first involved the generation of a dense genetic map of a cross between two A. rabiei isolates segregating for virulence on chickpea. This project is being conducted in collaboration with Dr. W. Chen, USDA-ARS who completed scoring approximately 80 progeny for virulence from this cross. We are currently developing a genetic map for this cross using AFLP, microsatellites and mating type. Parental isolates are being screened for fAFLPs in collaboration with Dr. N. Grunwald, USDA-ARS, Prosser. The second main area of research we have been pursuing is the genetics of host specificity of Ascochyta spp. associated with several legume hosts. This project is being conducted by a graduate student, Mr. Marco Hernandez-Bello. Marco has completed a series of greenhouse and growth chamber inoculation studies which have demonstrated that Ascochyta species are host-specific. He has also started to screen the progeny of several 'inter-specific' crosses for pathogenicity in the growth chamber and begun to develop AFLP markers for these crosses as well as 'intra-specific' crosses. Preliminary results support the hypothesis that host specificity may function as a pre-zygotic mating barrier and limit gene flow between 'species' adapted to each host. Related to the above project is a third major area of emphasis, a study of the host range of A. rabiei. During the past year we have found that A. rabiei can infect a wide range of hosts including wheat. The mechanism of fungal penetration, whether or not the fungus significantly colonizes these plants, and the epidemiological significance of this apparent wide host range remain to be determined. Due to funding constraints, we were unable to pursue this project further but are excited by these preliminary observations. Two additional research projects were finished during the past year dealing with the population structure, phylogeography and systematics of small-spored Alternaria species associated with 1) healthy and diseased citrus leaf tissue and 2) black rot, a post-harvest citrus pathogen. These manuscripts are currently being written by Dr. Lynne Carpenter-Boggs. This manuscript will be submitted to Molecular Ecology in the coming year. The hypothesis that all small-spored Alternaria taxa are able to cause black rot was tested by performing inoculation studies of citrus fruit. This manuscript will be submitted to Phytopathology within the next few months.

Impacts
Our study of the host specificity of Ascochyta spp. is significant because it represents one of the few studies of the genetics of host specificity at this level of biological organization. Preliminary results indicate that the progeny of an 'inter-specific' cross are not pathogenic on either parental host. This is extremely important because it supports the hypothesis that host specificity functions as a pre-zygotic mating barrier and limits gene flow among 'species' adapted to each host. The results of this project will be highly relevant to our understanding of the genetics of host specificity as well as fungal speciation processes. Our ecological study of Alternaria alternata on citrus is significant because it represents one of the few studies where large samples of pathogenic and non-pathogenic isolates of a fungus were sampled from different tissues of the same host plant and compared in a phylogeographic context. The results of this study suggest that closely related Alternaria taxa both colonize healthy citrus tissue and cause brown spot disease. Differences among isolates from each ecological niche are likely due to acquisition and/or loss of toxin sequences and toxin production. The significance of our phylogentic study of black rot isolates is that it revealed that several distinct phylogenetic lineages of small-spored Alternaria species are associated with black rot of citrus. Therefore, it is not appropriate for black rot isolates to hold taxonomic rank (ie. distinct species).

Publications

• Akimitsu, K., T.L. Peever, L.W. Timmer. 2003. Molecular, ecological and evolutionary approaches to understanding Alternaria diseases of citrus. Molecular Plant Pathology 4: 435-446.
• Barve, M.P., T. Arie, S.S. Salimath, F.J. Muehlbauer and T.L. Peever. 2003. Cloning and characterization of the mating type (MAT) locus from Ascochyta rabiei (teleomorph: Didymella rabiei) and a MAT phylogeny of legume-associated Ascochyta spp. Fungal Genetics and Biology 38: 151-167.
• Milgroom, M.G. and T.L. Peever. 2003. Population biology of plant pathogens: The synthesis of plant disease epidemiology and population genetics. Plant Disease 87: 608-617.
• Peever, T. L. and T. D. Murray. 2003. First report of tan spot of wheat in the U.S. Pacific Northwest caused by Pyrenophora tritici-repentis. Plant Disease 87:203. Published on-line as D-2002-1202-02N.

Progress 01/01/02 to 12/31/02

Outputs
We have completed cloning and characterization of the mating (MAT) genes from Ascochyta rabiei. Sequence data from the MAT idiomorphs allowed us to develop a mating type-specific PCR assay. This assay was used to determine mating type ratios in populations of A. rabiei. The population structure of A. rabiei in the US Pacific Northwest has been determined using molecular markers. Over 600 contemporary and historical isolates were screened for variation at 17 putative AFLP loci, 4 microsatellite loci and the mating type locus. Polymorphisms at these loci were used to infer population structure among isolates from commercial chickpea fields in the PNW as well as to infer the mating system of the fungus within sampled populations. We have completed a worldwide phylogeographic study of Alternaria alternata isolates from citrus using RAPD markers and endopolygalacturonase sequence data. Data were used to infer worldwide patterns of dispersal of the pathogen and to assist resistance breeding efforts in citrus. A phylogenetic analysis was performed on Phytophthora spp. sampled from irrigation canals in central Washington based on morphology, ITS-RFLP and ITS2 sequence data. The molecular data revealed the presence of several distinct taxa present in irrigation canals, all of which were pathogenic on pear. We completed a morphotaxonomic study of Alternaria spp. associated with grasses. This study will be continued in the coming year to include molecular data. We completed a phylogenetic analysis of Ascochyta species associated with legumes using ITS, glyceraldehyde-3-phosphate-dehydrogenase, chitinase, elongation factor alpha and MAT sequence data. Several closely related, host-specific species were identified and interspecific crosses of these species will be used to study the genetics of host specificity. We determined that A. rabiei isolates from chickpea are able to infect wheat. Isolates recovered from wheat were pathogenic on chickpea.

Impacts
Population structure studies of populations of Ascochyta rabiei on chickpea and Alternaria alternata on citrus have revealed substantial genetic variation among sampled populations. We found that populations of A. rabiei at the USDA-ARS Ascochyta blight disease nursery at WSU's Spillman Research Farm were distinct from those in commercial fields in the US Pacific Nothwest which means that early generation chickpea germplasm is currently not being screened against all potential variation in the pathogen. Similarly, our worldwide phylogeographic study of A. alternata populations from citrus revealed three major genetic lineages of the pathogen which has given important insights into how this pathogen has been spread around the globe on citrus and will also used to improve resistance breeding. Knowledge of the genetic variation in these pathogens has been used to redesign resistance screening procedures for both diseases so that breeding lines are exposed to the full range of genetic variation in each pathogen. This is necessary because plants which are resistant to one pathogen population may not be resistant to another and resistance selected in a location which does not reflect the true diversity in pathogen populations may fail when put into commercial use. The most environmentally and economically sound method for controlling plant disease is through the use of resistant plants and our research has contributed to the development of resistant chickpea and citrus germplasm by improving the procedures used for germplasm screening.

Publications

• Bhatia, A., T.L. Peever, K. Akimitsu, L. Carpenter-Boggs and L.W. Timmer. 2002. Ecology of Alternaria alternata on citrus. Phytopathology 92 (Suppl.): S7.
• Douhan, G.W., T.L. Peever and T.D. Murray. 2002. Multilocus population structure of Tapesia yallundae in Washington State. Molecular Ecology 11: 2229-2239.
• Dugan, F.M. and T.L. Peever. 2002. Morphological and cultural differentiation of described species of Alternaria from Poaceae. Mycotaxon 83: 229-264.
• Hoff, J.A., N.B. Klopfenstein, G.I. McDonald, J.R. Tonn, P.J. Zambino, J.D. Rogers, T.L. Peever, L.M. Carris. 2002. Fungal diversity in woody roots of east-slope Cascade Mountain Douglas-fir and ponderosa pine. Phytopathology 92 (Suppl.): S36.
• Peever, T.L., A. Ibanez, K. Akimitsu and L.W. Timmer. 2002. Worldwide phylogeography of the citrus brown spot pathogen, Alternaria alternata. Phytopathology 92: 794-802.
• Peever, T.L., Barve, M.P. and W.J. Kaiser. 2002. Molecular Systematics of Ascochyta spp. infecting legumes. 7th International Mycology Congress, Oslo, Norway, August 11-17, 2002.
• Peres, N.A.R., T.L. Peever, K.R. Chung and L.W. Timmer. 2003. Molecular characterization of isolates of Colletotrichum acutatum causing postbloom fruit drop of citrus and Key lime anthracnose in the Americas. 8th International Congress of Plant Pathology, Christchurch, NZ. February 2-7, 2003.
• Yamak, F, Peever, T.L., Grove, G.G., and Boal, R.J. 2002. Occurrence and identification of Phytophthora spp. pathogenic to pear fruit in irrigation water in the Wenatchee River Valley of Washington State. Phytopathology 92: 1210-1217.

Progress 01/01/01 to 12/31/01

Outputs
Isolates of A. rabiei sampled from commercial chickpea fields in the PNW and the Ascochyta blight screening nursery at the Spillman Farm were screened for genetic variation using two AFLP primer sets. Comparison of AFLP allele frequencies among locations has revealed substantial genetic differentiation among locations. Three genetically distinct groups, each with high bootstrap support, are evident from the data. Isolates collected by W.J. Kaiser from 1983-1990 are genetically distinct from isolates collected in the same region during 1998-2000. Other samples from this Cluster 1 included two samples from the same field of Spanish White chickpeas in the Walla Walla, WA area in 1998 and 2000 and a sample from Evans chickpea collected near Genesee, ID in 2000. All the isolates obtained from the resistant varieties Dwelley and Sanford fell into Cluster 2 which may indicate that Cluster 2 isolates are better adapted to these cultivars. Isolates falling into Cluster 3 were sampled exclusively from many different chickpea genotypes in the Ascochyta blight nursery at Spillman Farm in 1998 and 2000. Spillman isolates sampled in 1999 appear to be genetically distinct from those sampled in 1998 and 2000, however, the reasons for this difference are currently not clear. There are three main conclusions that can be drawn from this data. First, the isolates collected by W.J. Kaiser shortly after Ascochyta blight was first described in the PNW have largely been replaced by other genotypes in this area. This may be due to host selection as highly susceptible chickpeas were grown from 1983-1990 and these have been completely replaced by more resistant cultivars. Second, these results indicate that isolates sampled from the disease screening nursery at Spillman Farm are distinct from isolates sampled in commercial chickpea fields. This is significant because it means that early generation breeding lines may not be screened against all potential variation present among A. rabiei isolates in the PNW. Finally, the existence of 3 genetically distinct groups of A. rabiei in the PNW raises questions about potential biological differences among these 3 groups such as differences in virulence and adaptation to resistant cultivars. An improved understanding of the genetic diversity of A. rabiei in the PNW will allow us to design improved strategies for breeding resistant chickpeas and to maximize their useful life in agriculture. Contemporary isolates collected from resistant cultivars appear to be genetically distinct from those collected from susceptible cultivars during 1983-1990. This means that different genotypes of the pathogen are currently present in the PNW and these genotypes may be better adapted to resistant cultivars. In addition, isolates sampled from the blight screening nursery at WSU's Spillman Farm are genetically distinct from those isolates collected from most of the commercial chickpea fields in the PNW. This means that early-generation chickpea cultivars screened at Spillman have not been exposed to all potential variation in the pathogen.

Impacts
The results of this research have led to a modification in the way that chickpea germplasm is screened for resistance to Ascochyta blight and will lead to cultivars with improved levels of resistance. Development of chickpea cultivars with high levels of resistance to Ascochyta blight will allow reduction of fungicide use to control this disease. Starting this season, breeding lines will be spray-inoculated with a conidial suspension of A. rabiei consisting of several isolates representing all genetic variation present in the PNW. Using this approach, it is hoped that early generation breeding lines will be exposed to all potential variation present in the pathogen and that any potential pathogen genotype x host genotype interactions will be detected at an early stage of cultivar development. Our studies of the population biology of A. rabiei have other important practical implications for Ascochyta blight management and chickpea breeding. For example, if we find that certain pathotypes (ie. isolates able to cause disease on hosts with particular resistance genes) of the fungus are restricted to certain regions, it may be advantageous to deploy particular host resistant genes in some regions and not in others. We expect to see an immediate impact in the way that resistant chickpea germplasm is selected as a result of this research because for the first time we will have a detailed picture of how genetic and pathogenic variation is distributed in A. rabiei populations.

Publications

• Peever, T.L. and F.J. Muehlbauer. 2001. Integrated disease management of Ascochyta blight of chickpea in the Pacific Northwest of the United States: Contributions of fungal population biology to sustainable control. Proceedings of the 4th European Conference on Grain Legumes, Cracow, Poland, 8-12, July, 2001.
• Peever, T.L., A. Ibaannez and L.W. Timmer. 2001. Worldwide population structure of Alternaria sp. causing brown spot of tangerines and tangerine hybrids. Phytopathology 91(Suppl): S188.
• Peever, T.L., A. Masunaka, K. Akimitsu, T. Tsuge, A. Bhatia and L.W. Timmer. 2001. Phylogeography of Alternaria alternata on citrus. Phytopathology 91(Suppl): S70
• Peever, T.L., G. Su, and F.J. Muehlbauer. 2001. Population structure of Ascochyta rabiei in the US Pacific Northwest. North American Pulse Improvement Association Biennial Meeting, October 28-30, 2001, Fargo, ND.
• Su, G., T.L. Peever and L.W. Timmer. 2001. Molecular systematics of citrus-associated Alternaria spp. Phytopathology 91(Suppl): S190.
• Yamak, F., T.L. Peever and G.G. Grove. 2001. Phytophthora spp. isolated from irrigation water in the Wenatchee River Valley. Phytopathology 91 (Suppl): S192.

Progress 01/01/00 to 12/31/00

Outputs
The population biology of Ascochyta was studied using amplified fragment length polymorphism (AFLP) markers. During 2000, we continued to sample the pathogen from commercial chickpea fields as well as the Ascochyta blight nursery at WSU's Spillman Experimental Farm. In 2000, we sampled approximately 400 isolates of the pathogen, grew them up in liquid medium and extracted DNA from them. To date, we have sampled over 800 isolates and extracted DNA for AFLP analysis. We have identified one pair of AFLP primers which reveals significant genetic variation among our sampled isolates and which is suitable for population genetic analysis. This AFLP data is currently being analyzed to determine the population structure of A. rabiei in the PNW. We have been successful in making several genetic crosses with A. rabiei isolates from our collection in order to determine the inheritance of the AFLP markers. All AFLP markers segregate in a 1:1 ratio. The Mendelian inheritance of these markers indicates that they will be suitable for population genetics. We have cloned and sequenced approximately 1.3 kb of the MAT 1-2 idiomorph of A. rabiei using TAIL-PCR. This sequence will be used for further TAIL-PCR to obtain a full-length clone of the MAT 1-2 idiomorph. Primers will be designed to conserved flanking sequences to amplify, clone and sequence the MAT 1-1 idiomorph. Using these sequences, MAT-specific PCR primers will be designed for population studies of A. rabiei. In collaboration with K. Akimitsu at Kagawa University, Japan and L.W. Timmer at the University of Florida, we are studying the population genetics, host specificity and molecular systematics of Alternaria spp. on citrus. During 2000, we completed sequencing the beta-tubulin, mitochondrial large subunit rDNA and endo-polygalacturonase genes of approximately 100 Alternaria species and have performed phylogenetic analyses on the sequences. We have conclusively demonstrated that all pathogenic and endophytic Alternaria spp. associated with citrus are a single phylogenetic species which is indistinguishable from A. alternata. With endo-polygalacturonase sequence data, we have conclusively demonstrated that endophytic isolates of Alternaria sp. are closely related to the pathogenic species from brown spot lesions. We have demonstrated that host-specific toxin (ACT) sequences are present in non-pathogenic isolates of A. alternata. Using PCR and genomic Southern blots, we have determined that although the non-pathogens carry ACT sequences, they appear to be present in fewer copy number compared to the pathogenic isolates. Phylogeographic analysis of A. alternata brown spot isolates from different citrus-growing parts of the world has revealed that the brown spot isolates can be divided into three clades with high bootstrap support. One clade consists of isolates from Florida and Colombia, and the other of isolates from Turkey, Israel, South Africa and Australia. Similar differentiation is apparent with other types of genetic markers including RAPDs and ACT1 sequence data.

Impacts
We have made significant progress in our understanding of the population biology of Ascochyta rabiei in the Pacific Northwest (PNW) which is relevant to the breeding of resistant chickpea germplasm. By sampling commercial chickpea fields and WSU's Ascochyta blight screening nursery through several years and using molecular markers, we have determined that there is substantial genetic variation among A. rabiei populations in the PNW. Understanding how this variation is distributed will greatly improve our ability to develop resistant chickpea germplasm and to develop germplasm that will provide long-lasting resistance to Ascochyta blight. We have conclusively demonstrated that all the morphological Alternaria species associated with citrus are a single phylogenetic species, Alternaria alternata. This one species occupies a number of ecological niches on citrus exhibiting pathogenic, endophytic and saprophytic lifestyles. With phylogeographic, molecular systematic and population genetic approaches, we are beginning to understand how a single species of fungus is able to colonize such a diverse habitats. We are also starting to understand the role of host-specific toxin genes in the evolution of this fungus on citrus and our work represents the first evolutionary approach to the study of host-specific toxins in fungi.

Publications

• Masunaka, A., A. Tanaka, T. Tsuge, T.L. Peever, L.W. Timmer, M. Yamamoto, H. Yamamoto and K. Akimitsu. 2000. Distribution and characterization of AKT homologs in the tangerine pathotype of Alternaria alternata. Phytopathology 90: 762-768.
• Peever, T.L., L. Olsen, A. Ibanez and L.W. Timmer. 2000. Genetic Differentiation and Host Specificity among Populations of Alternaria spp. Causing Brown Spot of Grapefruit and Tangerine x Grapefruit Hybrids in Florida. Phytopathology 90: 407-414.
• Peever, T.L., R.S. Zeigler, A.E. Dorrance, F.J. Correa-Victoria and S. St. Martin. 2000. Pathogen Population Genetics and Breeding for Disease Resistance. On-line feature article for APSNet. American Phytopathological Society, St. Paul, MN. (http://www.scisoc.org/feature/pathpopgenetics/Top.html)
• Peever, T.L., Y.-C. Liu, P. Cortesi and M.G. Milgroom. 2000. Variation in Tolerance and Virulence in the Chestnut Blight Fungus-Hypovirus Interaction. Applied and Environmental Microbiology 66: 4863-4869.
• Pimentel, G., L.M. Carris and T.L. Peever. 2000. Characterization of interspecific hybrids between Tilletia controversa and T. bromi. Mycologia 92: 411-420.
• Pimentel, G., T.L. Peever and L.M. Carris. 2000. Genetic variation among natural populations of Tilletia controversa and T. bromi. Phytopathology 90: 376-383.
• Timmer, L. W., P.D. Roberts, H.M. Darhower, P.M. Bushong, E.W. Stover, T.L. Peever and A.M. Ibanez, A. M. 2000. Epidemiology and control of citrus greasy spot in different citrus-growing areas in Florida. Plant Disease 84: 1294-1298.
• Timmer, L.W., H.M. Darhower, S.E. Zitko, T.L. Peever, A.M. Ibanez and P.M. Bushong. 2000. Environmental factors affecting the severity of Alternaria brown spot of citrus and their potential use in timing fungicide applications. Plant Disease 84: 638-643.

Progress 01/01/99 to 12/31/99

Outputs
We have continued to study the population biology of Ascochyta rabiei, an important pathogen of legumes in the Pacific Northwest. An extensive sample of A. rabiei isolates was obtained from commercial chickpea fields and WSU's Spillman Research Farm in 1999. Approximately 300 isolates were collected, grown in liquid culture and DNA extracted for molecular analysis. We have identified several combinations of PCR primers in our amplified fragment length polymorphism (AFLP) analysis which reveal genetic variation among our sampled isolates and which are suitable for population genetic analysis. We are currently screening additional primers and isolates from the Pacific Northwest to ensure that the markers will reveal enough genetic variation to be useful for population genetic studies of the fungus in this area. We performed several genetic crosses with isolates from our collection in order to determine the inheritance of the AFLP markers. To date, we have been unsuccessful in producing any ascospores but are currently testing several additional approaches to producing crosses that we are confident will yield ascospores. In collaboration with L.W. Timmer at the University of Florida, I have continued to study the population genetics and host specificity of Alternaria spp. on citrus. We are working with mycologists Emory Simmons and Pedro Crous to try to determine the identity of citrus-associated Alternaria species. We have sequenced several genomic regions and are using the sequence data for phylogenetic analysis. To date, we have sequenced the mitochondrial large and small subunits and the 5' end of the beta tubulin gene and have been unable to differentiate any of the citrus-associated Alternarias. This data has confirmed our hypothesis that all Alternaria spp. associated with citrus are closely related and are likely A. alternata. In Fall 1999, I sampled approximately 300 isolates of A. alternata from healthy citrus tissue and from brown spot lesions from two hosts in Florida. Our finding of Alternaria endophytes in citrus is novel and we plan to compare these endophytic isolates to isolates obtained from brown spot lesions and black rot lesions by determining the phylogenetic relationships among them. We have also been pursuing research on the host-specific toxin genes in A. alternata on citrus and have found that one toxin sequence (ACT1) is present in endophytic and saprophytic isolates, a result which has not been previously reported. We have sequenced the toxin gene from 17 citrus isolates and performed a phylogenetic analysis on ACT1. This analysis has revealed that isolates fall into two clades with high bootstrap support. One clade consisted of isolates from Florida and Colombia, and the other of isolates from Turkey, Israel, South Africa and Australia demonstrating that the toxin gene is differentiated between Old World and New World isolates.

Impacts
(N/A)

Publications

• Peever, T.L., Canihos, Y., Olsen, L., Ibanez, A., Liu, Y,-C. and L.W. Timmer. 1999. Population Genetic Structure and Host Specificity of Alternaria spp. Causing Brown Spot of Minneola Tangelo and Rough Lemon in Florida. Phytopathology 89: 851-860.
• Canihos, Y., Peever, T.L. and L.W. Timmer. 1999. Temperature, leaf wetness and isolate effects on infection of Minneola tangelo leaves by Alternaria sp. Plant Disease 83: 429-433.
• Peever, T.L., L. Olsen, A. Ibanez and L.W. Timmer. 1999. Population structure and host specificity of Alternaria sp. causing brown spot of tangerines, grapefruit and tangerine hybrids in Florida. Phytopathology 89 (Suppl): S59.