Population Genetic Characteristics and Mating Type Frequency of Venturia effusa from Pecan in South America.

Scab, caused by the plant-pathogenic fungus Venturia effusa, is a major disease of pecan in South America, resulting in loss of quantity and quality of nut yield. Characteristics of the populations of V. effusa in South America are unknown. We used microsatellites to describe the genetic diversity and population structure of V. effusa in South America, and determined the mating type status of the pathogen. The four hierarchically sampled orchard populations from Argentina (AR), Brazil (BRC and BRS), and Uruguay (UR) had moderate to high genotypic and gene diversity. There was evidence of population differentiation (Fst = 0.196) but the correlation between geographic distance and genetic distance was not statistically significant. Genetic differentiation was minimal between the UR, BRC, and BRS populations, and these populations were more clearly differentiated from the AR population. The MAT1-1 and MAT1-2 mating types occurred in all four orchards and their frequencies did not deviate from the 1:1 ratio expected under random mating; however, multilocus linkage equilibrium was rejected in three of the four populations. The population genetics of South American populations of V. effusa has many similarities to the population genetics of V. effusa previously described in the United States. Characterizing the populations genetics and reproductive systems of V. effusa are important to establish the evolutionary potential of the pathogen and, thus, its adaptability-and can provide a basis for informed approaches to utilizing available host resistance and determining phytosanitary needs.

Mating Type Idiomorphs, Heterothallism, and High Genetic Diversity in Venturia carpophila, Cause of Peach Scab.

Scab (caused by Venturia carpophila) is a major disease affecting peach in the eastern United States. The aims of the study were to characterize the mating-type loci in V. carpophila, determine whether they are in equilibrium, and assess the population genetic diversity and structure of the pathogen. The mating-type gene MAT1-1-1 was identified in isolate JP3-5 in an available genome sequence, and the MAT1-2-1 gene was PCR amplified from isolate PS1-1, thus indicating a heterothallic structure. Mating-type loci structures were consistent with those of other Venturia spp. (V. effusa and V. inaequalis): the mating-type gene is positioned between APN2 encoding a DNA lyase and a gene encoding a Pleckstrin homology domain. Primers designed to each of the mating-type genes and a reference gene TUB2 were used as a multiplex PCR to screen a population (n = 81) of V. carpophila from various locations in the eastern United States. Mating types in five of the nine populations studied were in equilibrium. Among the 81 isolates, there were 69 multilocus genotypes. A population genetic analysis of the populations with >10 individuals (four populations) showed them to be genetically diverse. Linkage disequilibrium was found in five of nine populations with ≥4 isolates. A discriminant analysis of principal components indicated three genetic clusters, although extensive admixture was observed. Mating-type identification in V. carpophila provides a basis for understanding reproductive methods of the pathogen and can be a basis for further studies of the genetics of the peach scab pathogen.

Chromosome-Level Reference Genome of Venturia effusa, Causative Agent of Pecan Scab.

Pecan scab, caused by Venturia effusa, is a devastating disease of pecan (Carya illinoinensis), which results in economic losses on susceptible cultivars throughout the southeastern United States. To enhance our understanding of pathogenicity in V. effusa, we have generated a complete telomere-to-telomere reference genome of V. effusa isolate FRT5LL7-Albino. By combining Illumina MiSeq and Oxford Nanopore MinION data, we assembled a 45.2-Mb genome represented by 20 chromosomes and containing 10,820 putative genes, of which 7,619 have at least one functional annotation. The likely causative mutation of the albino phenotype was identified as a single base insertion and a resulting frameshift in the gene encoding the polyketide synthase ALM1. This genome represents the first full chromosome-level assembly of any Venturia sp.

Simulated folivory increases vertical transmission of fungal endophytes that deter herbivores and alter tolerance to herbivory in Poa autumnalis.

BACKGROUND AND AIMS: The processes that maintain variation in the prevalence of symbioses within host populations are not well understood. While the fitness benefits of symbiosis have clearly been shown to drive changes in symbiont prevalence, the rate of transmission has been less well studied. Many grasses host symbiotic fungi (Epichloë spp.), which can be transmitted vertically to seeds or horizontally via spores. These symbionts may protect plants against herbivores by producing alkaloids or by increasing tolerance to damage. Therefore, herbivory may be a key ecological factor that alters symbiont prevalence within host populations by affecting either symbiont benefits to host fitness or the symbiont transmission rate. Here, we addressed the following questions: Does symbiont presence modulate plant tolerance to herbivory? Does folivory increase symbiont vertical transmission to seeds or hyphal density in seedlings? Do plants with symbiont horizontal transmission have lower rates of vertical transmission than plants lacking horizontal transmission? METHODS: We studied the grass Poa autumnalis and its symbiotic fungi in the genus Epichloë. We measured plant fitness (survival, growth, reproduction) and symbiont transmission to seeds following simulated folivory in a 3-year common garden experiment and surveyed natural populations that varied in mode of symbiont transmission. KEY RESULTS: Poa autumnalis hosted two Epichloë taxa, an undescribed vertically transmitted Epichloë sp. PauTG-1 and E. typhina subsp. poae with both vertical and horizontal transmission. Simulated folivory reduced plant survival, but endophyte presence increased tolerance to damage and boosted fitness. Folivory increased vertical transmission and hyphal density within seedlings, suggesting induced protection for progeny of damaged plants. Across natural populations, the prevalence of vertical transmission did not correlate with symbiont prevalence or differ with mode of transmission. CONCLUSIONS: Herbivory not only mediated the reproductive fitness benefits of symbiosis, but also promoted symbiosis prevalence by increasing vertical transmission of the fungus to the next generation. Our results reveal a new mechanism by which herbivores could influence the prevalence of microbial symbionts in host populations.

Variation Among Orchardgrass (Dactylis glomerata) Germplasm for Choke Prevalence Caused by Epichloë typhina.

Orchardgrass, or cocksfoot (Dactylis glomerata L.), is a cool-season forage grass susceptible to the choke disease caused by Epichloë typhina. Choke has been reported in orchardgrass seed production fields across the temperate regions of the world, but fungicides have not been efficacious in reducing choke incidence or prevalence. To assess the potential for genetic resistance or tolerance of orchardgrass to choke, we evaluated the variation in orchardgrass cultivars and accessions for choke prevalence and characterized infected plants for endophyte secondary metabolite and mating type gene presence. Significant variation was detected across years and locations. Choke prevalence did not always increase with the age of the stand, nor did choke prevalence correlate with flowering time or swathing time of the entries. Both mating types of E. typhina were detected in approximately equal proportions, and no evidence for loline, ergot alkaloid, or indole-diterpene biosynthesis was found. Plants with multiple infected tillers often showed more than one mating type present in the plant, indicating multiple infection events rather than a single infection event that spread to multiple tillers. Both accessions and cultivars with significant choke, and no choke, were detected, which constitute sources of germplasm for further testing and breeding.

Fine-Scale Population Genetic Structure and Within-Tree Distribution of Mating Types of Venturia effusa, Cause of Pecan Scab in the United States.

Scab (caused by Venturia effusa) is the major disease of pecan in the southeastern United States. There is no information available on the fine-scale population genetic diversity or the occurrence of clonal types at small spatial scales that provides insight into inoculum sources and dispersal mechanisms, and potential opportunity for sexual reproduction. To investigate fine-scale genetic diversity, four trees of cultivar Wichita (populations) were sampled hierarchically: within each tree canopy, four approximately evenly spaced terminals (subpopulations) were selected and up to six leaflets (sub-subpopulations) were sampled from different compound leaves on each terminal. All lesions (n = 1 to 8) on each leaflet were sampled. The isolates were screened against a panel of 29 informative microsatellite markers and the resulting multilocus genotypes (MLG) subject to analysis. Mating type was also determined for each isolate. Of 335 isolates, there were 165 MLG (clonal fraction 49.3%). Nei's unbiased measure of genetic diversity for the clone-corrected data were moderate to high (0.507). An analysis of molecular variance demonstrated differentiation (P = 0.001) between populations on leaflets within individual terminals and between terminals within trees in the tree canopies, with 93.8% of variance explained among isolates within leaflet populations. Other analyses (minimum-spanning network, Bayesian, and discriminant analysis of principal components) all indicated little affinity of isolate for source population. Of the 335 isolates, most unique MLG were found at the stratum of the individual leaflets (n = 242), with similar total numbers of unique MLG observed at the strata of the terminal (n = 170), tree (n = 166), and orchard (n = 165). Thus, the vast majority of shared clones existed on individual leaflets on a terminal at the scale of 10s of centimeters or less, indicating a notable component of short-distance dispersal. There was significant linkage disequilibrium (P < 0.001), and an analysis of Psex showed that where there were multiple encounters of an MLG, they were most probably the result of asexual reproduction (P < 0.05) but there was no evidence that asexual reproduction was involved in single or first encounters of an MLG (P > 0.05). Overall, the MAT1-1-1 and MAT1-2-1 idiomorphs were at equilibrium (73:92) and in most populations, subpopulations, and sub-subpopulations. Both mating types were frequently observed on the same leaflet. The results provide novel information on the characteristics of populations of V. effusa at fine spatial scales, and provide insights into the dispersal of the organism within and between trees. The proximity of both mating idiomorphs on single leaflets is further evidence of opportunity for development of the sexual stage in the field.

Evidence for Sexual Reproduction: Identification, Frequency, and Spatial Distribution of Venturia effusa (Pecan Scab) Mating Type Idiomorphs.

Venturia effusa (syn. Fusicladium effusum), causal agent of pecan scab, is the most prevalent pathogen of pecan (Carya illinoinensis), causing severe yield losses in the southeastern United States. V. effusa is currently known only by its asexual (conidial) stage. However, the degree and distribution of genetic diversity observed within and among populations of V. effusa are typical of a sexually reproducing fungal pathogen, and comparable with other dothideomycetes with a known sexual stage, including the closely related apple scab pathogen, V. inaequalis. Using the mating type (MAT) idiomorphs from V. inaequalis, we identified a single MAT gene, MAT1-1-1, in a draft genome of V. effusa. The MAT1-1-1 locus is flanked by two conserved genes encoding a DNA lyase (APN2) and a hypothetical protein. The MAT locus spanning the flanking genes was amplified and sequenced from a subset of 14 isolates, of which 7 contained MAT1-1-1 and the remaining samples contained MAT1-2-1. A multiplex polymerase chain reaction screen was developed to amplify MAT1-1-1, MAT1-2-1, and a conserved reference gene encoding ß-tubulin, and used to screen 784 monoconidial isolates of V. effusa collected from 11 populations of pecan across the southeastern United States. A hierarchical sampling protocol representing region, orchard, and tree allowed for analysis of MAT structure at different spatial scales. Analysis of this collection revealed the frequency of the MAT idiomorphs is in a 1:1 equilibrium of MAT1-1:MAT1-2. The apparent equilibrium of the MAT idiomorphs provides impetus for a renewed effort to search for the sexual stage of V. effusa. [Formula: see text] Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Population Genetic Structure of Venturia effusa, Cause of Pecan Scab, in the Southeastern United States.

Venturia effusa is the most important pathogen of pecan in the southeastern United States. Little information exists on the population biology and genetic diversity of the pathogen. A hierarchical sampling of 784 isolates from 63 trees in 11 pecan orchards in the southeastern United States were screened against a set of 30 previously characterized microsatellite markers. Populations were collected from Georgia (n = 2), Florida (n = 1), Alabama (n = 2), Mississippi (n = 1), Louisiana (n = 1), Illinois (n = 1), Oklahoma (n = 1), Texas (n = 1), and Kansas (n = 1). Clonality was low in all orchard populations (≤10.1% of isolates), and there were consistently high levels of genotypic diversity (Shannon-Weiner's index = 3.49 to 4.59) and gene diversity (Nei's measure = 0.513 to 0.713). Analysis of molecular variance showed that, although 81% of genetic diversity occurred at the scale of the individual tree, 16% occurred between orchards and only 3% between trees within orchards. All populations could be differentiated from each other (P = 0.01), and various cluster analyses indicated that some populations were more closely related compared with other pairs of populations. This is indicative of some limited population differentiation in V. effusa in the southeastern United States. Bayesian and nearest-neighbor methods suggested eight clusters, with orchards from Georgia and Florida being grouped together. A minimum spanning tree of all 784 isolates also indicated some isolate identification with source population. Linkage disequilibrium was detected in all but one population (Kansas), although 8 of the 11 populations had <20% of loci at disequilibrium. A Mantel test demonstrated a relationship between physical and genetic distance between populations (Z = 11.9, r = 0.559, P = 0.001). None of the populations were at mutation-drift equilibrium. All but 3 of the 11 populations had a deficiency of gene diversity compared with that expected at mutation-drift equilibrium (indicating population expansion); the remaining populations had an excess of gene diversity compared with that expected at mutation-drift equilibrium (indicating a recent bottleneck). These observations are consistent with the known history of pecan and pecan scab, which is that V. effusa became an issue on cultivated pecan in the last approximately 120 years (recent population expansion). Recently reported mating type genes and the sexual stage of this fungus may help explain the observed population characteristics, which bear a strong resemblance to those of other well-characterized sexually reproducing ascomycete pathogens.

Interspecific and intraspecific hybrid Epichloë species symbiotic with the North American native grass Poa alsodes.

The endophyte presence and diversity in natural populations of Poa alsodes were evaluated along a latitudinal transect from the southern distribution range in North Carolina to New York. Two distinct Epichloë hybrid taxa were identified from 23 populations. Each taxon could easily be distinguished by polymerase chain reaction (PCR) genotyping with primers designed to mating type genes and alkaloid biosynthesis genes that encode key pathway steps for ergot alkaloids, indole-diterpenes, lolines, and peramine. The most commonly found Epichloë taxon, Poa alsodes Taxonomic Group-1 (PalTG-1), was detected in 22 populations at high infection frequencies (72-100%), with the exception of one population at high elevation (26% infection). The second taxon, PalTG-2, was observed only in five populations in Pennsylvania constituting 12% of infected samples. Phylogenetic analyses placed PalTG-1 as an interspecific hybrid of E. amarillans and E. typhina subsp. poae ancestors, and it is considered a new hybrid species, which the authors name Epichloë alsodes. PalTG-2 is an intraspecific hybrid of two E. typhina subsp. poae ancestors, similar to E. schardlii from the host Cinna arundinacea, which the authors propose as a new variety, Epichloë schardlii var. pennsylvanica. Epichloë alsodes isolates were all mating type MTA MTB and tested positive for dmaW, easC, perA, and some LOL genes, but only the alkaloid N-acetylnorloline was detected in E. alsodes-infected plant material. Epichloë schardlii var. pennsylvanica isolates were all mating type MTB MTB and tested positive for perA, but peramine was not produced. Both E. alsodes and E. schardlii var. pennsylvanica appeared to have complete perA genes, but point mutations were identified in E. alsodes that would render the encoded perA gene nonfunctional.

Long-term ungulate exclusion reduces fungal symbiont prevalence in native grasslands.

When symbionts are inherited by offspring, they can have substantial ecological and evolutionary consequences because they occur in all host life stages. Although natural frequencies of inherited symbionts are commonly <100 %, few studies investigate the ecological drivers of variation in symbiont prevalence. In plants, inherited fungal endophytes can improve resistance to herbivory, growth under drought, and competitive ability. We evaluated whether native ungulate herbivory increased the prevalence of a fungal endophyte in the common, native bunchgrass, Festuca campestris (rough fescue, Poaceae). We used large-scale (1 ha) and long-term (7-10 year) fencing treatments to exclude native ungulates and recorded shifts in endophyte prevalence at the scale of plant populations and for individual plants. We characterized the fungal endophyte in F. campestris, Epichloë species FcaTG-1 (F. campestris taxonomic group 1) for the first time. Under ungulate exclusion, endophyte prevalence was 19 % lower in plant populations, 25 % lower within plant individuals, and 39 % lower in offspring (seeds) than in ungulate-exposed controls. Population-level endophyte frequencies were also negatively correlated with soil moisture across geographic sites. Observations of high within-plant variability in symbiont prevalence are novel for the Epichloë species, and contribute to a small, but growing, literature that documents phenotypic plasticity in plant-endophyte symbiota. Altogether, we show that native ungulates can be an important driver of symbiont prevalence in native plant populations, even in the absence of evidence for direct mechanisms of mammal deterrence. Understanding the ecological controls on symbiont prevalence could help to predict future shifts in grasslands that are dominated by Epichloë host plants.

Plant-symbiotic fungi as chemical engineers: multi-genome analysis of the clavicipitaceae reveals dynamics of alkaloid loci.

The fungal family Clavicipitaceae includes plant symbionts and parasites that produce several psychoactive and bioprotective alkaloids. The family includes grass symbionts in the epichloae clade (Epichloë and Neotyphodium species), which are extraordinarily diverse both in their host interactions and in their alkaloid profiles. Epichloae produce alkaloids of four distinct classes, all of which deter insects, and some-including the infamous ergot alkaloids-have potent effects on mammals. The exceptional chemotypic diversity of the epichloae may relate to their broad range of host interactions, whereby some are pathogenic and contagious, others are mutualistic and vertically transmitted (seed-borne), and still others vary in pathogenic or mutualistic behavior. We profiled the alkaloids and sequenced the genomes of 10 epichloae, three ergot fungi (Claviceps species), a morning-glory symbiont (Periglandula ipomoeae), and a bamboo pathogen (Aciculosporium take), and compared the gene clusters for four classes of alkaloids. Results indicated a strong tendency for alkaloid loci to have conserved cores that specify the skeleton structures and peripheral genes that determine chemical variations that are known to affect their pharmacological specificities. Generally, gene locations in cluster peripheries positioned them near to transposon-derived, AT-rich repeat blocks, which were probably involved in gene losses, duplications, and neofunctionalizations. The alkaloid loci in the epichloae had unusual structures riddled with large, complex, and dynamic repeat blocks. This feature was not reflective of overall differences in repeat contents in the genomes, nor was it characteristic of most other specialized metabolism loci. The organization and dynamics of alkaloid loci and abundant repeat blocks in the epichloae suggested that these fungi are under selection for alkaloid diversification. We suggest that such selection is related to the variable life histories of the epichloae, their protective roles as symbionts, and their associations with the highly speciose and ecologically diverse cool-season grasses.

Disparate independent genetic events disrupt the secondary metabolism gene perA in certain symbiotic Epichloë species.

Peramine is an insect-feeding deterrent produced by Epichloë species in symbiotic association with C3 grasses. The perA gene responsible for peramine synthesis encodes a two-module nonribosomal peptide synthetase. Alleles of perA are found in most Epichloë species; however, peramine is not produced by many perA-containing Epichloë isolates. The genetic basis of these peramine-negative chemotypes is often unknown. Using PCR and DNA sequencing, we analyzed the perA genes from 72 Epichloë isolates and identified causative mutations of perA null alleles. We found nonfunctional perA-ΔR* alleles, which contain a transposon-associated deletion of the perA region encoding the C-terminal reductase domain, are widespread within the Epichloë genus and represent a prevalent mutation found in nonhybrid species. Disparate phylogenies of adjacent A2 and T2 domains indicated that the deletion of the reductase domain (R*) likely occurred once and early in the evolution of the genus, and subsequently there have been several recombinations between those domains. A number of novel point, deletion, and insertion mutations responsible for abolishing peramine production in full-length perA alleles were also identified. The regions encoding the first and second adenylation domains (A1 and A2, respectively) were common sites for such mutations. Using this information, a method was developed to predict peramine chemotypes by combining PCR product size polymorphism analysis with sequencing of the perA adenylation domains.

Alkaloid variation among epichloid endophytes of sleepygrass (Achnatherum robustum) and consequences for resistance to insect herbivores.

Epichloid endophytes are well known symbionts of many cool-season grasses that may alleviate environmental stresses for their hosts. For example, endophytes produce alkaloid compounds that may be toxic to invertebrate or vertebrate herbivores. Achnatherum robustum, commonly called sleepygrass, was aptly named due to the presence of an endophyte that causes toxic effects to livestock and wildlife. Variation in alkaloid production observed in two A. robustum populations located near Weed and Cloudcroft in the Lincoln National Forest, New Mexico, suggests two different endophyte species are present in these populations. Genetic analyses of endophyte-infected samples revealed major differences in the endophyte alkaloid genetic profiles from the two populations, which were supported with chemical analyses. The endophyte present in the Weed population was shown to produce chanoclavine I, paspaline, and terpendoles, so thus resembles the previously described Epichloë funkii. The endophyte present in the Cloudcroft population produces chanoclavineI, ergonovine, lysergic acid amide, and paspaline, and is an undescribed endophyte species. We observed very low survival rates for aphids feeding on plants infected with the Cloudcroft endophyte, while aphid survival was better on endophyte infected plants in the Weed population. This observation led to the hypothesis that the alkaloid ergonovine is responsible for aphid mortality. Direct testing of aphid survival on oat leaves supplemented with ergonovine provided supporting evidence for this hypothesis. The results of this study suggest that alkaloids produced by the Cloudcroft endophyte, specifically ergonovine, have insecticidal properties.

Deletion of the fungal gene soft disrupts mutualistic symbiosis between the grass endophyte Epichloë festucae and the host plant.

Hyphal anastomosis, or vegetative hyphal fusion, establishes the interconnection of individual hyphal strands into an integrated network of a fungal mycelium. In contrast to recent advances in the understanding of the molecular basis for hyphal anastomosis, knowledge of the physiological role of hyphal anastomosis in the natural habitats of filamentous fungi is still very limited. To investigate the role of hyphal anastomosis in fungal endophyte-plant interactions, we generated mutant strains lacking the Epichloë festucae soft (so) gene, an ortholog of the hyphal anastomosis gene so in the endophytic fungus E. festucae. The E. festucae Δso mutant strains grew similarly to the wild-type strain in culture but with reduced aerial hyphae and completely lacked hyphal anastomosis. The most striking phenotype of the E. festucae Δso mutant strain was that it failed to establish a mutualistic symbiosis with the tall fescue plant host (Lolium arundinaceum); instead, it killed the host plant within 2 months after the initial infection. Microscopic examination revealed that the death of the tall fescue plant host was associated with the distortion and disorganization of plant cells. This study suggests that hyphal anastomosis may have an important role in the establishment/maintenance of fungal endophyte-host plant mutualistic symbiosis.

Epichloe canadensis, a new interspecific epichloid hybrid symbiotic with Canada wildrye (Elymus canadensis).

Many Epichloë endophytes found in cool-season grasses are interspecific hybrids possessing much or all of the genomes of two or three progenitors. Here we characterize Epichloë canadensis sp. nov., a hybrid species inhabiting the grass species Elymus canadensis native to North America. Three distinct morphotypes were identified that were separated into two groups by molecular phylogenetic analysis. Sequence analysis of the translation elongation factor 1-α (tefA) and ß-tubulin (tubB) genes revealed two copies in all isolates examined. Phylogenetic analyses indicated that allele 1 of each gene was derived from Epichloë amarillans and allele 2 from Epichloë elymi. This is the first documentation of an interspecific hybrid endophyte derived from parents of strictly North American origins. Alkaloid gene profiling using primers specific to genes in the peramine, loline, indole-diterpene and ergot alkaloid pathways may indicate chemotypic variation in the ergot alkaloid and loline pathways between the assigned morphotypes. All isolates have the gene enabling the production of peramine but lack genes in the indole-diterpene biosynthesis pathway. Morphology and phylogenetic evidence support the designation of isolates from El. canadensis as a new interspecific hybrid species.

Prevalence of an intraspecific Neotyphodium hybrid in natural populations of stout wood reed (Cinna arundinacea L.) from eastern North America.

Members of genus Neotyphodium are asexual derivatives of sexual Epichloë species and maintain endophytic relationships with many cool-season grasses. Most Neotyphodium species analyzed so far are interspecific hybrids with combined or partial genomes of two or three ancestral species. In this study we characterized Neotyphodium isolates from Cinna arundinacea, a perennial cool-season grass from eastern North America. A total of 23 isolates grouping into two distinct morphotypes were obtained from five local populations of C. arundinacea. PCR amplification and cloning of translation-elongation factor 1-α (tefA) and ß-tubulin (tubB) genes of 10 isolates comprising both morphotypes (two isolates per location) revealed that all 10 contain two copies of tefA and tubB genes. Surprisingly phylogenetic analysis of mainly non-coding sequence from these genes revealed that both copies in each isolate were inherited from Epichloë typhina ancestors, indicating that the C. arundinacea endophytes arose through intraspecific hybridization between two E. typhina progenitors with extant relatives infecting hosts Poa nemoralis and Poa pratensis. Furthermore the tefA sequences were identical between isolates, as were tubB sequences, despite obvious morphological differences. Profiling of alkaloid biosynthetic genes from these isolates indicated the presence of the peramine biosynthetic gene (perA) and the absence of genes required for biosynthesis of lolines, indole-diterpenes and ergot alkaloids. Thus this endophyte is potentially capable of producing peramine in planta and providing protection to its host from insect pests. The absence of genes for indole-diterpenes and ergot alkaloid biosynthesis makes this endophyte a candidate for agricultural applications. Based on our phylogenetic analysis, alkaloid profiling and description of morphological characteristics, we propose the name Neotyphodium schardlii for these isolates from C. arundinacea, a new member of genus Neotyphodium and the first described to have arisen through intraspecific hybridization.

First description of the sexual stage of Venturia effusa, causal agent of pecan scab.

Pecan scab, caused by Venturia effusa, is the most prevalent disease of pecan in the southeastern United States. Recent characterization of the mating type (MAT) distribution of V. effusa revealed that the MAT idiomorphs are in equilibrium at various spatial scales, indicative of regular sexual recombination. However, the occurrence of the sexual stage of V. effusa has never been observed, and the pathogen was previously considered to rely entirely on asexual reproduction. We were able to generate the sexual stage by pairing isolates of opposite mating types on oatmeal culture media. Cultures were incubated at 24 C for 2 mo to allow hyphae from isolates of each mating type to interact. Culture plates were then incubated at 4 C for 4 mo, after which immature pseudothecia were observed. Following exposure to a 12-h photoperiod for 2 wk at 24 C, asci and ascospores readily developed. Pseudothecium and ascospore production was optimal when incubated for 4 mo at 4 C. We utilized progeny from a cross of an albino isolate and wild-type (melanized) isolates to determine that recombination had occurred. Multilocus genotyping using 32 microsatellite markers confirmed that progeny were the result of recombination, which was further supported by segregation of mating types and culture pigmentation. Albino progeny were all confirmed to contain the same mutation in the polyketide synthase (PKS1) melanin biosynthesis gene as the albino parent. The results of this study demonstrate the heterothallic nature of V. effusa. The impact of determining the source of the overwintering ascostroma will aid in management decisions to reduce the primary inoculum in the disease cycle.

Detection of double-stranded RNA elements in the plant pathogenic fungus Rhizoctonia solani.

Many species of fungi have been shown to harbor double-stranded RNA (dsRNA) elements. A single fungal isolate of Rhizoctonia solani may have as many as five different dsRNA elements within them. The presence of specific dsRNA elements influence pathogenicity in host plants.

Phylogenetic relatedness of the M2 double-stranded RNA in Rhizoctonia fungi.

Isolates from closely related fungi in the Rhizoctonia species complex were examined for the occurrence of the M2 double-stranded RNA (dsRNA) by amplifying a conserved 1000 nucleotide region of the dsRNA with reverse transcription PCR. The M2 dsRNA was detected in representative isolates belonging to three anastomosis groups (AG) of R. solani (AG-1-IA, AG-4 and AG-6; teleomorph = Thanatephorus) and four AGs of binucleate Rhizoctonia (AG-A, AG-F, AG-R and AG-U; teleomorph = Ceratobasidium). Amplified PCR products from the 3' region of the M2 dsRNA from a representative sample of 12 isolates from eight different AGs were sequenced and subjected to parsimony analysis and coalescent simulations to infer ancestral lineages and to reconstruct the ancestral history of haplotypes. Seven dsRNA haplotypes were inferred from the sample of 12 isolates. One haplotype was composed of only isolates of Ceratobasidium belonging to different AGs. The rooted gene genealogies from coalescent simulations suggested that the ancestral M2 dsRNA haplotype most likely evolved in Thanatephorus (anamorph = R. solani AG-1-IA) and has been acquired recently by isolates of Ceratobasidium. Reconstruction of the ancestral history of haplotypes with a parsimony-based approach that assumes both mutation and recombination suggested that four haplotypes recombined before coalescing to their most recent common ancestor, while three haplotypes coalesced without recombination in the recent past. There was no unique association of haplotype within a specific AG of either Ceratobasidium or Thanatephorus to support co-evolution of the M2 dsRNA within the fungal host. To our knowledge this is the first report of a dsRNA occurring in Ceratobasidium that also is present in Thanatephorus.

Molecular identification and characterization of endophytes from uncultivated barley.

Epichloë species (Clavicipitaceae, Ascomycota) are endophytic symbionts of many cool-season grasses. Many interactions between Epichloë and their host grasses contribute to plant growth promotion, protection from many pathogens and insect pests, and tolerance to drought stress. Resistance to insect herbivores by endophytes associated with Hordeum species has been previously shown to vary depending on the endophyte-grass-insect combination. We explored the genetic and chemotypic diversity of endophytes present in wild Hordeum species. We analyzed seeds of Hordeum bogdanii, H. brevisubulatum, and H. comosum obtained from the US Department of Agriculture's (USDA) National Plant Germplasm System (NPGS), of which some have been reported as endophyte-infected. Using polymerase chain reaction (PCR) with primers specific to Epichloë species, we were able to identify endophytes in seeds from 17 of the 56 Plant Introduction (PI) lines, of which only 9 lines yielded viable seed. Phylogenetic analyses of housekeeping, alkaloid biosynthesis, and mating type genes suggest that the endophytes of the infected PI lines separate into five taxa: Epichloë bromicola, Epichloë tembladerae, and three unnamed interspecific hybrid species. One PI line contained an endophyte that is considered a new taxonomic group, Epichloë sp. HboTG-3 (H. bogdanii Taxonomic Group 3). Phylogenetic analyses of the interspecific hybrid endophytes from H. bogdanii and H. brevisubulatum indicate that these taxa all have an E. bromicola allele but the second allele varies. We verified in planta alkaloid production from the five genotypes yielding viable seed. Morphological characteristics of the isolates from the viable Hordeum species were analyzed for their features in culture and in planta. In the latter, we observed epiphyllous growth and in some cases sporulation on leaves of infected plants.