Molecular and Alkaloid Characterization of Claviceps purpurea Sensu Lato From Grass Seed Production Areas of the U.S. Pacific Northwest.

Ergot, caused by Claviceps purpurea sensu lato, is an economically important seed replacement disease of Kentucky bluegrass (Poa pratensis) and perennial ryegrass (Lolium perenne) seed crops. C. purpurea sensu stricto is considered the primary Claviceps species responsible, but genetic diversity and cryptic species within C. purpurea sensu lato have previously been reported. Fifty-six C. purpurea sensu lato isolates collected from P. pratensis (n = 21) and L. perenne (n = 35) in Oregon and Washington between 2010 and 2014 were characterized via random amplified polymorphic DNA (RAPD), partial internal transcribed spacer (ITS), ß-tubulin and elongation factor-1α (EF-1α) sequences, conidial size, and ergot alkaloid chemotype. Based on RAPD analysis, seven isolates from P. pratensis and 33 isolates from L. perenne collected in Oregon corresponded to C. purpurea sensu stricto, and 13 isolates collected from P. pratensis in Washington and Oregon were identified as C. humidiphila. Partial ITS, ß-tubulin, and EF-1α sequences identified 10 isolates from P. pratensis as C. humidiphila, and seven isolates from P. pratensis and 33 isolates from L. perenne were identified as C. purpurea sensu stricto. Several isolates generated ambiguous RAPD bands or sequences that prevented identification. Ergot alkaloid chemotype profiling found that ergocornine and its epimer were predominant in sclerotia from P. pratensis, whereas ergotamine and its epimer were most abundant in sclerotia from L. perenne. This study confirms the presence of the C. purpurea sensu lato species complex in the U.S. Pacific Northwest and suggests that more research is needed to characterize and mitigate Claviceps spp. infection of grass seed crops in North America.

Detection and Quantification of Airborne Claviceps purpurea sensu lato Ascospores from Hirst-Type Spore Traps using Real-Time Quantitative PCR.

The U.S. Pacific Northwest states of Oregon and Washington are major producers of cool-season grass seed. Ergot, caused by fungi in the Claviceps purpurea sensu lato group, is an important seed replacement disease of grass worldwide. Microscopic methods that are currently used to quantify airborne Claviceps ascospores captured by spore traps are not currently rapid enough to allow for detecting and reporting of spore numbers in a timely manner, hindering growers from using this information to help manage ergot. We developed a SYBR Green real-time quantitative polymerase chain reaction (qPCR)-based assay for fast and efficient detection and quantification of C. purpurea sensu lato ascospores from Hirst-type spore traps. Species-specificity of the qPCR assay was confirmed against 41 C. purpurea sensu lato isolates collected from six hosts and six other Claviceps spp. Significant relationships were observed between cycle threshold (Ct) values and standard curves of serial dilutions of DNA ranging from 1 pg to 10 ng (R2 = -0.99; P = 0.0002) and DNA extracted from a conidial suspension representing 8 to 80,000 conidia (R2 = -0.99; P = 0.0004). Ct values from qPCR were significantly correlated with results from microscopic examination of spore trap samples from the field (r = -0.68; P < 0.0001) and the procedure was able to detect a single ascospore from spore trap tape samples. The qPCR procedure developed in this study provided a means for quantifying airborne Claviceps ascospores that was highly specific and useful over a wide range of spore densities, and could be performed in a matter of hours instead of days. The qPCR assay developed in this study could be part of an integrated pest management approach to help grass seed growers make risk-based fungicide application decisions for ergot management in grass grown for seed.

Identification of Environmental Factors Related to Claviceps purpurea Ascospore Production in Perennial Ryegrass Seed Fields and Development of Predictive Models.

Claviceps purpurea, the causal agent of ergot of perennial ryegrass seed crops, overwinters as sclerotia in the soil and releases airborne ascospores in the spring that infect flower ovaries and replace seed with sclerotia. Burkard spore traps were used to quantify the dispersal phenology and concentration of ascospores in perennial ryegrass seed fields in the Columbia Basin of Oregon. Weather factors were measured concurrently with spore trapping. Nonparametric regression, box-and-whisker plots, and univariate analysis were used to visualize and identify trends between ascospore concentrations and weather variables. Most ascospores (75.4%) were trapped when minimum soil temperatures were between 16.2 and 20.4°C. Over 67% of the total ascospores trapped were observed when minimum air temperatures were between 6.8 and 12.4°C and 64% of ascospores were trapped when daily mean dew point was between 3.7 and 8.2°C. Environmental favorability index (EFI) models were developed and validated based on their ability to predict ascospore occurrence. The EFI models were able to predict ascospore occurrence with an accuracy of 71.7 to 87.5% depending on the year. The models were up to 79.8% accurate when validated using three years of historical spore trap data not used in the EFI model development. Ninety-four percent of ascospores were trapped when cumulative air degree days, using lower and upper thresholds of 10 and 25°C, respectively, were between 230 and 403. These results suggest that weather parameters can be used to model C. purpurea ascospore occurrence and potentially improve the timing and efficacy of fungicide applications by identifying when plant protection is most needed.

Simple sequence repeat markers that identify Claviceps species and strains.

BACKGROUND: Claviceps purpurea is a pathogen that infects most members of Pooideae, a subfamily of Poaceae, and causes ergot, a floral disease in which the ovary is replaced with a sclerotium. When the ergot body is accidently consumed by either man or animal in high enough quantities, there is extreme pain, limb loss and sometimes death. RESULTS: This study was initiated to develop simple sequence repeat (SSRs) markers for rapid identification of C. purpurea. SSRs were designed from sequence data stored at the National Center for Biotechnology Information database. The study consisted of 74 ergot isolates, from four different host species, Lolium perenne, Poa pratensis, Bromus inermis, and Secale cereale plus three additional Claviceps species, C. pusilla, C. paspali and C.fusiformis. Samples were collected from six different counties in Oregon and Washington over a 5-year period. Thirty-four SSR markers were selected, which enabled the differentiation of each isolate from one another based solely on their molecular fingerprints. Discriminant analysis of principle components was used to identify four isolate groups, CA Group 1, 2, 3, and 4, for subsequent cluster and molecular variance analyses. CA Group 1 consisting of eight isolates from the host species P. pratensis, was separated on the cluster analysis plot from the remaining three groups and this group was later identified as C. humidiphila. The other three groups were distinct from one another, but closely related. These three groups contained samples from all four of the host species. These SSRs are simple to use, reliable and allowed clear differentiation of C. humidiphila from C. purpurea. Isolates from the three separate species, C. pusilla, C. paspali and C.fusiformis, also amplified with these markers. CONCLUSIONS: The SSR markers developed in this study will be helpful in defining the population structure and genetics of Claviceps strains. They will also provide valuable tools for plant breeders needing to identify resistance in crops or for researchers examining fungal movements across environments.

Spatial Patterns of Ergot and Quantification of Sclerotia in Perennial Ryegrass Seed Fields in Eastern Oregon.

Ergot, caused by Claviceps purpurea, is a major disease of perennial ryegrass grown for seed in eastern Oregon. The objective of this research was to quantify and describe the spatial patterns of ergot severity in each of three 50-ha commercial fields of perennial ryegrass grown for seed in 2012 and 2013. In total, 1,433 and 1,405 quadrats were sampled among the three fields in 2012 and 2013, respectively, and the percentage of quadrats with ergot ranged from 59 to 90%. The mean incidence of infected seed heads in each quadrat ranged between 13 and 29%, while mean severity in each quadrat ranged from 0.2 to 0.5 sclerotia per seed head. Significant autocorrelation and clustering were observed in all three fields in both years, as indicated by Moran's I and spatial analysis by distance indices of aggregation. The mean number of ergot sclerotia collected from each field after harvest ranged between 4 and 15 sclerotia m-2 in 2012 and 18 and 119 sclerotia m-2 in 2013. Sclerotia left in perennial fields after harvest are a significant source of inoculum that should be targeted for control. This is the first study to quantify spatial patterns of ergot in perennial ryegrass and provides insights into possible mechanisms that contribute to ergot etiology and epidemiology.

Afternoon Ascospore Release in Claviceps purpurea Optimizes Perennial Ryegrass Infection.

In Kentucky bluegrass (Poa pratensis), Claviceps purpurea, the causal agent of ergot, typically releases ascospores during the early-morning hours, between about midnight and 10:00 a.m., corresponding to time of flowering, when the unfertilized ovaries are most susceptible to infection. During aeromycology studies of C. purpurea in perennial ryegrass (Lolium perenne) in northeastern Oregon during 2008 to 2010 and 2013, a strain of C. purpurea was found that released ascospores in the afternoon, coinciding with flowering in perennial ryegrass. Under controlled environmental conditions, sclerotia from perennial ryegrass and Kentucky bluegrass released spores in the afternoon and morning, respectively, consistent with timing of spore release under field conditions. Internal transcribed spacer (ITS) sequences of single sclerotial isolates from Kentucky bluegrass and perennial ryegrass were consistent with C. purpurea, although minor variations in ITS sequences among isolates were noted. Differences between Kentucky bluegrass and perennial ryegrass isolates were observed in random amplified polymorphic DNA. Evidence is provided for adaptation of C. purpurea to perennial ryegrass by means of delayed spore release that coincides with afternoon flowering in perennial ryegrass.

Survival, Germination, and Growth of Epichloë typhina and Significance of Leaf Wounds and Insects in Infection of Orchardgrass.

Epichloë typhina is an important stroma-producing endophytic ascomycete that is responsible for significant yield loss in orchardgrass (Dactylis glomerata) seed production fields. Infection is presumed to occur through leaves or stems, although details of the infection process and conditions that favor leaf infection are not well understood. The primary objectives of this study were to investigate the early stages of infection, including the effect of temperature or water potential on ascospore germination and subsequent growth of E. typhina, the tolerance of ascospores to desiccation, the requirement of leaf wounds for infection of orchardgrass by E. typhina, and the potential for insects to facilitate infection. Ascospores tolerated dry conditions, with at least 40% surviving 12 days under desiccation. Germination and growth of E. typhina was greatest at 25°C, with little to no growth at 5 and 35°C. Mycelial growth decreased with decreasing water potential from -0.3 to -10 MPa. Ascospore germination on leaves was predominantly hyphal at wound sites and iterative (conidiogenous) at sites without wounds. E. typhina typically entered leaves through wounds. Direct penetration was rarely observed and appeared to be associated with ascospore clusters. Germ tubes were significantly longer at sites with honeydew deposits from the bird cherry-oat aphid than at sites without honeydew. Growth of E. typhina was also observed at feeding sites of eriophyid mites, suggesting that leaf-wounding or sap-excreting insects support epiphyllous growth of E. typhina on leaves.

A Reevaluation of the Host Range and Geographical Distribution of Claviceps Species in the United States.

A listing of host and state reports and distribution maps for 11 taxa of Claviceps occurring in the United States, including C. africana, C. cinerea, C. grohii, C. nigricans, C. paspali, C. pusilla, C. purpurea var. purpurea and var. spartinae, C. tripsaci, C. yanagawensis, and C. zizaniae, was prepared based on literature citations and examination of specimens from herbaria. The occurrence of C. ranunculoides is questioned based on examination of conidia and sclerotia from archived specimens. Collections of C. purpurea var. purpurea from grasses in the Pani-coideae were referred to other Claviceps spp. based on occurrence of macroconidia and micro-conidia. C. purpurea var. purpurea was found on 165 grass species within the continental United States and Alaska. The size of conidia of C. purpurea var. purpurea was found to be relatively stable across host and geographical regions. However, conidia of C. purpurea var. purpurea from hosts in the Aveneae and Meliceae (generally associated with wet habitats) were more variable in size and generally larger than those from other tribes in the Pooideae. Claviceps spp. in the continental United States occurred in diverse habitats, including temperate grasslands of the middle to northern latitudes (C. purpurea var. purpurea, C. nigricans) to the middle to southern latitudes (C. pusilla), coastal habitats (C. purpurea var. spartinae, C. ziza-niae), northern wetlands (C. grohii), southern temperate to subtropical grasslands (C. africana, C. paspali, C. tripsaci, C. yanagawensis), and arid southwestern grasslands (C. cinerea).

Occurrence and Distribution of Ergot and Estimates of Seed Loss in Kentucky Bluegrass Grown for Seed in Central Oregon.

In 1991 to 1993, ergot was detected in 36, 44, and 62%, respectively, of fields of Kentucky bluegrass grown for seed in central Oregon. Relatively few fields (2 to 13%) had a high (more than 20 sclerotia/gm seed) level of ergot. Ergot severity (percent sclerotia by weight) in 1991 to 1993 was estimated at 0.85, 0.07, and 0.34%, respectively. Percent seed replaced by ergot in 1991 to 1993 was estimated at 0.22, 0.02, and 0.08, respectively. Recleaning of seed to reduce ergot contamination and to meet purity standards resulted in a 7.8% reduction in marketable seed weight. Estimated value of seed replaced by ergot in 1991, 1992, and 1993, was $75,625, $4,758, and $17,076, respectively. Estimated value of seed lost during recleaning in 1991, 1992, and 1993, was $37,669, $8,171, and $21,964, respectively. Weed grasses supporting ergot at the time of harvest of Kentucky bluegrass were species of Bromus, Secale, Festuca, and Poa. However, very few seed heads of weed grasses were infected, and they appeared to contribute little to ergot development in central Oregon. Most infested fields (with more than one sclerotium/gm seed prior to recleaning) or weed grass sites with ergot were located in the southern range of the area of study.