Short-Term Host Selection Pressure Has Little Effect on the Evolution of a Monoclonal Population of Verticillium dahliae Race 1.

Understanding pathogen evolution over time is vital for plant breeding and deployment of host resistance. In the context of a soilborne pathogen, the potential of host-directed evolution of a Verticillium dahliae race 1 isolate and genotypic variation of V. dahliae associated with two major hosts (lettuce and tomato) were determined. In total, 427 isolates were recovered over 6 years from a resistance screening nursery infested with a single V. dahliae race 1 isolate. In a separate study, an additional 206 isolates representing 163 and 43 isolates from commercial lettuce and tomato fields, respectively, were collected. Analyses of isolates recovered from the screening nursery over 6 years revealed no changes in the race and mating type composition but did uncover seven simple sequence repeat (SSR) variant genotypes. No significant genotypic variation in V. dahliae was observed between or within fields of either lettuce or tomato but pathogen populations were significantly differentiated between these two hosts. Replicated virulence assays of variant SSR genotypes on lettuce differential cultivars suggested no significant difference in virulence from the wild-type race 1 isolate introduced into the field. This suggests that deployed race 1 host resistance will be robust against the widespread race 1 populations in lettuce-growing regions at least for 6 years unless novel pathogen genotypes or races are introduced into the system.

Clonal expansion of Verticillium dahliae in lettuce.

Few studies in population biology have documented how structure and diversity of pathogens evolve over time at local scales. With the historical samples of Verticillium dahliae available from lettuce, we investigated the structure and diversity of this pathogen in time and space. Three hundred twenty-nine V. dahliae isolates from lettuce fields collected over 18 years were characterized with polymorphic microsatellite markers and polymerase chain reaction tests for race and mating type. Genetic variation within and among commercial lettuce fields in a single season was also investigated using an additional 146 isolates. Sixty-two haplotypes (HTs) were observed among the 329 isolates. A single HT was frequently observed over multiple years and locations (61.40%). Genetic diversity, allelic richness, and private allelic richness suggested a relatively recent clonal expansion. Race 1 (93.63%) and MAT1-2-1 (99.69%) were overwhelmingly represented among the isolates. Linkage disequilibrium was significant (P < 0.001) for all populations, suggesting limited sexual recombination in the sampled populations from lettuce. Populations from 2006, 2009, and 2010 had higher numbers of unique HTs, implying a recent introduction of novel HTs. We conclude that V. dahliae population from lettuce evaluated in this study is expanding clonally, consistent with an asexually reproducing pathogen, and the movement of clonal genotypes locally occurs over time.

Molecular variation among isolates of Verticillium dahliae and polymerase chain reaction-based differentiation of races.

Verticillium dahliae is a soilborne fungal pathogen that causes vascular wilt in a variety of economically important crops worldwide. There are two races of V. dahliae that infect tomato and lettuce. Although race-1-specific resistance has been identified in both tomato and lettuce, no resistant sources are available for race 2. Molecular analyses were employed to characterize the genetic variability and race structure of 101 isolates of V. dahliae from a variety of hosts, mainly from central and coastal California, and 10 isolates exotic to this area. Analyses of the 16 simple sequence repeat (SSR) markers illustrated that tomato subpopulations from central California were distinct relative to the marigold subpopulations. In contrast, cotton and olive isolates showed admixture with tomato isolates. Analyses of both the ribosomal DNA intergenic spacer regions and SSR markers revealed high genetic variability among isolates but were unable to delineate races of V. dahliae. However, a polymerase chain reaction (PCR) assay was applied to amplify a race-1-specific amplicon from the isolates in many hosts from different geographic areas, and was coupled with virulence assays for validation of the data. Results of the PCR assay showed 100% concordance with the virulence assay to differentiate race 1 from race 2 of 48 isolates from tomato. The results indicate that the PCR assay can be applied to differentiate the two races to support our related aim of breeding host resistance, and further reveal insights into the distribution of races in tomato and lettuce cropping systems in California.

Real-time PCR assays for the quantification of Phialophora gregata f. sp. sojae IGS genotypes A and B.

Populations of Phialophora gregata f. sp. sojae, the causal agent of brown stem rot (BSR) of soybean, consist of two genotypes, designated A and B. These genotypes are differentiated by an insertion or deletion in the intergenic spacer region (IGS) of ribosomal DNA. The two genotypes differ in the type and severity of symptoms they cause and have displayed preferential host colonization. Methods to quantify populations of P. gregata f. sp. sojae and to distinguish between the two genotypes are essential to understanding this host-pathogen interaction and to improving control of BSR. A real-time, quantitative polymerase chain reaction (qPCR) assay was developed for the specific detection and quantification of P. gregata f. sp. sojae genotype A. This assay is specific to P. gregata f. sp. sojae genotype A, sensitive to 50 fg of DNA, and unaffected by the presence of soybean or soil DNA. When the P. gregata f. sp. sojae genotype A-specific primer/probe set is used in a multiplex qPCR assay with a previously developed primer/probe set which indiscriminately amplifies both genotypes, the quantity of P. gregata f. sp. sojae genotype B can be indirectly determined. This multiplex assay provides a rapid and robust method for studying both the population size and genetic structure of P. gregata f. sp. sojae in its soybean host and in the soil.

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 May 2009-31 July 2009.

This article documents the addition of 512 microsatellite marker loci and nine pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Alcippe morrisonia morrisonia, Bashania fangiana, Bashania fargesii, Chaetodon vagabundus, Colletes floralis, Coluber constrictor flaviventris, Coptotermes gestroi, Crotophaga major, Cyprinella lutrensis, Danaus plexippus, Fagus grandifolia, Falco tinnunculus, Fletcherimyia fletcheri, Hydrilla verticillata, Laterallus jamaicensis coturniculus, Leavenworthia alabamica, Marmosops incanus, Miichthys miiuy, Nasua nasua, Noturus exilis, Odontesthes bonariensis, Quadrula fragosa, Pinctada maxima, Pseudaletia separata, Pseudoperonospora cubensis, Podocarpus elatus, Portunus trituberculatus, Rhagoletis cerasi, Rhinella schneideri, Sarracenia alata, Skeletonema marinoi, Sminthurus viridis, Syngnathus abaster, Uroteuthis (Photololigo) chinensis, Verticillium dahliae, Wasmannia auropunctata, and Zygochlamys patagonica. These loci were cross-tested on the following species: Chaetodon baronessa, Falco columbarius, Falco eleonorae, Falco naumanni, Falco peregrinus, Falco subbuteo, Didelphis aurita, Gracilinanus microtarsus, Marmosops paulensis, Monodelphis Americana, Odontesthes hatcheri, Podocarpus grayi, Podocarpus lawrencei, Podocarpus smithii, Portunus pelagicus, Syngnathus acus, Syngnathus typhle,Uroteuthis (Photololigo) edulis, Uroteuthis (Photololigo) duvauceli and Verticillium albo-atrum. This article also documents the addition of nine sequencing primer pairs and sixteen allele specific primers or probes for Oncorhynchus mykiss and Oncorhynchus tshawytscha; these primers and assays were cross-tested in both species.

Evaluation of QoI Fungicide Application Strategies for Managing Fungicide Resistance and Potato Early Blight Epidemics in Wisconsin.

Potato early blight (Alternaria solani) is a yield-limiting disease and control depends primarily on multiple fungicide applications. Azoxystrobin, registered in the United States in 1999, initially provided outstanding early blight control. Within 3 years, approximately 80% of the total potato acreage was being treated with azoxystrobin and other quinone outside inhibitor (QoI), fungicides registered subsequently. Alternaria solani isolates with decreased in vitro sensitivity to azoxystrobin were detected in Wisconsin during 2001. Field experiments were conducted in 2001 to 2003 to evaluate season-long fungicide programs and test fungicide resistance management strategies. The fungicide program recommended to growers at that time, which consisted of three applications of azoxystrobin for weeks 1, 3, and 5 alternated with applications of chlorothalonil at label recommended rates, was effective in controlling early blight when conditions were conducive to disease development. Mean sensitivity in vitro of A. solani isolates from fungicide efficacy field experiments in 2001 to 2003 was numerically highest for isolates from the untreated control plots, chlorothalonil-alone plots, or plots treated with three applications of azoxystrobin alternated with chlorothalonil compared with other treatments tested. Three single-nucleotide polymorphisms (SNPs) can cause the F129L substitution (TTC to TTA, CTC, or TTG) that results in decreased sensitivity to azoxystrobin of A. solani. The TTA mutant was the most frequently recovered mutant type in the field experiments. The frequency of recovery of wild-type isolates in experiments was 22% in 2001, 4% in 2002, and 22% in 2003.

Monitoring and Tracking Changes in Sensitivity to Azoxystrobin Fungicide in Alternaria solani in Wisconsin.

Azoxystrobin is a common fungicide used by farmers of Solanaceous crops against Alternaria solani, but there was growing concern about decreased sensitivity with repeated applications. In 2002 and 2003, monitoring of A. solani from commercial potato fields in Wisconsin indicated increased frequency and a statewide distribution of isolates with decreased in vitro sensitivity to azoxystrobin. Mean effective concentration in inhibiting spore germination by 50% values gathered in 2002 and 2003 were approximately 20-fold higher than baseline isolates of A. solani collected in 1998 from fields that had never been treated with azoxystrobin. This sensitivity decrease was correlated with site-specific mutations in the cytochrome b detected by quantitative real-time polymerase chain reaction. The F129L and the G143A substitution have been shown to cause a reduction in sensitivity or resistance, respectively, to quinone outside inhibitors. All of the recovered A. solani isolates collected in 2002 and 2003 were wild type at position 143. However, all three mutations responsible for the F129L substitution (TTA, CTC, and TTG) were detected in our samples. In addition, the frequency of this amino acid substitution in A. solani isolates was statistically different across sampling sites and years, indicating that sensitivity changes depended on specific disease management practices.

Multiplex Real-Time Quantitative PCR to Detect and Quantify Verticillium dahliae Colonization in Potato Lines that Differ in Response to Verticillium Wilt.

ABSTRACT Potato early dying (PED), also known as Verticillium wilt, caused by Verticillium dahliae, is a seasonal yield-limiting disease of potato worldwide, and PED-resistant cultivars currently represent only a small percentage of potato production. In this study, we developed a real-time quantitative polymerase chain reaction (Q-PCR) approach to detect and quantify V. dahliae. The efficiency of the designed primer pair VertBt-F/VertBt-R, derived from the sequence of the beta-tubulin gene, was greater than 95% in monoplex Q-PCR and duplex (using Plexor technology) procedures with primers PotAct-F/PotAct-R, obtained from the sequence of the actin gene, designed for potato. As few as 148 fg of V. dahliae DNA were detected and quantified, which is equivalent to five nuclei. Q-PCR detected V. dahliae in naturally infected air-dried potato stems and fresh stems of inoculated plants. Spearman correlations indicated a high correlation (upward of 80%) between V. dahliae quantifications using Q-PCR and the currently used plating assays. Moreover, Q-PCR substantially reduced the variability compared with that observed in the plating assay, and allowed for the detection of V. dahliae in 10% of stem samples found to be pathogen free on the culture medium. The described Q-PCR approach should provide breeders with a more sensitive and less variable alternative to time-consuming plating assays to distinguish response of breeding lines to colonization by V. dahliae.

Colonization Dynamics and Spatial Progression of Verticillium dahliae in Individual Stems of Two Potato Cultivars with Differing Responses to Potato Early Dying.

Potato early dying (PED), caused by Verticillium dahliae, is a chronic yield-limiting disease of potato (Solanum tuberosum). In this study, we describe the colonization dynamics of V. dahliae in two potato cultivars with varying responses to PED. We utilized a quantitative real-time polymerase chain reaction (Q-PCR) assay to assess the colonization and spatial progression of V. dahliae in cvs. Ranger Russet (moderately resistant) and Russet Norkotah (highly susceptible). Ninety plants per cultivar were inoculated with a conidial suspension in the greenhouse. Every 2 weeks until week 10, we collected basal samples from 15 plants, and repeatedly sampled the growing apices of another 15 plants. The mean infection coefficient (IC) values in the basal and apical samples were significantly lower in cv. Ranger Russet at all five sampling dates. The pathogen was detected in basal samples of both cultivars by week 2, and in apical samples of cv. Russet Norkotah at week 4 and of cv. Ranger Russet at week 6. Colonization of cv. Russet Norkotah consistently increased in apical and basal samples during the 10 weeks, while it plateaued after week 6 in cv. Ranger Russet. Differences in response to PED appear associated with the speed of colonization and the establishment of a higher population density by V. dahliae in the plant.

First Report of Sudden Death Syndrome of Soybean in Wisconsin.

In August of 2006, soybean (Glycine max (L.) Merr.) plants collected from Columbia, Dane, Green Lake, Walworth, Jefferson, and Waushara counties in southern Wisconsin exhibited symptoms typical of sudden death syndrome (SDS) caused by Fusarium virguliforme O'Donnell & Aoki [synonym F. solani (Mart.) Sacc. f. sp. glycines] (1). Foliar symptoms ranged from chlorotic spots to severe interveinal chlorosis and necrosis. Taproots of symptomatic plants were necrotic and stunted and stems exhibited a light tan discoloration, but never the dark brown discoloration typical for brown stem rot, a disease with similar foliar symptoms. Isolations from root and crown tissue of symptomatic plants were made using one-quarter-strength potato dextrose agar (PDA) amended with 100 ppm of streptomycin. Slow-growing, white-to-cream fungal colonies with blue and turquoise sporodochia were observed. Spores produced in sporodochia grown on PDA ranged in size from 32.5 to 70 µm long (average 53.1 µm) and 3 to 6 µm wide (average 4.4 µm) and with 3-5 septa (mode of 3). Isolates were characteristic of F. virguliforme based on colony morphology, spore morphology and size, and the absence of microconidia (3). The identity of F. virguliforme was confirmed by PCR amplification and DNA sequencing of the ITS, BT1, Act, and EF1B regions. All isolate sequences exhibited single nucleotide polymorphisms that matched the sequences of these regions of F. virguliforme. Koch's postulates were conducted to confirm that the causal agent of the observed symptoms was F. virguliforme. Inoculum of single-spore isolates was produced on sterilized sorghum seed. After 14 days of incubation at 20 to 22°C and a 12-h photoperiod, the sorghum seed was assayed to determine colonization incidence by transferring seeds to PDA. In all trials, sorghum seed was 100% infested. Infested sorghum seeds (35) were placed in potting soil at 2 cm beneath each seed of the susceptible soybean cv. Williams 82 (4). Noninfested sorghum seed was used for a noninoculated control. Three trials were performed, each using 15 replicates of several fungal isolates and 15 replicates of the noninoculated control. Plants were grown in water baths located in a greenhouse (trial 1) and in a growth chamber (trial 2) and both maintained at an average temperature of 25°C with a 14-h photoperiod (2). The third trial was conducted in the growth chamber without a water bath with the same temperature and light regimen. In all environments, inoculated plants developed chlorotic spots 14 days after planting. After 21 days, symptoms progressed to a range of chlorotic mottling to interveinal chlorosis and necrosis. Foliar and root symptoms that resembled those on the original plant samples infected with F. virguliforme appeared on 88% of inoculated plants. Isolates that resembled the original F. virguliforme were recovered from 75% of inoculated plants and from 88% of plants showing symptoms. No symptoms were observed and no isolates were recovered from noninoculated plants. There was a statistically significant difference between inoculated and control plants (P < 0.001) based on the presence of symptoms and isolation success using the Goodman χ2 analysis. The confirmation of the presence of SDS in five counties suggests that the disease is widespread in Wisconsin and could become a serious threat to soybean production in the future. References: (1) T. Akoi et al. Mycoscience 46:162, 2005. (2) R. Y. Hashmi et al. Online publication. doi:10.1094/PHP-2005-0906-01-RS. Plant Health Progress, 2005. (3) K. W. Roy et al. Plant Dis. 81:259, 1997. (4) J. C. Rupe et al. Can. J. Bot. 79:829, 2001.

A methodology to detect and quantify five pathogens causing potato tuber decay using real-time quantitative polymerase chain reaction.

ABSTRACT Late blight (Phytophthora infestans), pink rot (Phytophthora erythroseptica), leak (Pythium ultimum), dry rot (Fusarium sambucinum), and soft rot (Erwinia carotovora subsp. carotovora and subsp. atroseptica) are particularly damaging diseases of stored potato tubers worldwide. In this study, we present a methodology to detect and quantify the causal agents of the five aforementioned diseases from whole potato tubers, using real-time quantitative-polymerase chain reaction. Six primer pairs were designed to amplify targets smaller than 150-bp DNA in single copy protein-coding gene targets of each of the pathogens and the potato host. Using a large collection of pure culture DNA samples, all primer pairs specifically detected the DNA target in the intended pathogenic species. Amplification efficiencies over a five-log dilution series ranged between 95 and 100% and were unaffected by the presence of large amounts of host DNA. The detection level of the primers reached 0.5 pg of target DNA. Pathogens were detected in 100 pg of total DNA extracted from 170 to 250 g of tubers, 4 days after inoculation, regardless of the presence of symptoms. The presence of P. erythroseptica, Pythium ultimum, or E. carotovora was also detected in 1 ng of DNA extracted from potato tubers collected from a commercial storage facility. This study provides the first step in a methodology to predict the storability of potato tubers following harvest.

Development of Sclerotinia Stem Rot in Potato Fields in South-Central Washington.

Sclerotinia stem rot, caused by Sclerotinia sclerotiorum, is a recurrent disease on potato in the Columbia Basin of Washington. The role of ascospores in association with disease onset and stem rot incidence in commercial fields and the role of flower blossoms on plant infection were investigated in 10 fields over 2 years. Ascospores of S. sclerotiorum were detected on a semiselective medium over several weeks, with a peak in number of ascospores near initial full bloom. A high proportion of blossoms at initial full bloom were contaminated with S. sclerotiorum prior to blossom fall in most fields. Stem lesions occurred after row closure and blossom drop, and were associated with mycelial mats growing from contaminated blossoms that had dropped on plant stems in the plant canopy or blossoms that dropped on the ground and stems contacting the ground. Incidence of Sclerotinia stem rot was reduced significantly when blossoms were removed from plants before blossom drop. Flower blossoms were shown to be a paramount bridge between airborne ascospores of S. sclerotiorum and stem infection in the potato canopy.

High genetic diversity, phenotypic uniformity, and evidence of outcrossing in sclerotinia sclerotiorum in the columbia basin of washington state.

ABSTRACT Sclerotinia sclerotiorum, the causal agent of potato stem rot, is prevalent and poorly managed on potatoes in the Columbia Basin of Washington. Because of the ubiquitous nature of the fungus and high crop diversity within the Columbia Basin, understanding the population structure and the potential for outcrossing of the pathogen would be helpful in developing disease management strategies. The population structure of S. sclerotiorum in the Columbia Basin from potato was examined using microsatellite markers and mycelial compatibility. Analysis of molecular variance revealed that 92% of the variability among 167 isolates was found within subpopulations, with limited, yet statistically significant impact of the collection date, but not the year or location of collection. Linkage disequilibrium and index of association analyses noted a potential for outcrossing in two locations, which was substantiated by the discovery of recombinant ascospores in three field-generated apothecia from the 12 apothecia examined. Microsatellite haplotypes were not correlated with mycelial compatibility groups. This high haplotypic diversity did not seem to impact pathologically important phenotypes. Greenhouse inoculations of potato plants exhibited no significant differences in aggressiveness on potato stems. Moreover, in vitro studies of response to fungicides and temperature stimuli yielded no significant differences among studied isolates. These findings illustrate the potential for outcrossing in warm temperate regions of North America, where a diversity of crops are planted simultaneously and in neighboring fields. This study also indicates that the unsatisfactory management of potato stem rot is likely not directly attributable to genetic factors, but to gaps in agricultural practices.