Many cool-season grasses (Poaceae subfam. Poöideae) host seed-transmissible symbionts (endophytes) in the fungal genus Epichloë, which can produce diverse alkaloids that protect against invertebrate and, in some cases, vertebrate herbivores. Rarely have population surveys been conducted to assess comprehensive alkaloid profiles and diversity of Epichloë in wild grasses. In this study, we surveyed Brachyelytrum erectum, which is a woodland grass in an early-diverging lineage of Poöideae, and commonly symbiotic with Epichloë brachyelytri. Analytical methods based on high-resolution UHPLC-MS/MS were refined to provide rapid, comprehensive detection, and quantitation of E. brachyelytri alkaloids, for six B. erectum populations in Kentucky. Chemotypes were identified with two or three of the alkaloids exo-1-acetamidopyrrolizidine (1), chanoclavine (2), and peramine (3). Both 1 and 2 are known as intermediates in biosynthetic pathways to more complex alkaloids, and chemotypes having both 1 and 2 as pathway end-products are novel. Such chemotypes were also identified in other species, and phylogenetic analysis indicated their multiple origins by a combination of convergent evolution, possible horizontal gene transfer, and interspecific hybridization. Alkaloid levels were comparable between most populations and at most plant developmental stages. Levels of 3 were compared between E. brachyelytri variants with and without 1, providing evidence for competition between the pathways in young shoots, but not in older leaves or seeds. Furthermore, levels of 1 and 2 were moderate to high compared with their respective alkaloid classes in other grass-Epichloë symbiotic systems. We conclude that production of the alkaloids likely represents an important metabolic investment by E. brachyelytri.IMPORTANCEDefensive mutualisms, symbioses of hosts with organisms that defend them against parasites or predators, play important ecological roles. A widespread example is protection of cool-season grasses by symbiotic Epichloë species, which are fungi that transmit in seeds and produce several kinds of anti-insect alkaloids. Profiles of alkaloids evolve due to shifting balances of their benefits and the costs of producing them. In this study, Epichloë brachyelytri symbiotic with the wild forest grass Brachyelytrum erectum produced three alkaloids, of which two have been rarely reported. Furthermore, variations in its alkaloid profiles and quantities of each alkaloid at different plant growth stages and tissues suggested that occasional loss of its most abundant alkaloid can be adaptive due to the metabolic load of producing it. Although rare, similar alkaloid profiles were identified in several other species in which they arose by a combination of convergent evolution, possible horizontal gene transfer, and interspecific hybridization.

A comprehensive survey of Epichloë fungal endophytes forming symbioses with the wild C3 forage grass Bromus auleticus across a transitional climate–soil zone in Uruguay was conducted. Among 83 accessions, we detected 70% were infected with Epichloë. Using PCR with 29 genetic markers targeting tefA, mating type and alkaloid biosynthesis genes we identified nine distinct Epichloë genotypes that varied in potential alkaloid production. Of these nine genotypes, 1, 2 and 3 were most commonly observed. All genotypes contained the genes required for the synthesis of pyrrolopyrazines (e.g., peramine, a known insect-feeding deterrent). Two genotypes (designated 1 and 7) contained genes required for the synthesis of pyrrolizidines (e.g. loline alkaloids, known for their broad spectrum insecticidal and insect-deterrent activity), but only genotype 1 was predicted to produce N-formylloline, a compound with strong insecticidal properties. Seven genotypes (2, 3, 4, 5, 6, 8 and 9) contained genes involved in indole diterpene (IDT) synthesis, however, only five genotypes (2, 3, 4, 5, and 6) were predicted to produce early pathway IDTs such as paspaline or terpendoles. Genotypes 8 and 9 lacked essential early pathway genes were not predicted to produce IDTs. Genotypes 3 and 7 contained EAS genes required for ergot alkaloid synthesis; however, these genotypes were only predicted to produce chanoclavine, as genes required for downstream ergovaline synthesis were absent. None of the Epichloë endophytes from this survey contained genes required for the synthesis of the mammalian toxins ergovaline or lolitrem B. As genotypes 1, 2, and 3 were the most prevalent in the collection, six plant accessions, two from each of these genotypes, were selected to evaluate the protective effects of their different Epichloë genotypes against the aphid Rhopalosiphum padi, a generalist insect, in laboratory-controlled bioassays. Notably, only the plant accessions associated with genotype 1, predicted to produce N-formylloline, significantly suppressed R. padi. Endophyte presence and distribution were strongly influenced by environmental factors, particularly water availability, soil organic matter, and temperature extremes. Additionally, plants associated with endophyte genotypes 1, 2 and 3 were grown in two contrasting environments, displayed genotype-specific effects on host performance emerged under different soil fertility conditions. These findings highlight the ecological and agronomic potential of Epichloë endophytes that would lack mammalian toxicity and could provide a foundation for developing sustainable forages with enhanced insect resistance and environmental resilience without compromising livestock health.

Fungi from the genus Epichloë form systemic and seed-transmissible endophytic infections of grasses in a symbiosis that is considered predominately mutualistic. During asymptomatic growth these Epichloë fungi produce a variety of secondary metabolites that benefit their host by providing protection against herbivory and disease. However, the Epichloë sexual cycle is antagonistic to its host, characterised by the proliferation of hyphae on reproductive tillers to form fungal stromata that encase and sterilise developing host inflorescences. Production of known host-protective Epichloë-derived secondary metabolites is suppressed in these stromata; however, we describe here the pyrrolodiazipic acids, a novel class of fungal alkaloids that are specifically produced in Epichloë stromata. Transcriptomic analysis identified the five-gene PZA cluster that, unlike other Epichloë biosynthetic gene clusters, is specifically expressed in stromata. Reconstruction of this PZA cluster in a heterologous host enabled the function of the encoded enzymes to be defined, key biosynthetic intermediates to be identified, and the final product characterised – stromatine, a pyrrolodiazepine with carboxylic acid functionality. Analysis of material from many different Epichloë-host associations revealed that stromatine was universally and exclusively found in stroma tissues. Stromatine was also produced in exceptionally large amounts, contributing almost 1% of the total stroma dry mass in some grass-endophyte associations. PZA gene deletion analyses across three different Epichloë species yielded interesting results; stromatine appears to be required for host infection by some Epichloë species, while the accumulation of PZA pathway intermediates appears to be detrimental to symbiosis. Genomic analyses reveal the PZA cluster is conserved among sexual Epichloë species but is dispensable for asexual strains. Combined with the tissue specificity and high levels of production, this suggests that stromatine plays a critical role in the Epichloë sexual cycle, with potential bioactivities and functions to be dissected further.

Scab of peach and almond is caused by Venturia carpophila, but little is known regarding the population genetics of the pathogen, which aids in understanding the epidemiology of the disease and guiding its management. Isolates (735) of V. carpophila were sampled from peach and almond orchards in the Eastern United States and California, respectively. The aims were to determine population genetic diversity and structure of V. carpophila, compare populations of the pathogen from each host, and determine mating type ratios. Measures of gene and genotypic diversity indicated greater diversity among peach as compared with almond populations of the pathogen. Mating types were consistently at equilibrium (clone corrected) for populations from peach, but none of the populations from almond were at equilibrium. The MAT1-1-1 idiomorph was at low frequency or absent from almond populations. All populations on both peach and almond exhibited linkage disequilibrium. Analysis of molecular variance showed structure (71.8% of variance at the scale of the tree and 25.3% at the scale of the geographic region or crop). A discriminant analysis of principal components showed that the peach and almond populations clustered as independent groups. The results suggest that on peach in the Southeastern United States, V. carpophila is a sexually reproducing pathogen, but on almond in California, it is likely asexual. The confounded factors of geographic location and crop host may have both contributed to population differentiation. Although more research is needed, these results have implications for our understanding of the disease epidemiology and management in the two regions.

Self-splicing group I and II introns are selfish genetic elements that are widely yet patchily distributed across the tree of life. Their selfish behavior comes from super-Mendelian inheritance behaviors, collectively called "homing", which allow them to rapidly spread within populations to the specific genomic sites they home into. Observations of self-splicing intron evolutionary dynamics have led to the formulation of an intron "lifecycle" model where, once fixed in a population, the introns lose selection for homing and undergo an extensive period of degradation until their eventual loss. Here, we find that self-splicing introns are common in the mitochondrial genomes of Epichloë species, endophytic fungi that live in symbioses with grasses. However, these introns show substantial intron presence-absence polymorphism, with our analyses suggesting that these result from a combination of vertical intron inheritance coupled with multiple invasion and loss events over the course of Epichloë evolution. Surprisingly, we find little evidence for the extensive intron degradation expected under the existing intron lifecycle model. Instead, these introns in Epichloë appear to be lost soon after fixation, suggesting that Epichloë self-splicing introns have a different lifecycle. However, rapid intron loss alone cannot explain our results, indicating that additional factors, such as the evolution of homing suppressors, also contribute to Epichloë self-splicing intron dynamics. This work shows that self-splicing introns have more diverse evolutionary dynamics than previously appreciated.

Epichlo & euml; species are systemic, often seed-transmissible symbionts (endophytes) of cool-season grasses (Poaceae subfam. Po & ouml;ideae) that produce up to four classes of bioprotective alkaloids. Whereas haploid Epichlo & euml; species may reproduce sexually and transmit between host plants (horizontally), many Epichlo & euml; species are polyploid hybrids that are exclusively transmitted via seeds (vertically). Therefore, the generation of, and selection on, chemotypic (alkaloid) profiles and diversity should differ between haploids and hybrids. We undertook a genome-level analysis of haploids and polyploid hybrids, emphasizing hybrids that produce lolines, which are potent broad-spectrum anti-invertebrate alkaloids that can accumulate to levels up to 2% of plant dry mass. Prior phylogenetic analysis had indicated that loline alkaloid gene clusters (LOL) in many hybrids are from the haploid species Epichlo & euml; bromicola, but no LOL-containing E. bromicola strains were previously identified. We discovered LOL-containing E. bromicola from host grasses Bromus tomentellus and Melica persica in a Mediterranean-climate region (MCR) in Isfahan Province, Iran, and from Thinopyrum intermedium in Poland. The isolates from B. tomentellus and M. persica were closely related and had nearly identical alkaloid gene profiles, and their LOL clusters were most closely related to those of several Epichlo & euml; hybrids. In contrast, several LOL genes in the isolate from T. intermedium were phylogenetically more basal in genus Epichlo & euml;, indicating trans-species polymorphism. While identifying likely hybrid ancestors, this study also revealed novel host ranges in central Iran, with the first observation of E. bromicola in host tribe Meliceae and of Epichlo & euml; festucae in host tribe Bromeae. We discuss the possibility that MCRs may be hotspots for diversification of grass-Epichlo & euml; symbioses via extended host ranges and interspecific hybridization of the symbionts.

The genus Epichloë consists of fungal endophytes that have co-evolved with many Pooideae grass species. Epichloë species display variability in endophytic lifestyle and the propensity to hybridize, and these features make them an interesting group to study the dynamics of selfish elements. Self-splicing group I and II introns, selfish genetic elements that are widely yet patchily distributed across the tree of life, are found in Epichloë species, particularly in their mitochondrial genomes. These introns are considered selfish as they display super-Mendelian inheritance as a result of processes, collectively termed ‘homing’, that allow them to rapidly spread, both within and between populations, into the specific genomic sites they occupy. This study found that the mitochondrial genomes of Epichloë species contain a wide variety of group I and group II self-splicing introns, but these introns show substantial presence-absence polymorphism. Strikingly, the distribution of these introns does not correlate with the known phylogeny of Epichloë, nor does it correlate with Epichloë sexuality/asexuality or hybridity. Moreover, phylogenies made using Epichloë mitochondrial genomes do not correlate with those made nuclear genes. This study showed that the best explanation for the distribution of these self-splicing introns is ongoing invasions and losses over the course of Epichloë evolution. Unexpectedly, however, little evidence was found for the extensive intron degradation that the current self-splicing intron model predicts should occur prior to complete intron loss. Instead, results suggest Epichloë mitochondrial self-splicing introns are rapidly lost following their fixation. However, analysis suggests that additional factors, such as the evolution of homing suppressors, likely contribute to Epichloë self-splicing intron dynamics. Collectively, the results from this study show that self-splicing introns have more diverse evolutionary dynamics than previously appreciated. However, the results also present a conundrum as they suggest there has been relatively frequent exchange of mitochondrial introns between these endophytic Epichloë lineages over the course of genus evolution. The nature of these exchanges and whether they underlie the inconsistency between mitochondrial and nuclear Epichloë phylogenies are unknown and deserve further attention.

Pecan scab, caused by Venturia effusa, is the most destructive disease of pecan in the south-eastern United States. The fungus was assumed to perpetuate solely through asexually produced conidia. Yet the identification of heterothallic mating type idiomorphs has led to successful crosses producing pseudothecia in vitro. To investigate factors influencing the development of pseudothecia, three isolates of each mating type (MAT1-1 x MAT1-2) were crossed pairwise on oatmeal agar. Pseudothecial production and maturation were assessed at 4, 8, 12, 16 and 22 degrees C, with incubation periods of 2, 3 or 4 months, either with or without light (0 or 12 h photoperiod) during maturation. There were significant effects of temperature, month and cross on the production of pseudothecia. Similar numbers of pseudothecia were produced at 4, 8, 12 and 16 degrees C (1.8-2.3 pseudothecia), but significantly fewer at 22 degrees C (0.1 pseudothecia). Pseudothecia remained immature at 22 degrees C suggesting a cold period is required for ascospore maturation. The isolate and mating type cross affected the number of pseudothecia, with both MAT1-1 and MAT1-2 contributing to the number produced, which interacted with time. MAT1-1 had the greatest effect on the number of pseudothecia produced, but this was modulated by MAT2-2. At the highest temperature there were no differences among mating type crosses. Light had no effect on maturation of pseudothecia in this study. The results indicate the likely temperatures and durations required to produce pseudothecia in the field; ascospores may play a role as primary inoculum in the disease cycle of pecan scab.

Pecan scab is a devastating disease that causes damage to pecan ( Carya illinoinensis (Wangenh.) K. Koch) fruit and leaves. The disease is caused by the fungus Venturia effusa (G. Winter) and the main management practice for controlling the disease is by application of fungicides at 2-to-3-week intervals throughout the growing season. Besides disease-related yield loss, application of fungicides can result in considerable cost and increases the likelihood of fungicide resistance developing in the pathogen. Resistant cultivars are available for pecan growers; although, in several cases resistance has been overcome as the pathogen adapts to infect resistant hosts. Despite the importance of host resistance in scab management, there is little information regarding the molecular basis of genetic resistance to pecan scab. The purpose of this study was to elucidate mechanisms of natural pecan scab resistance by analyzing transcripts that are differentially expressed in pecan leaf samples from scab resistant and susceptible trees. The leaf samples were collected from trees in a provenance collection orchard that represents the natural range of pecan in the US and Mexico. Trees in the orchard have been exposed to natural scab infections since planting in 1989, and scab ratings were collected over three seasons. Based on this data, ten susceptible trees and ten resistant trees were selected for analysis. RNA-seq data was collected and analyzed for diseased and non-diseased parts of susceptible trees as well as for resistant trees. A total of 313 genes were found to be differentially expressed when comparing resistant and susceptible trees without disease. For susceptible samples showing scab symptoms, 1,454 genes were identified as differentially expressed compared to non-diseased susceptible samples. Many genes involved in pathogen recognition, defense responses, and signal transduction were up-regulated in diseased samples of susceptible trees, whereas differentially expressed genes in pecan scab resistant samples were generally down-regulated compared to non-diseased susceptible samples. Our results provide the first account of candidate genes involved in resistance/susceptibility to pecan scab under natural conditions in a pecan orchard. This information can be used to aid pecan breeding programs and development of biotechnology-based approaches for generating pecan cultivars with more durable scab resistance.

Dr. Young has been internationally recognized for research on seed transmitted fungal endophytes and their impact on forage grazing systems, endophyte diversity in native grasses and crop wild relatives, pecan scab and a root rot pathogen of alfalfa. She has discovered genes for the synthesis of bioprotective alkaloids, identified and described new endophyte species, and developed genomic and culture resources. Dr. Young will share research on utilizing genomic techniques to better understand the various endophyte species in forage grazing systems.

Scab (Venturia effusa) is the major cause of economic loss in pecan in the south-eastern USA. We explored population genetic diversity and structure among orchards of cv. Desirable and native seedlings, and within-orchard variability among trees of all cultivars sampled. We compared the ability of 30, 15 and 7 previously developed microsatellites to characterize the population genetic diversity and structure of V. effusa. Analyses of molecular variance (AMOVA) provided little evidence of structure dependent on cultivar, but there was some evidence of structure between orchards of a cultivar based on distance. Individual orchard AMOVA showed that three of 11 orchards had between-tree population structure. Among six populations from cv. Desirable, a Mantel test showed that geographic distance was related to the pairwise genetic divergence (R-2 = 0.84). Among 11 orchards of various cultivars there was little difference in diversity using 30, 15 or 7 markers, or population structure based on AMOVA. Some minor differences in population structure were seen based on discriminant analysis of principal components, or dendrograms. Thus, depending on the objectives, future studies may use as few as 15 or 7 markers without losing ability to discern population genetic diversity or structure. More populations exhibited linkage disequilibrium when using 15 or 30 markers compared to when using seven markers. Knowledge of population genetics of V. effusa in relation to host genotype is needed to understand pathogen population interactions and gene flow, knowledge that will help underpin future breeding efforts to develop durable resistance in this long-lived orchard tree.

Genome rearrangements in filamentous fungi are prevalent but little is known about the modalities of their evolution, in part because few complete genomes are available within a single genus. To address this, we have generated and compared 15 complete telomere-to-telomere genomes across the phylogeny of a single genus of filamentous fungi, Epichloë. We find that the striking distinction between gene-rich and repeat-rich regions previously reported for isolated species is ubiquitous across the Epichloë genus. We built a species phylogeny from single-copy gene orthologs to provide a comparative framing to study chromosome composition and structural change through evolutionary time. All Epichloë genomes have exactly seven nuclear chromosomes, but despite this conserved ploidy, analyses reveal low synteny and substantial rearrangement of gene content across the genus. These rearrangements are highly lineage-dependent, with most occurring over short evolutionary distances, with long periods of structural stasis. Quantification of chromosomal rearrangements shows they are uncorrelated with numbers of substitutions and evolutionary distances, suggesting that different modes of evolution are acting to create nucleotide and chromosome-scale changes.

Abstract Fungi from the genus Epichloë form systemic endobiotic infections of cool season grasses, producing a range of host-protective natural products in return for access to nutrients. These infections are asymptomatic during vegetative host growth, with associations between asexual Epichloë spp. and their hosts considered mutualistic. However, the sexual cycle of Epichloë spp. involves virulent growth, characterized by the envelopment and sterilization of a developing host inflorescence by a dense sheath of mycelia known as a stroma. Microscopic analysis of stromata revealed a dramatic increase in hyphal propagation and host degradation compared with asymptomatic tissues. RNAseq was used to identify differentially expressed genes in asymptomatic vs stromatized tissues from 3 diverse Epichloë–host associations. Comparative analysis identified a core set of 135 differentially expressed genes that exhibited conserved transcriptional changes across all 3 associations. The core differentially expressed genes more strongly expressed during virulent growth encode proteins associated with host suppression, digestion, adaptation to the external environment, a biosynthetic gene cluster, and 5 transcription factors that may regulate Epichloë stroma formation. An additional 5 transcription factor encoding differentially expressed genes were suppressed during virulent growth, suggesting they regulate mutualistic processes. Expression of biosynthetic gene clusters for natural products that suppress herbivory was universally suppressed during virulent growth, and additional biosynthetic gene clusters that may encode production of novel host-protective natural products were identified. A comparative analysis of 26 Epichloë genomes found a general decrease in core differentially expressed gene conservation among asexual species, and a specific decrease in conservation for the biosynthetic gene cluster expressed during virulent growth and an unusual uncharacterized gene.

Drought stress reduces crop biomass yield and the profitability of rainfed agricultural systems. Evaluation of populations or accessions adapted to diverse geographical and agro-climatic environments sheds light on beneficial plant responses to enhance and optimize yield in resource-limited environments. This study used the morphological and physiological characteristics of leaves and roots from two different alfalfa subspecies during progressive drought stress imposed on controlled and field conditions. Two different soils (Experiments 1 and 2) imposed water stress at different stress intensities and crop stages in the controlled environment. Algorithm-based image analysis of leaves and root systems revealed key morphological and physiological traits associated with biomass yield under stress. The Medicago sativa subspecies (ssp.) sativa population, PI478573, had smaller leaves and maintained higher chlorophyll content (CC), leaf water potential, and osmotic potential under water stress. In contrast, M. sativa ssp. varia, PI502521, had larger leaves, a robust root system, and more biomass yield. In the field study, an unmanned aerial vehicle survey revealed PI502521 to have a higher normalized difference vegetation index (vegetation cover and plant health characteristics) throughout the cropping season, whereas PI478573 values were low during the hot summer and yielded low biomass in both irrigated and rainfed treatments. RhizoVision Explorer image analysis of excavated roots revealed a smaller diameter and a narrow root angle as target traits to increase alfalfa biomass yield irrespective of water availability. Root architectural traits such as network area, solidity, volume, surface area, and maximum radius exhibited significant variation at the genotype level only under limited water availability. Different drought-adaptive strategies identified across subspecies populations will benefit the plant under varying levels of water limitation and facilitate the development of alfalfa cultivars suitable across a broad range of growing conditions. The alleles from both subspecies will enable the development of drought-tolerant alfalfa with enhanced productivity under limited water availability.

The objective of this study was to analyze animal performance following renovation of toxic-infected tall fescue (TF) to novel endophyte tall fescue (NE) in order to increase producer adoption of NE. In 2018, three renovation strategies were implemented in a randomized complete block design. Strategies included: 1) control (C), 2) renovation to NE after one season of a single-specie cover crop (1-SM), 3) renovation to NE after three seasons of a single-specie cover crop (3-SM), and 4) renovation to NE after three seasons of a multi-specie cover crop (3-CM). Forty-eight pregnant (30-90-d) Angus heifers (initial BW 383 kg ± 32) were randomly assigned to rotationally graze spring growth for 56-d, two years after NE establishment. Pre- and post-grazing forage mass was measured every 7-d. Animal measurements were collected every 14-d. Data were analyzed using procGLIMMIX of SASv9.4. Average daily gain was greatest (P < 0.0001) for cattle grazing 3-CM (0.74 kg/d), 1-SM (0.72 kg/d), and 3-SM (0.62 kg/d) and least for C (0.33 kg/d). Pre-grazing forage biomass was greatest (P = 0.0157) in 3-CM (3261 kg/ha) and lowest in C (2523 kg/ha), with 3-SM (2813 kg/ha) and 1-SM (2779 kg/ha) being intermediate. Post-grazing forage biomass was greatest (P = 0.0063) in 3-CM (1982 kg/ha), C (1916 kg/ha), 1-SM (1900 kg/ha), but lower in 3-SM (1592 kg/ha). Prior to grazing, body condition score (BCS) was higher (P = 0.0029) for animals grazing C (5.48), but change in BCS of animals within treatments was not different (P = 0.798). Hair shedding scores (HS) were not different on d-0, with all treatments near 5. However, HS were highest (P < 0.0001) in C (4.75), and lowest in 1-SM (1.75), 3-SM (2), and 3-CM (2) after 56-d. Data suggest animal performance for NE was improved compared to TF during spring grazing in years following renovation.

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.

Phymatotrichopsis omnivora is a member of Pezizomycetes and causes root rot disease on a broad range of dicotyledonous plants. Using recently generated draft genome sequence data from four P. omnivora isolates, we developed simple sequence repeat (SSR) markers and identified both mating type genes (MAT1-1-1 and MAT1-2-1) in this fungus. To understand the genetic diversity of P. omnivora isolates (n = 43) and spore mats (n = 29) collected from four locations (Oklahoma, Texas, Arizona, and Mexico) and four host crops (cotton, alfalfa, peach, and soybean), we applied 24 SSR markers and showed that of the 72 P. omnivora isolates and spore mats tested, 41 were distinct genotypes. Furthermore, the developed SSR markers did not show cross-transferability to other close relatives of P. omnivora in the class Pezizomycetes. A multiplex PCR detecting both mating type idiomorphs and a reference gene (TUB2) was developed to screen P. omnivora isolates. Based on the dataset we tested, P. omnivora is a heterothallic fungus with both mating types present in the United States in a ratio close to 1:1. We tested P. omnivora spore mats obtained from spatially distinct disease rings that developed in a center-pivot alfalfa field and showed that both mating types can be present not only in the same field but also within a single spore mat. This study shows that P. omnivora has the genetic toolkit for generating sexually diverse progeny, providing impetus for future studies that focus on identifying sexual morphs in nature.

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.

Epichloë festucae is a common symbiont of the perennial and widely distributed cool season grass, Festuca rubra. The symbiosis is highly integrated involving systemic growth of the fungus throughout above-ground host parts and vertical transmission from plant to its offspring via host seeds. However, the nature of symbiosis is labile ranging from antagonistic to mutualistic depending on prevailing selection pressures. Both the loss of fungus in the maternal host lineage and horizontal transmission through sexual spores within the host population may partly explain the detected variation in symbiosis in wild grass populations. Epichloë species are commonly considered as pathogens when they produce sexual spores and partly castrate their host plant. This is the pathogenic end of the continuum from antagonistic to mutualistic interactions. Here we examined the population genetic structure of E. festucae to reveal the gene flow, importance of reproduction modes, and alkaloid potential of the symbiotic fungus in Europe. Epichloë-species are highly dependent on the host in survival and reproduction whilst benefits to the host are largely linked to defensive mutualism attributable to fungal-origin bioactive alkaloids that negatively affect vertebrate and/or invertebrate herbivores. We detected decreased genetic diversity in previously glaciated areas compared to non-glaciated regions during the last glacial maximum period and found three major genetic clusters in E. festucae populations: southern, northeastern and northwestern Europe. Sexual reproduction may have a higher role than expected in Spanish E. festucae populations due to the predominance of unique genotypes and presence of both mating types in the region. In contrast, asexual reproduction via host seeds predominates in the Faroe Island and Finland in northern Europe due to the presence of biased mating-type ratios and large dominant genotypes in the E. festucae populations within the region. A substantially larger variation of alkaloid genotypes was observed in the fungal populations than expected, although the variability of the alkaloid genotypes within populations is considerably lower in northern than Spanish populations in southern Europe. E. festucae populations consist of different combinations of alkaloid classes from the gene clusters of ergot alkaloid and indole-terpenes, and from pyrrolopyrazine alkaloid gene. We suggest that the postglacial distribution history of the host grass, prevailing reproduction strategies of E. festucae, and local selection pressures likely explain a large part of the genetic variation observed in fungal populations among geographic regions. The identified alkaloid genotypes can be used by turfgrass breeders to improve resistance against herbivores in red fescue varieties and to develop new sustainable cultivars in Europe.