Identification and Characterization of Pythium, Globisporangium, and Phytopythium Species Present in Floricultural Crops from Long Island, New York.

Several Pythium, Globisporangium, and Phytopythium species cause Pythium diseases in greenhouse floricultural crops, resulting in significant seasonal losses. Four hundred and eighteen Pythium, Globisporangium, and Phytopythium isolates from flowering crops, growing media, or bench and floor debris were collected from Long Island greenhouses or clinic samples between 2002 and 2013. Isolates were identified to species based on morphology and internal transcribed spacer barcoding. Twenty-two species of Pythium, Phytopythium, and Globisporangium were identified, with Globisporangium irregulare sensu lato (s.l.) being the most common. To determine the origin of inoculum during the 2011 cropping season, 11 microsatellite loci were analyzed in 124 G. irregulare s.l. isolates collected in four greenhouses and six previously collected from clinic samples. Cluster analyses grouped G. irregulare s.l. isolates into four groups: G. irregulare sensu stricto, plus three G. cryptoirregulare clusters. The population structure defined by greenhouse and host was found in two clades. Additionally, the population dynamics of G. irregulare s.l. isolates associated with Pelargonium spp. from 2011 to 2013 were examined using 85 isolates and nine informative microsatellite loci to assess inoculum survival over multiple cropping seasons. Although most isolates had unique genotypes, closely related genotypes were found in the same locations over different years. Our results indicate that G. irregulare s.l. inocula have local as well as remote origins. Isolates may be initially brought into ornamental operations from common sources, such as infected plant materials or infested potting mixes. Our results support the hypothesis that established strains can serve as inocula and survive in greenhouse facilities over multiple seasons.

A Step Towards Validation of High-Throughput Sequencing for the Identification of Plant Pathogenic Oomycetes.

The advancement in high-throughput sequencing (HTS) technology allows the detection of pathogens without the need for isolation or template amplification. Plant regulatory agencies worldwide are adopting HTS as a prescreening tool for plant pathogens in imported plant germplasm. The technique is a multipronged process and, often, the bioinformatic analysis complicates detection. Previously, we developed E-probe diagnostic nucleic acid analysis (EDNA), a bioinformatic tool that detects pathogens in HTS data. EDNA uses custom databases of signature nucleic acid sequences (e-probes) to reduce computational effort and subjectivity when determining pathogen presence in a sample. E-probes of Pythium ultimum and Phytophthora ramorum were previously validated only using simulated HTS data. However, HTS samples generated from infected hosts or pure culture may vary in pathogen concentration, sequencing bias, and data quality, suggesting that each pathosystem requires further validation. Here, we used metagenomic and genomic HTS data generated from infected hosts and pure culture, respectively, to further validate and curate e-probes of Pythium ultimum and Phytophthora ramorum. E-probe length was found to be a determinant of diagnostic sensitivity and specificity; 80-nucleotide e-probes increased the diagnostic specificity to 100%. Curating e-probes to increase specificity affected diagnostic sensitivity only for 80-nucleotide Pythium ultimum e-probes. Comparing e-probes with alternative databases and bioinformatic tools in their speed and ability to find Pythium ultimum and Phytophthora ramorum demonstrated that, although pathogen sequence reads were detected by other methods, they were less specific and slower when compared with e-probes.

Genetic Diversity of Phymatotrichopsis omnivora Based on Mating Type and Microsatellite Markers Reveals Heterothallic Mating System.

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.

Multiplex End-Point PCR for the Detection of Three Species of Ophiosphaerella Causing Spring Dead Spot of Bermudagrass.

A multiplex end-point polymerase chain reaction (PCR) assay was developed for identifying the three-fungal species in the genus Ophiosphaerella that cause spring dead spot (SDS), a devastating disease of bermudagrass. These fungi are difficult to identify by morphology because they seldom produce pseudothecia. To achieve species-specific diagnosis, three pairs of primers were designed to identify fungal isolates and detect the pathogen in infected roots. The internal transcribed spacer region, the translation elongation factor 1-α, and the RNA polymerase II second-largest subunit were selected as targets and served as templates for the design of each primer pair. To achieve uniform melting temperatures, three to five random nucleotide extensions (flaps) were added to the 5' terminus of some of the designed specific primers. Temperature cycling conditions and PCR components were standardized to optimize specificity and sensitivity of the multiplex reaction. Primers were tested in multiplex on DNA extracted from axenic fungal cultures and from field-collected infected and uninfected roots. A distinct amplicon was produced for each Ophiosphaerella sp. tested. The DNA from Ophiosphaerella close relatives and other common bermudagrass pathogens did not amplify during the multiplex assay. Metagenomic DNA from infected bermudagrass produced species-specific amplicons while DNA extracted from noninfected roots did not. This multiplex end-point PCR approach is a sensitive and specific molecular technique that allows for correct identification of SDS-associated Ophiosphaerella spp. from field-collected roots.

Hormetic Effects of Thiophanate-Methyl in Multiple Isolates of Sclerotinia homoeocarpa.

Twenty-eight isolates of Sclerotinia homoeocarpa, causal agent of dollar spot disease in turf, were assessed for fungicide hormesis at sublethal concentrations of thiophanate-methyl (T-methyl). Each isolate was grown in corn meal agar amended with 11 concentrations of T-methyl (30,500 to 0.047 µg/liter), and the area of mycelial growth was determined relative to the control. Three replicates were used per concentration, and the experiment was repeated three to five times for each isolate. Reference isolates (EC50 > 20 µg/liter), with no prior history of T-methyl exposure, were highly sensitive and not stimulated by low doses. Likewise, no stimulation was observed in two highly sensitive isolates (EC50 > 30 µg/liter) that had been preconditioned by exposure to T-methyl, or in four T-methyl-tolerant isolates. Seventeen (81%) preconditioned T-methyl-tolerant isolates (EC50 = 294 to1,550 µg/liter) had statistically significant growth stimulation, in the range of 2.8 to 19.7% relative to the control. These results support that hormesis (low-dose stimulation, high-dose inhibition) is a common dose response in preconditioned S. homoeocarpa, particularly in response to subtoxic doses of T-methyl.

Inferring the presence of aflatoxin-producing Aspergillus flavus strains using RNA sequencing and electronic probes as a transcriptomic screening tool.

E-probe Diagnostic for Nucleic acid Analysis (EDNA) is a bioinformatic tool originally developed to detect plant pathogens in metagenomic databases. However, enhancements made to EDNA increased its capacity to conduct hypothesis directed detection of specific gene targets present in transcriptomic databases. To target specific pathogenicity factors used by the pathogen to infect its host or other targets of interest, e-probes need to be developed for transcripts related to that function. In this study, EDNA transcriptomics (EDNAtran) was developed to detect the expression of genes related to aflatoxin production at the transcriptomic level. E-probes were designed from genes up-regulated during A. flavus aflatoxin production. EDNAtran detected gene transcripts related to aflatoxin production in a transcriptomic database from corn, where aflatoxin was produced. The results were significantly different from e-probes being used in the transcriptomic database where aflatoxin was not produced (atoxigenic AF36 strain and toxigenic AF70 in Potato Dextrose Broth).

Identification and characterization of simple sequence repeats (SSRs) for population studies of Puccinia novopanici.

Switchgrass (Panicum virgatum L.) can be severely affected by rust disease. Recently switchgrass rust caused by P. emaculata (now confirmed to be Puccinia novopanici) has received most of the attention by the research community because this pathogen is responsible for reducing the biomass production and biofuel feedstock quality of switchgrass. Microsatellite markers found in the literature were either not informative (no allele frequency) or showed few polymorphisms in the target populations, therefore additional markers are needed for future studies of the genetic variation and population structure of P. novopanici. This study reports the development and characterization of novel simple sequence repeat (SSR) markers from a Puccinia emaculata s.l. microsatellite-enriched library and expressed sequence tags (ESTs). Microsatellites were evaluated for polymorphisms on P. emaculata s.l. urediniospores collected in Iowa (IA), Mississippi (MS), Oklahoma (OK), South Dakota (SD) and Virginia (VA). Puccinia novopanici single spore whole genome amplifications were used as templates to validate the SSR reactions protocol and to assess a preliminary population genetics statistics of the pathogen. Eighteen microsatellite markers were polymorphic (average PIC=0.72) on individual urediniospores, with an average of 8.3 alleles per locus (range 3 to 17). Of the 49 SSRs loci initially identified in P. emaculata s.l., 18 were transferable to P. striiformis f. sp. tritici, 23 to P. triticina, 20 to P. sorghi and 31 to P. andropogonis. Thus, these markers could be useful for DNA fingerprinting and population structure analysis for population genetics, epidemiology and ecological studies of P. novopanici and potentially other related Puccinia species.

Population Structure of Pythium irregulare, P. ultimum, and P. sylvaticum in Forest Nursery Soils of Oregon and Washington.

Pythium species are important soilborne pathogens occurring in the forest nursery industry of the Pacific Northwest. However, little is known about their genetic diversity or population structure and it is suspected that isolates are moved among forest nurseries on seedling stock and shared field equipment. In order to address these concerns, a total of 115 isolates of three Pythium species (P. irregulare, P. sylvaticum, and P. ultimum) were examined at three forest nurseries using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers. Analyses revealed distinct patterns of intraspecific variation for the three species. P. sylvaticum exhibited the most diversity, followed by P. irregulare, while substantial clonality was found in P. ultimum. For both P. irregulare and P. sylvaticum, but not P. ultimum, there was evidence for significant variation among nurseries. However, all three species also exhibited at least two distinct lineages not associated with the nursery of origin. Finally, evidence was found that certain lineages and clonal genotypes, including fungicide-resistant isolates, are shared among nurseries, indicating that pathogen movement has occurred.

Discriminatory simplex and multiplex PCR for four species of the genus Sclerotinia.

Sclerotinia sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Eriks, and S. homoeocarpa F.T. Benn are the most relevant plant pathogenic species within the genus Sclerotinia because of their large range of economically important hosts, including tomato, peanut, alfalfa, and turfgrass, among others. Species identification based on morphological characteristics is challenging and time demanding, especially when one crop hosts multiple species. The objective of this study was to design specific primers compatible with multiplexing, for rapid, sensitive and accurate detection and discrimination among four Sclerotinia species. Specific primers were designed for the aspartyl protease gene of S. sclerotiorum, the calmodulin gene of S. trifoliorum, the elongation factor-1 alpha gene of S. homoeocarpa, and the laccase 2 gene of S. minor. The specificity and sensitivity of each primer set was tested individually and in multiplex against isolates of each species and validated using genomic DNA from infected plants. Each primer set consistently amplified DNA of its target gene only. DNA fragments of different sizes were amplified: a 264 bp PCR product for S. minor, a 218 bp product for S. homoeocarpa, a 171 bp product for S. sclerotiorum, and a 97 bp product for S. trifoliorum. These primer sets can be used individually or in multiplex for identification of Sclerotinia spp. in pure culture or from infected plants. The multiplex assay had a lower sensitivity limit than the simplex assays (0.0001 pg/µL DNA of each species). The multiplex assay developed is an accurate and rapid tool to differentiate between the most relevant plant pathogenic Sclerotinia species in a single PCR reaction.

Detection and assessment of chemical hormesis on the radial growth in vitro of oomycetes and fungal plant pathogens.

Although plant diseases can be caused by bacteria, viruses, and protists, most are caused by fungi and fungus-like oomycetes. Intensive use of fungicides with the same mode of action can lead to selection of resistant strains increasing the risk of unmanageable epidemics. In spite of the integrated use of nonchemical plant disease management strategies, agricultural productivity relies heavily on the use of chemical pesticides and biocides for disease prevention and treatment and sanitation of tools and substrates. Despite the prominent use of fungi in early hormesis studies and the continuous use of yeast as a research model, the relevance of hormesis in agricultural systems has not been investigated by plant pathologists, until recently. A protocol was standardized for detection and assessment of chemical hormesis in fungi and oomycetes using radial growth as endpoint. Biphasic dose-responses were observed in Pythium aphanidermatum exposed to sub-inhibitory doses of ethanol, cyazofamid, and propamocarb, and in Rhizoctonia zeae exposed to ethanol. This report provides an update on chemical hormesis in fungal plant pathogens and a perspective on the potential risks it poses to crop productivity and global food supply.

Sublethal Doses of Mefenoxam Enhance Pythium Damping-off of Geranium.

The effect of sublethal doses of fungicides on fungicide-resistant Pythium isolates is unknown but potentially relevant to disease management. Occasional grower reports of Pythium disease increases after fungicide applications and our observations of greater radial growth in vitro on fungicide-amended media than on nonamended media suggests that Pythium isolates may be stimulated by sublethal doses of fungicides. The objectives of this study were to determine whether Pythium isolates were stimulated by sublethal doses of mefenoxam in vitro and whether this stimulation had any influence on Pythium damping-off of geranium seedlings. A mefenoxam-resistant isolate of Pythium aphanidermatum displayed 10% mean radial growth increase in vitro with mefenoxam at 1 × 10-10 µg/ml compared with growth on nonamended agar (nonsignificant). Geranium seedlings treated with one of eight mefenoxam concentrations were inoculated with 5-mm-diameter colonized agar plugs and evaluated for disease severity every 24 h. The area under the disease progress curve and the survival curve were estimated for each treatment and compared. Significant increases in damping-off were observed with mefenoxam at 1 × 10-6 and 1 × 10-10 µg/ml. Our data indicate that a Pythium isolate with resistance to mefenoxam can be stimulated by sublethal doses of the fungicide, and that this stimulation can result in significantly higher rates of Pythium damping-off of geranium seedlings.

Genetic structure and distribution of pythium aphanidermatum populations in Pennsylvania greenhouses based on analysis of AFLP and SSR markers.

Pythium aphanidermatum is one of the most aggressive species in the genus and has a wide host range, but little is known about its population genetic structure. We tested 123 P. aphanidermatum isolates with six AFLP primer combinations and four SSR markers. The genetic diversity of P. aphanidermatum was 0.34 with AFLP and 0.55 with SSR markers. SSR genotypes totaled 3-8 for each locus, and a total of 14 SSR genotypes were found among all isolates. Three major genetic groups were identified with the combination of AFLP and SSR marker data. The genetic structure observed among P. aphanidermatum isolates was related to location and mefenoxam fungicide resistance instead of host. Four genotypes (PA1, PA2, PA5 and PA7) were found in the population from a commercial greenhouse, and the genetic diversity of a greenhouse population was similar to that found in the whole sample. The molecular tools for P. aphanidermatum isolates identified the possible gene flow within and among populations in Pennsylvania greenhouses.

Pythium cryptoirregulare, a new species within the P. irregulare complex.

Pythium identification is based on several characteristics with considerable variation, particularly in Pythium irregulare Buis. as currently recognized. Thirty-one isolates of Pythium irregulare Buis. from various hosts and geographic regions were compared by genetic analysis of multiloci DNA fingerprints, sequence analysis of nuclear and mitochondrial genes and morphological and growth rate studies. Previous research indicated two distinct groupings within the species, P. irregulare sensu stricto and a clade referred to here as Pythium sp. Parsimony analyses of 338 AFLP markers divided P. irregulare s.l. into two clades. Comparison of the allele frequencies of 236 polymorphic AFLP loci revealed significant differences between them. The two clades differed in the frequencies of 182 (77%) alleles. P. irregulare s.s. had 122 (52%) polymorphic loci while Pythium sp. had 205 (87%). Pythium sp. had one fixed allele and 79 polymorphic loci absent in P. irregulare s.s. P. irregulare s.s. displayed 16 polymorphic loci absent in Pythium sp. Parsimony and distance analyses of the ribosomal intergenic transcribed spacers (ITS) and the cox II gene sequences support the separation of P. irregulare s.s. and Pythium sp. Amplicon length in P. irregulare s.s. ITS sequences were 936-938 bp and 936-949 bp in Pythium sp. The two clades were separated by two fixed insertion/deletion mutations, nine fixed nucleotide substitutions in the ITS region and three fixed single nucleotide substitutions in the cox II sequences. Average growth rates of the groups differed at 10, 30 and 36 C but not at 15, 21 or 25 C. Statistically significant differences were found in oogonium, oospore and ooplast diameters, antheridial cell length and in ooplast index. We propose that a new species, Pythium cryptoirregulare, be delineated from Pythium irregulare sensu stricto.

Amplified Fragment Length Polymorphism Analysis and Internal Transcribed Spacer and coxII Sequences Reveal a Species Boundary Within Pythium irregulare.

ABSTRACT Pythium irregulare is a plant-pathogenic oomycete that causes significant damage to a variety of crops, including ornamentals and vegetables. Morphological as well as molecular studies have reported high levels of genetic diversity within P. irregulare sensu lato which has raised the question as to whether it is a single species or is actually a complex of morphologically similar (cryptic) species. In this study, we used amplified fragment length polymorphism (AFLP) fingerprinting and DNA sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal genes (ITS region) and a portion of the mitochondrial cytochrome oxidase II gene and the spacer region between coxI and coxII to characterize 68 isolates of P. irregulare from the United States. The ITS sequence of a P. irregulare neotype at the CBS collection as well as ITS and coxII sequences for P. irregulare, P. spinosum, and P. sylvaticum from previous studies were included in our analysis. Cluster analysis identified a 19-isolate group (IR-II) that separated itself from the rest of the sample (IR-I). Population structure and sequence analyses supported the distinction of IR-I and IR-II and identified IR-II as P. irregulare sensu stricto. IR-I was designated Pythium sp. clade IR-I. Two insertion/deletion mutations and nine nucleotide substitutions in the ITS region and three in the sequence of coxII and the adjacent spacer region separated the two species. Additionally, they differed significantly (P > 0.01) in the frequency of 182 (77%) AFLP alleles. Gene flow results suggested that P. irregulare sensu stricto and Pythium sp. clade IR-I are cryptic species capable of exchanging favorable alleles (Nm = 0.72).

Diagnosis and Population Analysis of Pythium Species Using AFLP Fingerprinting.

Accurate identification of Pythium species, the causal agents of Pythium root rot and dampingoff of seedlings, and characterization of populations within the species would greatly assist in selecting and implementing control strategies for these pathogens. Several molecular techniques offer methods for accurate and rapid identification of species, but provide little information about their populations. In this study, amplified fragment length polymorphism (AFLP) fingerprinting was used to characterize plant-pathogenic Pythium species and intraspecific populations. Species-diagnostic AFLP fingerprints for Pythium aphanidermatum, P. irregulare, and P. ultimum, and tentative fingerprints for six other species, were identified. Intraspecific distance analyses of P. aphanidermatum, P. ultimum, and P. irregulare revealed distinct patterns of intraspecific variation among the three species. P. aphanidermatum showed the smallest mean distance among isolates (15%), followed by P. ultimum (37%). P. irregulare had the largest mean distance among isolates (64%), which were divided into two populations with great genetic differentiation (FST = 0.2), suggesting the presence of a cryptic species boundary within this species.