DNA barcoding of phytopathogens for disease diagnostics and bio-surveillance.

DNA barcoding has proven to be a versatile tool for plant disease diagnostics in the genomics era. As the mass parallel and next generation sequencing techniques gained importance, the role of specific barcodes came under immense scrutiny. Identification and accurate classification of phytopathogens need a universal approach which has been the main application area of the concept of barcode. The present review entails a detailed description of the present status of barcode application in plant disease diagnostics. A case study on the application of Internal Transcribed Spacer (ITS) as barcode for Aspergillus and Fusarium spp. sheds light on the requirement of other potential candidates as barcodes for accurate identification. The challenges faced while barcoding novel pathogens have also been discussed with a comprehensive outline of integrating more recent technologies like meta-barcoding and genome skimming for detecting plant pathogens.

Fungi, bacteria and oomycota opportunistically isolated from the seagrass, Zostera marina.

Fungi in the marine environment are often neglected as a research topic, despite that fungi having critical roles on land as decomposers, pathogens or endophytes. Here we used culture-dependent methods to survey the fungi associated with the seagrass, Zostera marina, also obtaining bacteria and oomycete isolates in the process. A total of 108 fungi, 40 bacteria and 2 oomycetes were isolated. These isolates were then taxonomically identified using a combination of molecular and phylogenetic methods. The majority of the fungal isolates were classified as belonging to the classes Eurotiomycetes, Dothideomycetes, and Sordariomycetes. Most fungal isolates were habitat generalists like Penicillium sp. and Cladosporium sp., but we also cultured a diverse set of rare taxa including possible habitat specialists like Colletotrichum sp. which may preferentially associate with Z. marina leaf tissue. Although the bulk of bacterial isolates were identified as being from known ubiquitous marine lineages, we also obtained several Actinomycetes isolates and a Phyllobacterium sp. We identified two oomycetes, another understudied group of marine microbial eukaryotes, as Halophytophthora sp. which may be opportunistic pathogens or saprophytes of Z. marina. Overall, this study generates a culture collection of fungi which adds to knowledge of Z. marina associated fungi and highlights a need for more investigation into the functional and evolutionary roles of microbial eukaryotes associated with seagrasses.

Leptolegnia chapmanii como alternativa biológica para el control de Aedes aegypti

Leptolegnia chapmanii is a facultative pathogen of many species of mosquitoes, among which species of the genus Aedes, Culex and Anopheles stand out for their medical and sanitary importance. The potential of L. chapmanii as an alternative to control lies in its virulence, pathogenicity and specificity against the larval stages of mosquitoes, and because of its harmlessness to non-target species such as fish and amphibians, among others. The natural presence of L. chapmanii had been reported in Argentina, Brazil and the United States. Its presence is possible in other countries throughout the American continent. The development of protocols to produce, formulate, store and apply products based on this microorganism is one of the objectives proposed for the group of Entomopathogenic Fungi at the Centro de Estudios Parasitológicos y de Vectores, Universidad Nacional de La Plata. The efficacy of L. chapmanii as controller is affected by external factors such as temperature, pH, salinity and radiation among others. The process of transfer from the research centers to industry implies many phases. In this way, our project with L. chapmanii is in an initial phase, where we are working on a laboratory scale in proof of concept. We hope to begin soon with the efficacy, efficiency, stability and ecotoxicological safety tests, at the laboratory, semi-field and field scale.

Leptolegnia chapmanii es un microorganismo patógeno facultativo de diversas especies de mosquito, entre las que se destacan, por su importancia médica y sanitaria, especies de los géneros Aedes, Culex y Anopheles. El potencial de L. chapmanii como alternativa de control radica en la virulencia, capacidad patógena y grado de especificidad que presenta hacia los estadios larvales de las diferentes especies de mosquito, y por su inocuidad frente a organismos acuáticos no blanco como, por ejemplo, peces y anfibios. Su presencia natural ha sido reportada en Argentina, Brasil, y Estados Unidos, pensándose como posible en otros países dentro del continente americano. La eficacia de L. chapmanii como controlador se ve influenciada por factores externos, como la temperatura, la radiación y el pH, entre otros. Uno de los objetivos de trabajo del Grupo de Hongos Entomopatógenos del Centro de Estudios Parasitológicos y de Vectores de la Universidad Nacional de La Plata, corresponde al desarrollo de protocolos para la producción, formulación, almacenamiento y aplicación de productos basados en este microorganismo. Con este referente, estamos desarrollando un proyecto con L. chapmanii que se encuentra en la fase inicial, en la que se está trabajando la prueba de concepto a escala de laboratorio. Se espera continuar en el futuro con estudios de eficacia, eficiencia, estabilidad y seguridad ecotoxicológica, a diferentes escalas.

Relative Performance of MinION (Oxford Nanopore Technologies) versus Sequel (Pacific Biosciences) Third-Generation Sequencing Instruments in Identification of Agricultural and Forest Fungal Pathogens.

Culture-based molecular identification methods have revolutionized detection of pathogens, yet these methods are slow and may yield inconclusive results from environmental materials. The second-generation sequencing tools have much-improved precision and sensitivity of detection, but these analyses are costly and may take several days to months. Of the third-generation sequencing techniques, the portable MinION device (Oxford Nanopore Technologies) has received much attention because of its small size and possibility of rapid analysis at reasonable cost. Here, we compare the relative performances of two third-generation sequencing instruments, MinION and Sequel (Pacific Biosciences), in identification and diagnostics of fungal and oomycete pathogens from conifer (Pinaceae) needles and potato (Solanum tuberosum) leaves and tubers. We demonstrate that the Sequel instrument is efficient for metabarcoding of complex samples, whereas MinION is not suited for this purpose due to a high error rate and multiple biases. However, we find that MinION can be utilized for rapid and accurate identification of dominant pathogenic organisms and other associated organisms from plant tissues following both amplicon-based and PCR-free metagenomics approaches. Using the metagenomics approach with shortened DNA extraction and incubation times, we performed the entire MinION workflow, from sample preparation through DNA extraction, sequencing, bioinformatics, and interpretation, in 2.5 h. We advocate the use of MinION for rapid diagnostics of pathogens and potentially other organisms, but care needs to be taken to control or account for multiple potential technical biases.IMPORTANCE Microbial pathogens cause enormous losses to agriculture and forestry, but current combined culturing- and molecular identification-based detection methods are too slow for rapid identification and application of countermeasures. Here, we develop new and rapid protocols for Oxford Nanopore MinION-based third-generation diagnostics of plant pathogens that greatly improve the speed of diagnostics. However, due to high error rate and technical biases in MinION, the Pacific BioSciences Sequel platform is more useful for in-depth amplicon-based biodiversity monitoring (metabarcoding) from complex environmental samples.

Viruses of fungi and oomycetes in the soil environment.

Soils support a myriad of organisms hosting highly diverse viromes. In this minireview, we focus on viruses hosted by true fungi and oomycetes (members of Stamenopila, Chromalveolata) inhabiting bulk soil, rhizosphere and litter layer, and representing different ecological guilds, including fungal saprotrophs, mycorrhizal fungi, mutualistic endophytes and pathogens. Viruses infecting fungi and oomycetes are characterized by persistent intracellular nonlytic lifestyles and transmission via spores and/or hyphal contacts. Almost all fungal and oomycete viruses have genomes composed of single-stranded or double-stranded RNA, and recent studies have revealed numerous novel viruses representing yet unclassified family-level groups. Depending on the virus-host combination, infections can be asymptomatic, beneficial or detrimental to the host. Thus, mycovirus infections may contribute to the multiplex interactions of hosts, therefore likely affecting the dynamics of fungal communities required for the functioning of soil ecosystems. However, the effects of fungal and oomycete viruses on soil ecological processes are still mostly unknown. Interestingly, new metagenomics data suggest an extensive level of horizontal virus transfer between plants, fungi and insects.

Growth and fatty acid profiles of Halophytophthora vesicula and Salispina spinosa from Philippine mangrove leaves.

Studies on marine-sourced fatty acids have gathered significant interest recently as an important component of aquaculture feeds and of biofuel production. Of the organisms capable of producing fatty acids, marine oomycetes are promising model organisms. One group of marine oomycetes are the Halophytophthora spp. which is known to have an important role in leaf decomposition, thereby changing the plant debris into exudates which are usable to consumers in the mangrove ecosystems. This study reports the three mangrove oomycetes isolated from Philippine mangrove forests, identified herein as Halophytophthora vesicula AK1YB2 (Aklan), H. vesicula PQ1YB3 (Quezon) and Salispina spinosa ST1YB3 (Davao del Norte). These isolates were subjected to growth analyses using varying incubation parameters (salinity level and pH), and for fatty acid production. Results revealed the presence of different fatty acids such as Arachidonic acid, Linoleic acid and Vaccenic acid when grown on V8S and PYGS media. This study is the first observation of fatty acids from S. spinosa and H. vesicula from the Philippines. SIGNIFICANCE AND IMPACT OF THE STUDY: Tropical Philippines straddling west of the Pacific Ocean and East of South China Sea is rich in marine and estuarine oomycetes. These micro-organisms, hitherto poorly known and unstudied in the country, play an important role in the nutritive cycle of the mangrove ecosystem. Due to the increasing demand for an alternative source of fatty acids, species of Oomycetes isolated from select mangrove forests in Luzon, Visayas and Mindanao were analysed for their fatty acid contents. Prospects for industrially-important fatty acids make these Oomycetes all-important to study in applied microbiology in the Philippine setting where these structurally simple micro-organisms abound.

BjuWRR1, a CC-NB-LRR gene identified in Brassica juncea, confers resistance to white rust caused by Albugo candida.

KEY MESSAGE: BjuWRR1, a CNL-type R gene, was identified from an east European gene pool line of Brassica juncea and validated for conferring resistance to white rust by genetic transformation. White rust caused by the oomycete pathogen Albugo candida is a significant disease of crucifer crops including Brassica juncea (mustard), a major oilseed crop of the Indian subcontinent. Earlier, a resistance-conferring locus named AcB1-A5.1 was mapped in an east European gene pool line of B. juncea-Donskaja-IV. This line was tested along with some other lines of B. juncea (AABB), B. rapa (AA) and B. nigra (BB) for resistance to six isolates of A. candida collected from different mustard growing regions of India. Donskaja-IV was found to be completely resistant to all the tested isolates. Sequencing of a BAC spanning the locus AcB1-A5.1 showed the presence of a single CC-NB-LRR protein encoding R gene. The genomic sequence of the putative R gene with its native promoter and terminator was used for the genetic transformation of a susceptible Indian gene pool line Varuna and was found to confer complete resistance to all the isolates. This is the first white rust resistance-conferring gene described from Brassica species and has been named BjuWRR1. Allelic variants of the gene in B. juncea germplasm and orthologues in the Brassicaceae genomes were studied to understand the evolutionary dynamics of the BjuWRR1 gene.

TLC Screening Profile of Secondary Metabolites and Biological Activities of Salisapilia tartarea S1YP1 Isolated from Philippine Mangroves.

The Salisapilia species are estuarine oomycetes of the mangrove and saltmarsh ecosystem. To date, reports on the secondary metabolites and biological activities of these microorganisms are wanting. In this study, secondary metabolites in broth ethyl acetate extracts (BEAE) and mycelial ethyl acetate extracts (MEAE) of Salisapilia tartarea S1YP1 isolated from yellow senescent mangrove leaves were screened by Thin Layer Chromatography (TLC). Extracts were assayed for antioxidant, antibacterial, α- glucosidase inhibition, and cytotoxic activity. The TLC detected anthraquinones, anthrones, flavonoids, phenols, and triterpenes in both BEAE and MEAE. Coumarins were detected in BEAE but not in MEAE. Quantifying the total phenolics and total flavonoids content of the extracts in terms of gallic acid and quercetin equivalents, respectively shows that BEAE has higher total phenolic and flavonoid contents than MEAE. BEAE exhibited significant antioxidant activities through measurements of free radical scavenging activity against DPPH, hydroxyl, nitric oxide, and superoxide anion radicals as well as the ability to chelate Fe2+ metal ion. BEAE significantly inhibited in a dose-dependent manner α-glucosidase activity and selectively inhibited HepG2 cell proliferation. Antioxidant, α- glucosidase inhibitory, and cytotoxic activities have not been observed for MEAE. Both BEAE and MEAE do not have antibacterial activity.

Occurrence of Foliar Pathogens of Watermelon on Commercial Farms in South Carolina Estimated with Stratified Cluster Sampling.

A survey of foliar pathogens of watermelon based on two-stage cluster sampling was conducted on commercial farms in South Carolina in spring 2015, spring and fall 2016, and fall 2017. In total, 60 fields from 27 different growers in seven counties representing the main watermelon-producing areas in the state were sampled, using a stratified two-stage cluster sampling approach. In the sampling design, counties corresponded to strata, growers to first-stage clusters, and fields to second-stage clusters. In each field, 100 symptomatic leaves were collected at five equidistant sampling points along four transects encompassing a square shape of 2,500 m2. After collection, pathogens were identified based on reproductive structures formed on leaves during >12 h incubation. Estimates were obtained for the statewide probability of pathogen occurrence and associations between pathogen pairs. Six fungal pathogens, Stagonosporopsis spp., Podosphaera xanthii, Cercospora citrullina, Colletotrichum orbiculare, Myrothecium sensu lato (s.l.), and Corynespora cassiicola; the oomycete Pseudoperonospora cubensis; and three viral pathogens were identified on the examined leaves. With the exception of fall 2017, Stagonosporopsis spp. was the most prevalent pathogen in every season, followed by P. xanthii. The highest occurrence of P. cubensis was in spring 2015; it did not occur in 2016. The highest occurrence of C. orbiculare was in spring 2016; it did not occur in spring 2015. Myrothecium s.l. was the most common pathogen in fall 2017 and the second most common pathogen occurring by itself in fall 2016. The third most common pathogen in fall 2017, Corynespora cassiicola, was not observed in any other season. Eight of the 80 isolates of Stagonosporopsis spp. collected were identified as S. caricae, the rest as S. citrulli. All isolates of S. caricae were found in spring 2015 and originated from two fields in different counties. A total of three positive and five negative associations were found between pathogen pairs co-occurring on the same leaf. A positive association between Stagonosporopsis spp. and C. citrullina was the only significant association between pathogens found in two seasons, spring 2015 and spring 2016. Based on estimates of probability of pathogen occurrence across seasons, Stagonosporopsis spp. and P. xanthii are the most common pathogens on watermelons in South Carolina. This is the first report of C. cassiicola, S. caricae, and Myrothecium s.l. on watermelon in South Carolina.

Arabidopsis downy mildew effector HaRxL106 suppresses plant immunity by binding to RADICAL-INDUCED CELL DEATH1.

The oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) causes downy mildew disease on Arabidopsis. To colonize its host, Hpa translocates effector proteins that suppress plant immunity into infected host cells. Here, we investigate the relevance of the interaction between one of these effectors, HaRxL106, and Arabidopsis RADICAL-INDUCED CELL DEATH1 (RCD1). We use pathogen infection assays as well as molecular and biochemical analyses to test the hypothesis that HaRxL106 manipulates RCD1 to attenuate transcriptional activation of defense genes. We report that HaRxL106 suppresses transcriptional activation of salicylic acid (SA)-induced defense genes and alters plant growth responses to light. HaRxL106-mediated suppression of immunity is abolished in RCD1 loss-of-function mutants. We report that RCD1-type proteins are phosphorylated, and we identified Mut9-like kinases (MLKs), which function as phosphoregulatory nodes at the level of photoreceptors, as RCD1-interacting proteins. An mlk1,3,4 triple mutant exhibits stronger SA-induced defense marker gene expression compared with wild-type plants, suggesting that MLKs also affect transcriptional regulation of SA signaling. Based on the combined evidence, we hypothesize that nuclear RCD1/MLK complexes act as signaling nodes that integrate information from environmental cues and pathogen sensors, and that the Arabidopsis downy mildew pathogen targets RCD1 to prevent activation of plant immunity.

Halioticida noduliformans infection in eggs of lobster (Homarus gammarus) reveals its generalist parasitic strategy in marine invertebrates.

A parasite exhibiting Oomycete-like morphology and pathogenesis was isolated from discoloured eggs of the European lobster (Homarus gammarus) and later found in gill tissues of adults. Group-specific Oomycete primers were designed to amplify the 18S ribosomal small subunit (SSU), which initially identified the organism as the same as the 'Haliphthoros' sp. NJM 0034 strain (AB178865.1) previously isolated from abalone (imported from South Australia to Japan). However, in accordance with other published SSU-based phylogenies, the NJM 0034 isolate did not group with other known Haliphthoros species in our Maximum Likelihood and Bayesian phylogenies. Instead, the strain formed an orphan lineage, diverging before the separation of the Saprolegniales and Pythiales. Based upon 28S large subunit (LSU) phylogeny, our own isolate and the previously unidentified 0034 strain are both identical to the abalone pathogen Halioticida noduliformans. The genus shares morphological similarities with Haliphthoros and Halocrusticida and forms a clade with these in LSU phylogenies. Here, we confirm the first recorded occurrence of H. noduliformans in European lobsters and associate its presence with pathology of the egg mass, likely leading to reduced fecundity.

Isolation of Saprolegnia aenigmatica oomycetes and protocol for experimental infection of pacu (Piaractus mesopotamicus) / Isolamento do oomiceto Saprolegnia aenigmatica e protocolo de infecção experimental em pacu (Piaractus mesopotamicus)

Successful disease treatment depends on molecular studies under indoor conditions with experimental infection protocols that facilitate understanding the disease and the drug`s efficacy. The internal transcribed spacer (ITS) region was sequenced from three isolates, which were identified as Saprolegnia aenigmatica. Subsequently, healthy fish were immunosuppressed with dexamethasone (1.2 mg kg-1) and descaled to the skin using a sharp scalpel. These individuals were isolated in individual aquariums maintained at 22°C. Individuals in one group were subcutaneously inoculated with 9,000 zoospores (DDZ treatment), a second group was exposed to oomycetes in water with three colonized baits (DDB), a third group was maintained in water without zoospores (DD), and a control group (C) consisted of healthy animals. After 48 and 96 hours, two animals from each group were euthanized for fungal reisolation. The fish from groups DD and C did not show clinical signs, and no oomycetes were isolated. The animals from the DDZ and DDB groups showed cotton-wool-like masses on the skin, and S. aenigmatica was re-isolated. Thus, for infection using zoospores or baits parasitized by S. aenigmatica, an immunosuppressor (dexamethasone) and a sharp scalpel can be used effectively to establish an experimental infection in P. mesopotamicus.

O sucesso do tratamento de uma enfermidade depende do estudo de moléculas em condições de laboratório por meio de protocolos de infecção experimental que viabilizam a compreensão da doença e da eficácia dos fármacos. Pela sequência ITS foram identificadas três cepas de Saprolegnia aenigmatica. Dessa forma, pacus sadios foram submetidos à imunossupressão com dexametasona (1,2 mg kg-1), esfoliados com auxílio de bisturi e distribuídos em aquários a 22ºC. Após este procedimento, um grupo de animais foi inoculado com 9.000 zoósporos/peixe subcutâneo (DEZ), a outro foram adicionadas três iscas colonizadas com o oomiceto na água (DEI), um terceiro grupo foi mantido sem contato com o oomiceto (DE) e um quarto grupo, de animais sadios, representaram o controle (C). Após 48 e 96h deste procedimento, foram eutanaziados animais de cada grupo para reisolamento. Os animais do grupo DE e C não apresentaram sinais clínicos e não foi reisolado o oomiceto. Porém, tanto os animais do grupo DEZ quanto como os animais do grupo DEI apresentaram micélio branco na pele e foi reisolado Saprolegnia aenigmatica. Assim, a infecção com zoósporos ou com iscas colonizadas por S. aenigmatica, com o uso de dexametasona e abrasivo epitelial são formas eficazes de infecção experimental em P. mesopotamicus.

Protists are an integral part of the Arabidopsis thaliana microbiome.

Although protists occupy a vast range of habitats and are known to interact with plants among other things via disease suppression, competition or growth stimulation, their contributions to the 'phytobiome' are not well described. To contribute to a more comprehensive picture of the plant holobiont, we examined cercozoan and oomycete taxa living in association with the model plant Arabidopsis thaliana grown in two different soils. Soil, roots, leaves and wooden toothpicks were analysed before and after surface sterilization. Cercozoa were identified using 18S rRNA gene metabarcoding, whereas the Internal Transcribed Spacer 1 was used to determine oomycetes. Subsequent analyses revealed strong spatial structuring of protist communities between compartments, although oomycetes appeared more specialized than Cercozoa. With regards to oomycetes, only members of the Peronosporales and taxa belonging to the genus Globisporangium were identified as shared members of the A. thaliana microbiome. This also applied to cercozoan taxa belonging to the Glissomonadida and Cercomonadida. We identified a strong influence by edaphic factors on the rhizosphere, but not for the phyllosphere. Distinct differences of Cercozoa found preferably in wood or fresh plant material imply specific niche adaptations. Our results highlight the importance of micro-eukaryotes for the plant holobiont.

Direct colorimetric detection of unamplified pathogen DNA by dextrin-capped gold nanoparticles.

The interaction between gold nanoparticles (AuNPs) and nucleic acids has facilitated a variety of diagnostic applications, with further diversification of synthesis match bio-applications while reducing biotoxicity. However, DNA interactions with unique surface capping agents have not been fully defined. Using dextrin-capped AuNPs (d-AuNPs), we have developed a novel unamplified genomic DNA (gDNA) nanosensor, exploiting dispersion and aggregation characteristics of d-AuNPs, in the presence of gDNA, for sequence-specific detection. We demonstrate that d-AuNPs are stable in a five-fold greater salt concentration than citrate-capped AuNPs and the d-AuNPs were stabilized by single stranded DNA probe (ssDNAp). However, in the elevated salt concentrations of the DNA detection assay, the target reactions were surprisingly further stabilized by the formation of a ssDNAp-target gDNA complex. The results presented herein lead us to propose a mechanism whereby genomic ssDNA secondary structure formation during ssDNAp-to-target gDNA binding enables d-AuNP stabilization in elevated ionic environments. Using the assay described herein, we were successful in detecting as little as 2.94 fM of pathogen DNA, and using crude extractions of a pathogen matrix, as few as 18 spores/µL.

Metagenomic analysis of oomycete communities from the rhizosphere of field pea on the Canadian prairies.

Oomycetes are a diverse group of microorganisms; however, little is known about their composition and biodiversity in agroecosystems. Illumina MiSeq was used to determine the type and abundance of oomycetes associated with pea root rot in the Canadian prairies. Additional objectives of the study were to identify differences in oomycete communities associated with pea root health and compare oomycete communities among the 3 prairie provinces, where field peas are commonly cultivated. Samples of soil from the rhizosphere of field pea (Pisum sativum L.) were collected from patches of asymptomatic or diseased plants from 26 commercial fields in 2013 and 2014. Oomycete communities were characterized using metagenomic analysis of the ITS1 region on Illumina MiSeq. From 105 identified operational taxonomic units (OTUs), 45 and 16 oomycete OTUs were identified at species and genus levels, respectively. Pythium was the most prevalent genus and Pythium heterothallicum the most prevalent species in all 3 provinces in both 2013 and 2014. Aphanomyces euteiches, a very important pea root rot pathogen in regions of the prairies, was detected in 57% of sites but at very low abundance (<0.2%). Multivariate analysis revealed differences in the relative abundance of species in oomycete communities between asymptomatic and diseased sites, and among years and provinces. This study demonstrated that deep amplicon sequencing can provide information on the composition and diversity of oomycete communities in agricultural soils.

Assessment of Cytokinin-Induced Immunity Through Quantification of Hyaloperonospora arabidopsidis Infection in Arabidopsis thaliana.

Cytokinins have been shown to regulate plant immunity. Application of high levels of cytokinin to plants leads to decreased susceptibility to pathogens. In this chapter, we describe a fast and accurate protocol for assessment of cytokinin-induced immunity in Arabidopsis plants against an oomycete plant pathogen.

Oomycete Species Associated with Soybean Seedlings in North America-Part I: Identification and Pathogenicity Characterization.

Oomycete pathogens are commonly associated with soybean root rot and have been estimated to reduce soybean yields in the United States by 1.5 million tons on an annual basis. Limited information exists regarding the frequency and diversity of oomycete species across the major soybean-producing regions in North America. A survey was conducted across 11 major soybean-producing states in the United States and the province of Ontario, Canada. In 2011, 2,378 oomycete cultures were isolated from soybean seedling roots on a semiselective medium (CMA-PARPB) and were identified by sequencing of the internal transcribed spacer region of rDNA. Sequence results distinguished a total of 51 Pythium spp., three Phytophthora spp., three Phytopythium spp., and one Aphanomyces sp. in 2011, with Pythium sylvaticum (16%) and P. oopapillum (13%) being the most prevalent. In 2012, the survey was repeated, but, due to drought conditions across the sampling area, fewer total isolates (n = 1,038) were collected. Additionally, in 2012, a second semiselective medium (V8-RPBH) was included, which increased the Phytophthora spp. isolated from 0.7 to 7% of the total isolates. In 2012, 54 Pythium spp., seven Phytophthora spp., six Phytopythium spp., and one Pythiogeton sp. were recovered, with P. sylvaticum (14%) and P. heterothallicum (12%) being recovered most frequently. Pathogenicity and virulence were evaluated with representative isolates of each of the 84 species on soybean cv. Sloan. A seed-rot assay identified 13 and 11 pathogenic species, respectively, at 13 and 20°C. A seedling-root assay conducted at 20°C identified 43 species as pathogenic, having a significantly detrimental effect on the seedling roots as compared with the noninoculated control. A total of 15 species were pathogenic in both the seed and seedling assays. This study provides a comprehensive characterization of oomycete species present in soybean seedling roots in the major production areas in the United States and Ontario, Canada and provides a basis for disease management and breeding programs.

Occurrence and Distribution of Mating Types of Pseudoperonospora cubensis in the United States.

During the past two decades, a resurgence of cucurbit downy mildew has occurred around the world, resulting in severe disease epidemics. In the United States, resurgence of the disease occurred in 2004 and several hypotheses, including introduction of a new genetic recombinant or pathotype of the pathogen, have been suggested as potential causes for this resurgence. Occurrence and distribution of mating types of Pseudoperonospora cubensis in the United States were investigated using 40 isolates collected from cucurbits across 11 states from 2005 to 2013. Pairing of unknown isolates with known mating-type tester strains on detached leaves of cantaloupe or cucumber resulted in oospore formation 8 to 10 days after inoculation. Isolates differed in their ability to form oospores across all coinoculation pairings, with oospore numbers ranging from 280 to 1,000 oospores/cm2 of leaf tissue. Oospores were hyaline to golden-yellow, spherical, and approximately 36 µm in diameter. Of the 40 isolates tested, 24 were found to be of the A1 mating type, while 16 were of the A2 mating type. Mating type was significantly (P < 0.0001) associated with host type, whereby all isolates collected from cucumber were of the A1 mating type, while isolates from squash and watermelon were of the A2 mating type. Similarly, mating type was significantly (P = 0.0287) associated with geographical region, where isolates from northern-tier states of Michigan, New Jersey, New York, and Ohio were all A1, while isolates belonging to either A1 or A2 mating type were present in equal proportions in southern-tier states of Alabama, Florida, Georgia, North Carolina, South Carolina, and Texas. Viability assays showed that oospores were viable and, on average, approximately 40% of the oospores produced were viable as determined by the plasmolysis method. This study showed that A1 and A2 mating types of P. cubensis are present and the pathogen could potentially reproduce sexually in cucurbits within the United States. In addition, the production of viable oospores reported in this study suggests that oospores could have an important role in the biology of P. cubensis and could potentially influence the epidemiology of cucurbit downy mildew in the United States.

Oomycete Species Associated with Soybean Seedlings in North America-Part II: Diversity and Ecology in Relation to Environmental and Edaphic Factors.

Soybean (Glycine max (L.) Merr.) is produced across a vast swath of North America, with the greatest concentration in the Midwest. Root rot diseases and damping-off are a major concern for production, and the primary causal agents include oomycetes and fungi. In this study, we focused on examination of oomycete species distribution in this soybean production system and how environmental and soil (edaphic) factors correlate with oomycete community composition at early plant growth stages. Using a culture-based approach, 3,418 oomycete isolates were collected from 11 major soybean-producing states and most were identified to genus and species using the internal transcribed spacer region of the ribosomal DNA. Pythium was the predominant genus isolated and investigated in this study. An ecology approach was taken to understand the diversity and distribution of oomycete species across geographical locations of soybean production. Metadata associated with field sample locations were collected using geographical information systems. Operational taxonomic units (OTU) were used in this study to investigate diversity by location, with OTU being defined as isolate sequences with 97% identity to one another. The mean number of OTU ranged from 2.5 to 14 per field at the state level. Most OTU in this study, classified as Pythium clades, were present in each field in every state; however, major differences were observed in the relative abundance of each clade, which resulted in clustering of states in close proximity. Because there was similar community composition (presence or absence) but differences in OTU abundance by state, the ordination analysis did not show strong patterns of aggregation. Incorporation of 37 environmental and edaphic factors using vector-fitting and Mantel tests identified 15 factors that correlate with the community composition in this survey. Further investigation using redundancy analysis identified latitude, longitude, precipitation, and temperature as factors that contribute to the variability observed in community composition. Soil parameters such as clay content and electrical conductivity also affected distribution of oomycete species. The present study suggests that oomycete species composition across geographical locations of soybean production is affected by a combination of environmental and edaphic conditions. This knowledge provides the basis to understand the ecology and distribution of oomycete species, especially those able to cause diseases in soybean, providing cues to develop management strategies.

Detection and Quantification of Bremia lactucae by Spore Trapping and Quantitative PCR.

Bremia lactucae is an obligate, oomycete pathogen of lettuce that causes leaf chlorosis and necrosis and adversely affects marketability. The disease has been managed with a combination of host resistance and fungicide applications with success over the years. Fungicide applications are routinely made under the assumption that inoculum is always present during favorable environmental conditions. This approach often leads to fungicide resistance in B. lactucae populations. Detection and quantification of airborne B. lactucae near lettuce crops provides an estimation of the inoculum load, enabling more judicious timing of fungicide applications. We developed a quantitative polymerase chain reaction (qPCR)-based assay using a target sequence in mitochondrial DNA for specific detection of B. lactucae. Validation using amplicon sequencing of DNA from 83 geographically diverse isolates, representing 14 Bremia spp., confirmed that the primers developed for the TaqMan assays are species specific and only amplify templates from B. lactucae. DNA from a single sporangium could be detected at a quantification cycle (Cq) value of 32, and Cq values >35 were considered to be nonspecific. The coefficient of determination (R2) for regression between sporangial density derived from flow cytometry and Cq values derived from the qPCR was 0.86. The assay was deployed using spore traps in the Salinas Valley, where nearly half of U.S. lettuce is produced. The deployment of this sensitive B. lactucae-specific assay resulted in the detection of the pathogen during the 2-week lettuce-free period as well as during the cropping season. These results demonstrate that this assay will be useful for quantifying inoculum load in and around the lettuce fields for the purpose of timing fungicide applications based on inoculum load.