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.

Strain-Specific Resistance to Potato virus Y (PVY) in Potato and Its Effect on the Relative Abundance of PVY Strains in Commercial Potato Fields.

Potato virus Y (PVY) is a serious threat to potato production due to effects on tuber yield and quality, in particular, due to induction of potato tuber necrotic ringspot disease (PTNRD), typically associated with recombinant strains of PVY. These recombinant strains have been spreading in the United States for the past several years, although the reasons for this continuing spread remained unclear. To document and assess this spread between 2011 and 2015, strain composition of PVY isolates circulating in the Columbia Basin potato production area was determined from hundreds of seed lots of various cultivars. The proportion of nonrecombinant PVYO isolates circulating in Columbia Basin potato dropped ninefold during this period, from 63% of all PVY-positive plants in 2011 to less than 7% in 2015. This drop in PVYO was concomitant with the rise of the recombinant PVYN-Wi strain incidence, from less than 27% of all PVY-positive plants in 2011 to 53% in 2015. The proportion of the PVYNTN recombinant strain, associated with PTNRD symptoms in susceptible cultivars, increased from 7% in 2011 to approximately 24% in 2015. To further address the shift in strain abundance, screenhouse experiments were conducted and revealed that three of the four most popular potato cultivars grown in the Columbia Basin exhibited strain-specific resistance against PVYO. Reduced levels of systemic movement of PVYO in such cultivars would favor spread of recombinant strains in the field. The negative selection against the nonrecombinant PVYO strain is likely caused by the presence of the Nytbr gene identified in potato cultivars in laboratory experiments. Presence of strain-specific resistance genes in potato cultivars may represent the driving force changing PVY strain composition to predominantly recombinant strains in potato production areas.

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.

Diversity and evolution of potato mop-top virus.

Nearly complete sequences of RNA-CP and 3'-proximal RNA-TGB were determined for 43 samples of potato mop-top virus (PMTV) originating from potato tubers and field soil from Sweden, Denmark and the USA. The results showed limited diversity and no strict geographical grouping, suggesting only a few original introductions of PMTV from the Andes. Two distinguishable types of RNA-CP and RNA-TGB were found in the samples, but no specific combination of them correlated with spraing symptoms in tubers. Lack of positive selection in the coding sequences indicates that there is no specific molecular adaptation of PMTV to new vectors or hosts.

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.

Incidence and impact of Verticillium dahliae in soil associated with certified potato seed lots.

Verticillium dahliae causes Verticillium wilt of potato and can be found in soil associated with potato seed tubers. The purpose of this research was to quantify V. dahliae in soil associated with certified seed tubers and determine if this potential inoculum source is related to disease development in the field. Approximately 68% of seed lots assayed contained V. dahliae-infested soil on seed tuber surfaces (seed tuber soil). Over 82% of seed lots contained V. dahliae in loose seed lot soil obtained from bags and trucks used to transport seed tubers. Most samples contained ≤50 CFU/g but some contained >500 CFU/g. Most isolates (93%) were vegetative compatibility group 4A. Populations of V. dahliae in stem sap increased with increasing inoculum densities in field soils only when V. dahliae concentrations in seed tuber soil were low. High concentrations of V. dahliae in seed tuber soil resulted in greater stem sap colonization when V. dahliae inoculum densities in field soil were low (P < 0.01) and resulted in greater pathogen inoculum densities in postharvest field soils (P = 0.04). Seed tubers contaminated with V. dahliae-infested soils may introduce the pathogen into fields not previously cropped to potato or recontaminate those which have received preplant management practices. Long-term management of V. dahliae requires reducing propagules in soil associated with seed lots.

Complete genomic sequence of a Tobacco rattle virus isolate from Michigan-grown potatoes.

Tobacco rattle virus (TRV) causes stem mottle on potato leaves and necrotic arcs and rings in potato tubers, known as corky ringspot disease. Recently, TRV was reported in Michigan potato tubers cv. FL1879 exhibiting corky ringspot disease. Sequence analysis of the RNA-1-encoded 16-kDa gene of the Michigan isolate, designated MI-1, revealed homology to TRV isolates from Florida and Washington. Here, we report the complete genomic sequence of RNA-1 (6,791 nt) and RNA-2 (3,685 nt) of TRV MI-1. RNA-1 is predicted to contain four open reading frames, and the genome structure and phylogenetic analyses of the RNA-1 nucleotide sequence revealed significant homologies to the known sequences of other TRV-1 isolates. The relationships based on the full-length nucleotide sequence were different from than those based on the 16-kDa gene encoded on genomic RNA-1 and reflect sequence variation within a 20-25-aa residue region of the 16-kDa protein. MI-1 RNA-2 is predicted to contain three ORFs, encoding the coat protein (CP), a 37.6-kDa protein (ORF 2b), and a 33.6-kDa protein (ORF 2c). In addition, it contains a region of similarity to the 3' terminus of RNA-1, including a truncated portion of the 16-kDa cistron. Phylogenetic analysis of RNA-2, based on a comparison of nucleotide sequences with other members of the genus Tobravirus, indicates that TRV MI-1 and other North American isolates cluster as a distinct group. TRV M1-1 is only the second North American isolate for which there is a complete sequence of the genome, and it is distinct from the North American isolate TRV ORY. The relationship of the TRV MI-1 isolate to other tobravirus isolates is discussed.

Potato Silver Scurf Affected by Tuber Seed Treatments and Locations, and Occurrence of Fungicide Resistant Isolates of Helminthosporium solani.

The effectiveness of various seed-tuber treatments was evaluated for control of silver scurf on potato (Solanum tuberosum), caused by Helminthosporium solani, at four locations in Washing-ton and Oregon using seed-tubers from the same source. Disease incidence was determined at harvest and following storage, and differed significantly among locations. The highest incidence of disease was observed at Redmond, OR and the lowest was at Hermiston, OR. Significantly less silver scurf occurred on progeny-tubers, regardless of location, when seed was treated with the fungicide treatments fludioxonil, fludioxonil + quintozene, azoxystrobin, or thiophanate-methyl + mancozeb compared with the nontreated control. A sample of H. solani from seed planted in the Columbia Basin of Washington and Oregon was assessed for resistance to thia-bendazole (TBZ) and thiophanate-methyl (TPM). Sensitivity of 20 isolates of H. solani to TBZ was determined on V8 media amended with TBZ. Four isolates, selected as a subset from the 20 isolates tested for TBZ sensitivity, were tested for resistance to TPM, mancozeb, and TPM + mancozeb on amended V8 media. Isolates differed significantly in sensitivity to both chemicals. Thirteen isolates out of the 20 tested were resistant to TBZ. One out of the four subsets of isolates was resistant to both TBZ and TPM. TBZ sensitivity was not related to geographic origin of the isolates. Variation in resistance was evident on a small scale as seen with one resistant and one sensitive isolate collected from the same tuber. This is the first report that silver scurf incidence is affected by location despite the same tuber-seed source and also the first report of TBZ and TPM resistant isolates of H. solani from the Columbia Basin (Oregon and Washington) production area.

Aerial photography used for spatial pattern analysis of late blight infection in irrigated potato circles.

ABSTRACT Spatial and temporal dynamics of late blight were investigated from color, infrared aerial photographs of five commercial potato fields in the Columbia Basin during epidemics in 1993, 1995, and 1998. Aerial photographs were taken one to four times at 6- to 21-day intervals. Photographs were scanned and pixels, representing approximately 1 m(2) in the field, were used in the analysis. Late blight-infected plants were aggregated as indicated by runs analysis. Significant z-tests were computed for four directions during each sampling date in each of the five fields. Absolute z-values for runs analysis increased, indicating increasing aggregation in the four directions, as disease incidence increased in the early and midphases of the epidemics in each field. Variograms indicated the existence of autocorrelation among infected plants in four directions; the range of influence increased as disease incidence increased except at the highest levels of disease. Late blight was observed to spread in fields as foci. Late blight foci enlarged in size, produced distinct daughter foci, and coalesced. A field where initial inoculum likely originated from infected seed tubers exhibited less initial aggregation than the other fields, perhaps due to a different source of primary inoculum. Aerial photography coupled with spatial analyses of late blight-infected plants was an effective technique to quantitatively assess disease patterns in relatively large fields and was useful in quantifying an intensification of aggregation during the epidemic process on a large scale.

Comparison of Application Methods on Deposition and Redistribution of Chlorothalonil in a Potato Canopy and Potential for Control of Late Blight.

The effect of application method by aircraft (fixed wing), ground, and chemigation on deposition of chlorothalonil in a potato canopy was compared over 2 years. Initial chlorothalonil deposition was greatest with ground application, less by aircraft, and least with chemigation. After application by aircraft or ground, the upper canopy had more chlorothalonil than the middle, and the lower canopy had the least. With chemigation, the upper canopy also had more chlorothalonil, but amounts in the lower and middle canopy were similar. Redistribution occurred with time as material from the upper canopy moved downward. With a 9-day application frequency using ground or aircraft methods, residue levels throughout the treated potato canopy were maintained, even if distribution of the fungicide within the canopy was poor from the initial application. With chemigation, low initial chlorothalonil levels were further decreased due to redistribution, increasing the likelihood that levels could fall below that which may be needed to adequately control late blight, particularly beyond 7 days after application.

Cost of Fungicides Used to Manage Potato Late Blight in the Columbia Basin: 1996 to 1998.

The cost of managing potato late blight with fungicides in the Columbia Basin of Washington and Oregon in 1996 to 1998 was documented and compared with the cost of managing the epidemic in 1995. Mean number of fungicide applications on late-season potatoes from 1996 to 1998 ranged from 5.3 to 8.8 in the north Columbia Basin of Washington and 8.5 to 12.3 in the southern basin of Washington and Oregon. Mean cost per hectare of fungicides and application on late-season potatoes in 1998 was $316 per hectare ($128 per acre) in Washington's north basin and $472 per hectare ($191 per acre) in Washington's south basin. Even though the price of most fungicides had increased since 1995, total cost of control per hectare over the season was less during 1996 to 1998 than in 1995 because of altered management practices. These included fewer fungicide applications, a shift toward lower cost fungicides, substitution of aerial application by chemigation, and a reduction in the number of fields chemically desiccated before harvest. Total cost of managing late blight and tuber rot loss was $22.3 million in 1998, whereas it was $30 million in 1995. The 1998 cost included $19.8 million for fungicide applications and materials, $1.1 million for canopy desiccation, and $1.4 million loss due to tuber rot in storage.

Expansion of Potato Late Blight Forecasting Models for the Columbia Basin of Washington and Oregon.

A regional potato late blight forecasting system for irrigated potatoes in the semiarid environment of the Columbia Basin was expanded by developing specific forecasting models for four vicinities throughout the Basin. Relationships between weather and outbreaks of late blight at the locations over a 27-year period were examined using logistic regression analysis. The response variable was a year either with or without a late blight outbreak. An indicator variable representing the occurrence of an outbreak during the preceding year (Yp) and number of days of rain during April and May (Ram) correctly classified the disease status (presence or absence of late blight) of 89, 82, 78, and 78% of the years at Prosser, Washington, Hermiston, Oregon, and Hanford and Othello, Washington, respectively. The percentage of years with disease outbreaks correctly classified was 93, 85, 79, and 79% at the four respective locations. All years with late blight outbreaks and 96% of the total years were correctly classified using data from at least one of the four locations. These predictors are particularly important early in the growing season and can be used to make area forecasts. A second set of predictors, Yp and number of days of rain in July and August (Rja), for Hermiston and Hanford, and a third set, Yp, Ram, and Rja, for Prosser and Othello were found effective for making additional late blight forecasts later in the growing season.

Fungicide Application for Late Blight Management: A Boom Attached to a Center Pivot Irrigation System.

Chlorothalonil was applied to commercial potato fields of cvs. Russet Norkotah and Russert Burbank in replicated trials through either a center-pivot irrigation system or a spray-boom-attached-to-the-pivot system. Water rates were 26,192 and 675 liters/ha for the center pivot and attached boom, respectively. Three crop canopy levels were sampled within 2 h and again after 6 days following fungicide applications made at 7-day intervals. Efficacy of the two application methods was tested by inoculation of individual excised leaves with an isolate of strain US-1 or US-8 of Phytophthora infestans and by chemical residue analysis. Severity of late blight did not differ between the two fungicide application methods 2 h after application but, after 6 days, disease severity was significantly less on leaves where chlorothalonil was applied by the attached boom. Fungicide residues were three times higher on leaves where chlorothalonil was applied by the attached boom than with the center-pivot system. Significantly more disease occurred on fungicide-treated and non-treated leaves inoculated with the US-8 than the US-1 strain. Severity of late blight on inoculated leaves was inversely related to the amount of chlorothalonil residue on leaves.

Soil Fumigant Effects on Three Genera of Potential Soilborne Pathogenic Fungi and Their Effect on Potato Yield in the Columbia Basin of Oregon.

During three years of trials in commercial production fields, populations of three genera of potato pathogens, Fusarium spp, Pythium spp, and Verticillium dahliae, were followed before and after the single or combination use of 1,3-dichloropropene (1,3-D), 1,3-D + 17% chloropicrin (1,3-D + chloropicrin), or metam sodium (MS). Populations of these fungi did not always increase during the growing season but the relative population at a soil depth of 0 to 30 cm was nearly always higher than at 30 to 60 cm, regardless of year, sampling time, or fungal pathogen. The use of MS alone or in combination with 1,3-D generally suppressed recovery of all three genera and also increased yields. 1,3-D or 1,3-D + chloropicrin did not reduce fungal populations, but 1,3-D increased yield in 1 of 3 years. Reduced rates of MS and 1,3-D used in combination were as effective as higher rates of MS used alone. Multiple regression analysis comparing yield with fungal populations before planting indicated that population size was correlated negatively with yield. Propagules of V. dahliae had the greatest impact in reducing yield, but propagules of Pythium spp. and Fusarium spp. may have been important when populations of V. dahliae were low. Soil populations of Fusarium spp. and Pythium spp. have not been reported previously to be associated with yield reductions in potato grown in the Columbia Basin. Threshold estimates suggested that yield of number one tubers was reduced by 1 metric ton/ha for each 0.6 to 3.0 V. dahliae CFU/g dry soil present at planting.