First confirmed case of equine pythiosis in Northern Veracruz, Mexico.

A clinical case of an adult horse with invasive, ulcerative, proliferative, pyogranulomatous disease of the skin (tumor) in the shoulder region is presented. The mass had a granulomatous and crater-shaped appearance, with serosanguinous discharge and the presence of fistulas with caseous material. The tumor was removed by surgery and sent to the laboratory for diagnosis. Histopathology was performed using Grocott-Gomori methenamine silver stain. The presence of necrotic material, fibrosis, infiltrated cells, and brown-colored hyphae, characteristic of members of the genus Pythium, were observed. To identify the infecting species, conventional PCRs for the amplification of the ITS-1 was carried out. Histopathological and PCR tests confirmed infection by a Pythium insidiosum strain closely associated with previous records from the US and Central America. Our report represents the first molecularly confirmed case of equine pythiosis in Mexico.

Generation of protoplasts provides a powerful experimental research tool for biological and pathogenicity studies of Pythium insidiosum.

INTRODUCTION: Pythiosis is a high-mortality infectious condition in humans and animals. The etiologic agent is Pythium insidiosum. Patients present with an ocular, vascular, cutaneous/subcutaneous, or gastrointestinal infection. Antifungal medication often fails to fight against P. insidiosum. The effective treatment is limited to radical surgery, resulting in organ loss. Fatal outcomes are observed in advanced cases. Pythiosis needs to be studied to discover novel methods for disease control. Genome data of P. insidiosum is publicly available. However, information on P. insidiosum biology and pathogenicity is still limited due to the lack of a cost-effective animal model and molecular tools. MATERIALS AND METHODS: We aimed to develop a high-efficiency protocol for generating P. insidiosum protoplast, and used it to set up an animal model, in vitro drug susceptibility assay, and DNA transformation for this pathogen. RESULTS: P. insidiosum protoplast was successfully generated to establish a feasible pythiosis model in embryonic chicken eggs and an efficient in vitro drug susceptibility assay. DNA transformation is a critical method for gene manipulation necessary for functional genetic studies in pathogens. Attempts to establish a DNA transformation method for P. insidiosum using protoplast were partly successful. Significant work needs to be done for genetically engineering a more robust selection marker to generate stable transformants at increased efficiency. CONCLUSION: This study is the first to report an efficient P. insidiosum protoplast production for clinical and research applications. Such advances are crucial to speeding up the pathogen's biology and pathogenicity exploration.

Volatile Organic Compounds Emitted by the Biocontrol Agent Pythium oligandrum Contribute to Ginger Plant Growth and Disease Resistance.

The oomycete Pythium oligandrum is a potential biocontrol agent to control a wide range of fungal and oomycete-caused diseases, such as Pythium myriotylum-caused rhizome rot in ginger, leading to reduced yields and compromised quality. Previously, P. oligandrum has been studied for its plant growth-promoting potential by auxin production and induction of disease resistance by elicitors such as oligandrin. Volatile organic compounds (VOCs) play beneficial roles in sustainable agriculture by enhancing plant growth and resistance. We investigated the contribution of P. oligandrum-produced VOCs on plant growth and disease suppression by initially using Nicotiana benthamiana plants for screening. P. oligandrum VOCs significantly enhanced tobacco seedling and plant biomass contents. Screening of the individual VOCs showed that 3-octanone and hexadecane promoted the growth of tobacco seedlings. The total VOCs from P. oligandrum also enhanced the shoot and root growth of ginger plants. Transcriptomic analysis showed a higher expression of genes related to plant growth hormones and stress responses in the leaves of ginger plants exposed to P. oligandrum VOCs. The concentrations of plant growth hormones such as auxin, zeatin, and gibberellic acid were higher in the leaves of ginger plants exposed to P. oligandrum VOCs. In a ginger disease biocontrol assay, the VOC-exposed ginger plants infected with P. myriotylum had lower levels of disease severity. We conclude that this study contributes to understanding the growth-promoting mechanisms of P. oligandrum on ginger and tobacco, priming of ginger plants against various stresses, and the mechanisms of action of P. oligandrum as a biocontrol agent. IMPORTANCE Plant growth promotion plays a vital role in enhancing production of agricultural crops, and Pythium oligandrum is known for its plant growth-promoting potential through production of auxins and induction of resistance by elicitors. This study highlights the significance of P. oligandrum-produced VOCs in plant growth promotion and disease resistance. Transcriptomic analyses of leaves of ginger plants exposed to P. oligandrum VOCs revealed the upregulation of genes involved in plant growth hormone signaling and stress responses. Moreover, the concentration of growth hormones significantly increased in P. oligandrum VOC-exposed ginger plants. Additionally, the disease severity was reduced in P. myriotylum-infected ginger plants exposed to P. oligandrum VOCs. In ginger, P. myriotylum-caused rhizome rot disease results in severe losses, and biocontrol has a role as part of an integrated pest management strategy for rhizome rot disease. Overall, growth enhancement and disease reduction in plants exposed to P. oligandrum-produced VOCs contribute to its role as a biocontrol agent.

Genome of Pythium myriotylum Uncovers an Extensive Arsenal of Virulence-Related Genes among the Broad-Host-Range Necrotrophic Pythium Plant Pathogens.

The Pythium (Peronosporales, Oomycota) genus includes devastating plant pathogens that cause widespread diseases and severe crop losses. Here, we have uncovered a far greater arsenal of virulence factor-related genes in the necrotrophic Pythium myriotylum than in other Pythium plant pathogens. The genome of a plant-virulent P. myriotylum strain (~70 Mb and 19,878 genes) isolated from a diseased rhizome of ginger (Zingiber officinale) encodes the largest repertoire of putative effectors, proteases, and plant cell wall-degrading enzymes (PCWDEs) among the studied species. P. myriotylum has twice as many predicted secreted proteins than any other Pythium plant pathogen. Arrays of tandem duplications appear to be a key factor of the enrichment of the virulence factor-related genes in P. myriotylum. The transcriptomic analysis performed on two P. myriotylum isolates infecting ginger leaves showed that proteases were a major part of the upregulated genes along with PCWDEs, Nep1-like proteins (NLPs), and elicitin-like proteins. A subset of P. myriotylum NLPs were analyzed and found to have necrosis-inducing ability from agroinfiltration of tobacco (Nicotiana benthamiana) leaves. One of the heterologously produced infection-upregulated putative cutinases found in a tandem array showed esterase activity with preferences for longer-chain-length substrates and neutral to alkaline pH levels. Our results allow the development of science-based targets for the management of P. myriotylum-caused disease, as insights from the genome and transcriptome show that gene expansion of virulence factor-related genes play a bigger role in the plant parasitism of Pythium spp. than previously thought. IMPORTANCE Pythium species are oomycetes, an evolutionarily distinct group of filamentous fungus-like stramenopiles. The Pythium genus includes several pathogens of important crop species, e.g., the spice ginger. Analysis of our genome from the plant pathogen Pythium myriotylum uncovered a far larger arsenal of virulence factor-related genes than found in other Pythium plant pathogens, and these genes contribute to the infection of the plant host. The increase in the number of virulence factor-related genes appears to have occurred through the mechanism of tandem gene duplication events. Genes from particular virulence factor-related categories that were increased in number and switched on during infection of ginger leaves had their activities tested. These genes have toxic activities toward plant cells or activities to hydrolyze polymeric components of the plant. The research suggests targets to better manage diseases caused by P. myriotylum and prompts renewed attention to the genomics of Pythium plant pathogens.

Canine gastrointestinal pythiosis: a case report in Brazilian Pantanal from a diagnostic approach / Pitiose gastrointestinal canina: relato de caso no Pantanal brasileiro a partir de uma abordagem diagnóstica

Pythiosis is caused by an aquatic fungus-like organism (Pythium insidiosum). It is considered an important public health issue as it can affect both animals and humans. This paper reports a case of gastrointestinal pythiosis in a dog. The patient was hospitalized for four days, during which the animal received supportive and symptomatic treatment. But the applied treatment was unsuccessful and the dog's clinical condition worsened, culminating in death. Complementary imaging tests such as radiography and ultrasonography, as well as hematological tests, were performed during the hospitalization period. The definitive diagnosis was reached in the postmortem as macroscopic and microscopic characteristics suggested the presence of intestinal granuloma and accentuated multifocal suppurative necrotic enteritis. Additionally, the histological evaluation revealed morphological structures compatible with P. insidiosum. Also, the results of nested PCR performed showed partial amplification (105 bp) of the ITS1 region of the ribosomal gene of P. insidiosum.(AU)

A pitiose é causada por um organismo aquático semelhante a um fungo (Pythium insidiosum) e considerada um importante problema de saúde pública, pois pode afetar animais e humanos. Este artigo relata um caso de pitiose gastrointestinal em um cão. O paciente ficou internado por quatro dias, período em que o animal recebeu tratamento de suporte e sintomático. No entanto, o tratamento aplicado não teve sucesso e o quadro clínico do cão piorou, culminando com a morte. Exames de imagem complementares, como radiografia e ultrassonografia, bem como exames hematológicos, foram realizados durante o período de internação. O diagnóstico definitivo foi feito na autópsia, pois as características macroscópicas e microscópicas sugeriam a presença de granuloma intestinal e acentuada enterite necrótica multifocal supurativa. Além disso, a avaliação histológica revelou estruturas morfológicas compatíveis com P. insidiosum. Além disso, a nested PCR foi realizada e mostrou amplificação parcial (105 pb) da região ITS1 do gene ribossomal de P. insidiosum.(AU)

Geographic distribution of Pythium insidiosum infections in the United States.

OBJECTIVE: To describe the geographic distribution of infections caused by Pythium insidiosum in dogs, horses, and other animal species in the US. ANIMALS: For the last 20 years, we have collected data from cases of pythiosis in 1,150 horses, 467 dogs, and other species (59) from various geographic locations in the US. PROCEDURES: Due to lost data (from 2006 to 2016), the selected cases include years 2000 to 2005 and 2016 to 2020. The selection of cases was based on infected host clinical features, serum samples demonstrating strong positive anti-P insidiosum IgG titers in serologic assays, and positive results on ≥ 1 of the following diagnostic modalities: microbial culture on 2% Sabouraud dextrose agar, histologic evaluation, PCR assay, and wet mount cytologic evaluation (with potassium hydroxide). RESULTS: Most confirmed P insidiosum infections were found in horses and dogs in the southeastern US. Interestingly, in Texas, no cases were found west of longitude 100°W. Few pythiosis cases were diagnosed in west-coast states. Equine cases were more often diagnosed during summer and fall months, but canine cases were more often diagnosed between September and February. Cases in other species were discovered in the same geographic areas as those in dogs and horses. CLINICAL RELEVANCE: To our knowledge, this is the first report providing the ecological distribution of P insidiosum infection in affected species in the US. Results of this study illustrated the importance of including P insidiosum in the differential diagnostic scheme of nonhealing skin lesions or intestinal granulomatous masses, particularly in dogs and horses inhabiting or having visited endemic areas.

Nep1-like proteins as a target for plant pathogen control.

The lack of efficient methods to control the major diseases of crops most important to agriculture leads to huge economic losses and seriously threatens global food security. Many of the most important microbial plant pathogens, including bacteria, fungi, and oomycetes, secrete necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs), which critically contribute to the virulence and spread of the disease. NLPs are cytotoxic to eudicot plants, as they disturb the plant plasma membrane by binding to specific plant membrane sphingolipid receptors. Their pivotal role in plant infection and broad taxonomic distribution makes NLPs a promising target for the development of novel phytopharmaceutical compounds. To identify compounds that bind to NLPs from the oomycetes Pythium aphanidermatum and Phytophthora parasitica, a library of 587 small molecules, most of which are commercially unavailable, was screened by surface plasmon resonance. Importantly, compounds that exhibited the highest affinity to NLPs were also found to inhibit NLP-mediated necrosis in tobacco leaves and Phytophthora infestans growth on potato leaves. Saturation transfer difference-nuclear magnetic resonance and molecular modelling of the most promising compound, anthranilic acid derivative, confirmed stable binding to the NLP protein, which resulted in decreased necrotic activity and reduced ion leakage from tobacco leaves. We, therefore, confirmed that NLPs are an appealing target for the development of novel phytopharmaceutical agents and strategies, which aim to directly interfere with the function of these major microbial virulence factors. The compounds identified in this study represent lead structures for further optimization and antimicrobial product development.

Prediction and Characterization of RXLR Effectors in Pythium Species.

RXLR effectors, a class of secreted proteins that are transferred into host cells to manipulate host immunity, have been reported to widely exist in oomycetes, including those from genera Phytophthora, Hyaloperonospora, Albugo, and Saprolegnia. However, in Pythium species, no RXLR effector has yet been characterized, and the origin and evolution of such virulent effectors are still unknown. Here, we developed a modified regular expression method for de novo identification of RXLRs and characterized 359 putative RXLR effectors in nine Pythium species. Phylogenetic analysis revealed that all oomycetous RXLRs formed a single superfamily, suggesting that they might have a common ancestor. RXLR effectors from Pythium and Phytophthora species exhibited similar sequence features, protein structures, and genome locations. In particular, there were significantly more RXLR proteins in the mosquito biological control agent P. guiyangense than in the other eight Pythium species, and P. guiyangense RXLRs might be the result of gene duplication and genome rearrangement events, as indicated by synteny analysis. Expression pattern analysis of RXLR-encoding genes in the plant pathogen P. ultimum detected transcripts of the majority of the predicted RXLR genes, with some RXLR effectors induced in infection stages and one RXLR showing necrosis-inducing activity. Furthermore, all predicted RXLR genes were cloned from two biocontrol agents, P. oligandrum and P. periplocum, and three of the RXLR genes were found to induce a defense response in Nicotiana benthamiana. Taken together, our findings represent the first evidence of RXLR effectors in Pythium species, providing valuable information on their evolutionary patterns and the mechanisms of their interactions with diverse hosts.

Convergent Evolution of C239S Mutation in Pythium spp. ß-Tubulin Coincides with Inherent Insensitivity to Ethaboxam and Implications for Other Peronosporalean Oomycetes.

Ethaboxam is a benzamide antioomycete chemical (oomicide) used in corn and soybean seed treatments. Benzamides are hypothesized to bind to ß-tubulin, thus disrupting microtubule assembly. Recently, there have been reports of corn- and soybean-associated oomycetes that are insensitive to ethaboxam despite never having been exposed. Here, we investigate the evolutionary history and molecular mechanism of ethaboxam insensitivity. We tested the sensitivity of 194 isolates representing 83 species across four oomycete genera in the Peronosporalean lineage that were never exposed to ethaboxam. In all, 84% of isolates were sensitive to ethaboxam (effective concentration to reduce optical density at 600 nm by 50% when compared with the nonamended control [EC50] < 5 µg ml-1), whereas 16% were insensitive (EC50 > 11 µg ml-1). Of the insensitive isolates, two different transversion mutations were present in the 239th codon in ß-tubulin within three monophyletic groups of Pythium spp. The transversion mutations lead to the same amino acid change from an ancestral cysteine to serine (C239S), which coincides with ethaboxam insensitivity. In a treated soybean seed virulence assay, disease severity was not reduced on ethaboxam-treated seed for an isolate of Pythium aphanidermatum containing a S239 but was reduced for an isolate of P. irregulare containing a C239. We queried publicly available ß-tubulin sequences from other oomycetes in the Peronosporalean lineage to search for C239S mutations from other species not represented in our collection. This search resulted in other taxa that were either homozygous or heterozygous for C239S, including all available species within the genus Peronospora. Evidence presented herein supports the hypothesis that the convergent evolution of C239S within Peronosporalean oomycetes occurred without selection from ethaboxam yet confers insensitivity. We propose several evolutionary hypotheses for the repeated evolution of the C239S mutation.

Clinical profile, risk factors and outcome of medical, surgical and adjunct interventions in patients with Pythiuminsidiosum keratitis.

PURPOSE: To report clinical profile and compare management options for Pythium keratitis. METHOD: Retrospective interventional study of 46 patients diagnosed as Pythium keratitis by PCR DNA sequencing from January 2014 to July 2017. Interventions were categorised into medical management (MM) (topical azithromycin and linezolid with oral azithromycin at presentation), surgery (S) (therapeutic penetrating keratoplasty, TPK), surgical adjunct (SA) (cryotherapy±alcohol with TPK) and medical adjunct (MA) (MM after TPK). RESULTS: Primary treatment included MM (1 eye), SA (3 eyes) and S (42 eyes). Recurrence occurred in 27/43 eyes (MM+S group). Second surgery (S) was required in 11 eyes (TPK-2), with additional procedures (SA) in 10 eyes and evisceration in five eyes. 8/43 eyes received MA after TPK-1. One eye required TPK-3. Recurrence occured in all eyes that received MA (100%) and in 28 of 54 TPKs (51.8%) (TPK 1+2+3) in 42 eyes. Recurrence was noted in 1/14 (7.1%) that underwent SA. CONCLUSION: The currently available and recommended treatment for Pythium keratitis is surgical by means of a TPK and in worse cases evisceration. In our study, MM/MA measures showed no benefit with recurrence or worsening of infection requiring resurgery. Almost 50% of TPKs had a recurrence requiring resurgery. However, adjunctive procedures during TPK appear to have additional benefit with low risk of recurrence and could be included as routine care.

Phylogenomic analysis supports multiple instances of polyphyly in the oomycete peronosporalean lineage.

The study of biological diversification of oomycetes has been a difficult task for more than a century. Pioneer researchers used morphological characters to describe this heterogeneous group, and physiological and genetic tools expanded knowledge of these microorganisms. However, research on oomycete diversification is limited by conflicting phylogenies. Using whole genomic data from 17 oomycete taxa, we obtained a dataset of 277 core orthologous genes shared among these genomes. Analyses of this dataset resulted in highly congruent and strongly supported estimates of oomycete phylogeny when we used concatenated maximum likelihood and coalescent-based methods; the one important exception was the position of Albugo. Our results supported the position of Phytopythium vexans (formerly in Pythium clade K) as a sister clade to the Phytophthora-Hyaloperonospora clade. The remaining clades comprising Pythium sensu lato formed two monophyletic groups. One group was composed of three taxa that correspond to Pythium clades A, B and C, and the other group contained taxa representing clades F, G and I, in agreement with previous Pythium phylogenies. However, the group containing Pythium clades F, G and I was placed as sister to the Phytophthora-Hyaloperonospora-Phytopythium clade, thus confirming the lack of monophyly of Pythium sensu lato. Multispecies coalescent methods revealed that the white blister rust, Albugo laibachii, could not be placed with a high degree of confidence. Our analyses show that genomic data can resolve the oomycete phylogeny and provide a phylogenetic framework to study the evolution of oomycete lifestyles.

Evolution of the Sterol Biosynthetic Pathway of Pythium insidiosum and Related Oomycetes Contributes to Antifungal Drug Resistance.

Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum Direct exposure to Py. insidiosum zoospores can initiate infections of the eye, limb, gastrointestinal tract, or skin/subcutaneous tissue. Treatments for pythiosis have mostly relied on surgery. Antifungal drugs are generally ineffective against Py. insidiosum However, one patient with an invasive Py. insidiosum infection recovered completely following treatment with terbinafine and itraconazole. Additionally, the drug target sterol biosynthetic enzymes have been identified in the oomycete Aphanomyces euteiches It remains an open question whether Py. insidiosum is susceptible to the antifungal drugs and harbors any of the known drug target enzymes. Here, we determined the in vitro susceptibilities of terbinafine and itraconazole against 30 isolates of Py. insidiosum We also analyzed endogenous sterols and searched for genes encoding the sterol biosynthetic enzymes in the genomes of Py. insidiosum and related oomycetes. The susceptibility assay showed that the growth of each of the Py. insidiosum isolates was inhibited by the antifungal agents, but only at difficult-to-achieve concentrations, which explains the clinical resistance of the drugs in the treatment of pythiosis patients. Genome searches of Py. insidiosum and related oomycetes demonstrated that these organisms contained an incomplete set of sterol biosynthetic enzymes. Gas chromatographic mass spectrometry did not detect any sterol end products in Py. insidiosum In conclusion, Py. insidiosum possesses an incomplete sterol biosynthetic pathway. Resistance to antifungal drugs targeting enzymes in the ergosterol biosynthetic pathway in Py. insidiosum was due to modifications or losses of some of the genes encoding the drug target enzymes.

Cutaneous pythiosis in a donkey (Equus asinus) in Brazil.

Our study describes the clinical, epidemiologic, pathologic, immunohistochemical, and molecular aspects PCR of a case of cutaneous pythiosis in a donkey (Equus asinus) from Brazil. During a dry period, the animal grazed for 4 months around a pond where the vegetation remained green. Skin lesions were nodular, multifocal, and disseminated, mainly involving the legs, ventral chest, and mammary gland. On cut surface, there were multifocal to coalescent discrete yellow foci, and occasional small cavitations with a few kunkers. Ulcerative nodular pyogranulomatous and eosinophilic dermatitis with folliculitis and furunculosis were observed histologically. Hyphae were observed in sections stained with Gomori methenamine silver. Immunohistochemistry with Pythium insidiosum antibodies yielded strong immunostaining of hyphae. P. insidiosum DNA was extracted from tissues in paraffin blocks by amplification of a fragment of 105 bp, which targets the 5.8S ribosomal gene. After the diagnosis of pythiosis, the larger skin lesions were excised and treated as second intention healing wounds, which were completely healed 30 days after resection. Small skin lesions regressed spontaneously in ~60 days. The granulomatous inflammation and outcome of the disease in this donkey were similar to cases of pythiosis in cattle.

Outbreak of Pythium keratitis during rainy season: a case series.

PURPOSE: To describe typical clinical and laboratory characteristics of severe fungal keratitis caused by Pythium insidiosum during the rainy season in Northeast Thailand and to report the efficacy of P. insidiosum vaccine in the treatment of Pythium keratitis. METHODS: A series of hospital-based consecutive cases of Pythium keratitis were diagnosed and treated at Srinagarind Hospital (Khon Kaen University, Khon Kaen, Thailand). The clinical presentations, diagnostic tests, and management are described. RESULTS: Severe fungal keratitis caused by P. insidiosum was diagnosed in 5 eyes of 4 patients between May 2009 and July 2009. All cases had a history of fungal keratitis after being exposed to contaminated water. Upon slit-lamp examination, subepithelial and superficial stromal opacities were observed in a reticular pattern in all cases. Pythium insidiosum was identified and confirmed by both microbiological culture and polymerase chain reaction. Clinical worsening was detected after conventional treatment with antifungal agents. Therapeutic penetrating keratoplasty with either donor cornea or scleral graft was performed together with topical antifungal administration and P. insidiosum vaccination. Subsequent evisceration was performed in 1 eye. CONCLUSIONS: An outbreak of Pythium keratitis in Northeast Thailand was reported. Distinctive clinical features are a suggestive clue for early diagnosis. Combination treatment including topical antifungal agents, radical surgery, and P. insidiosum vaccine may be considered for the management of Pythium keratitis.

Equine pythiosis: report in crossed bred (Criole Venezuelan) horses.

Pythium insidiosum is a pathogenic oomycete known since 1890 that causes pythiosis in mammals. In this report, seven P. insidiosum isolates were recovered from Venezuelan horses and were characterized. The strains were recovered from biopsied tissues and kunkers collected from granulomatous masses located on the hind limb and from a nodular lesion in the left upper eyelid, which decrease the ability of the horses to be used for working purposes. The methods used to identify P. insidiosum isolates were based on the production of sporangia and zoospores, histopathology and PCR assay. To further characterize these strains, portions of the 18S rRNA genes of the seven isolates were sequenced. The sequences showed high homology to previously described P. insidiosum DNA sequences available in GenBank. Similar studies based on the morphological, histological and molecular data identified the etiological agent in samples of granulomatous lesions in these equines as P. insidiosum. In America, the infection has been diagnosed more frequently in equines of Brazil, Colombia, Costa Rica and the United States of America.

Insights into the molecular mechanism of tolerance to carboxylic acid amide (CAA) fungicides in Pythium aphanidermatum.

BACKGROUND: Tolerance to the oomycete-specific carboxylic acid amide (CAA) fungicides is a poorly understood mechanism in Pythium species. The root-rot and damping-off causative agent Pythium aphanidermatum and the CAA fungicide mandipropamid (MPD) were used to investigate the molecular basis of CAA tolerance. RESULTS: Five genes putatively involved in carbohydrate synthesis were identified and characterised: one chitin synthase gene, PaChs, and four cellulose synthase genes PaCesA1 to PaCesA4, of which PaCesA3 encodes the MPD target enzyme. These genes were differentially expressed throughout the life cycle of P. aphanidermatum. Mycelium treated with MPD concentrations slightly affecting mycelial growth did not cause a change in PaCesA3 expression nor a strong upregulation of PaCesA homologues. The high tolerance level of P. aphanidermatum and the lack of PaCesA upregulation imply that MPD tolerance is the result of a specific amino acid configuration in the cellulose synthase 3 (CesA3) target enzyme. Indeed, P. aphanidermatum displays the amino acid L1109 which is also associated with MPD resistance in artificial mutants of Phytophthora species. CONCLUSION: It is concluded that MPD tolerance in P. aphanidermatum is not caused by compensatory mechanisms but most likely by an inherent target-site configuration in PaCesA3 that hinders MPD binding to the enzyme pocket.

Molecular analyses of Pythium irregulare isolates from grapevines in South Africa suggest a single variable species.

The Pythium irregulare species complex is the most common and widespread Pythium spp. associated with grapevines in South Africa. This species complex has been subdivided into several morphological and phylogenetic species that are all highly similar at the sequence level [internal transcribed spacer (ITS) and cytochrome c oxidase (cox) regions]. The complex includes Pythium regulare and Pythium cylindrosporum, which are morphologically distinct, and P. irregulare sensu stricto (s.s.) and Pythium cryptoirregulare, which are morphologically similar. The aim of the current study was to determine whether 50 South African grapevine P. irregulare isolates represented more than one phylogenetically distinct species. The isolates were characterised using nuclear (ITS and ß-tubulin) and mitochondrial (cox1 and cox2) gene region phylogenies and two isozyme loci [glucose-6-phosphate isomerase (Gpi) and malate dehydrogenase (Mdh-1)]. Some of the gene sequence data were difficult to interpret phylogenetically, since some isolates contained two or more polymorphic ITS copies within the same isolate (intra-isolate variation) that clustered into different ITS sub-clades, i.e. the P. irregulare s.s. and P. cryptoirregulare sub-clades. The molecular data furthermore only revealed the presence of one phylogenetic species, P. irregulare. Morphological analyses of a subset of the isolates confirmed that the isolates were P. irregulare, and further showed that the P. cylindrosporum ex-type strain formed typical P. irregulare oogonia, and not the previously reported distinct elongated oogonia. Some of the molecular analyses suggested the occurrence of outcrossing events and possibly the formation of aneuploids or polyploids since (i) the nuclear and mitochondrial gene data sets were incongruent, (ii) polymorphic ITS copies were present within the same isolate, (iii) heterozygosities were observed in the ß-tubulin gene and Gpi and Mdh-1 loci in some isolates and (iv) more than two ß-tubulin alleles were detected in some isolates. Altogether, the data suggest that P. irregulare, P. cryptoirregulare, P. cylindrosporum, and possibly P. regulare should be synonimised under the name P. irregulare.

Implications of oligomeric forms of POD-1 and POD-2 proteins isolated from cell walls of the biocontrol agent Pythium oligandrum in relation to their ability to induce defense reactions in tomato.

The cell wall protein fraction (CWP) isolated from the biocontrol agent Pythium oligandrum induces defense reactions in tomato. CWP contains two novel elicitin-like proteins, POD-1 and POD-2, both with seven cysteines. To determine the essential structure in the defense-eliciting components of CWP, five fractions (F1, F2, F3, F4 and F5) were fractionated from CWP using cation chromatography and their components and disulfide bond compositions were analyzed. The expression levels of three defense-related genes (PR-6, LeCAS and PR-2b) were determined in tomato roots treated with each of the five fractions. Of the five fractions, F4 containing a heterohexamer of POD-1 and POD-2, and F5 containing a homohexamer of POD-1, both with disulfide bonds formed between all cysteine residues, induced the expression of three genes. F4 treatment also induced the accumulation of ethylene in tomato. The predicted three-dimensional structures of POD-1 and POD-2, and the results of SEC and MALDI-TOF MS analyses suggest that F4 consists of three POD-1 and POD-2 disulfide-bonded heterodimers that interleave into a hexameric ring through noncovalent association. These results suggest that this structure, which F5 also appears to form, is essential for stimulating defense responses in tomato.

Pythium insidiosum keratitis in Israel.

PURPOSE: To report with morphologic and phylogenetic speciation the first case from Israel of Pythium insidiosum keratitis associated with contact-lens wear. METHODS: Case report and literature review. RESULTS: A 21-year-old man with a history of contact-lens use and water exposure was hospitalized in Israel for a corneal ulcer. The ulcer progressed despite intensive antibiotics. He flew home to the United States for further care. Examination revealed a corneal ulcer with hypopyon. The infection progressed despite intensive medical therapy, and a therapeutic penetrating keratoplasty was performed. Histology and cornea cultures from the host cornea revealed sparsely septate, branching hyphae, consistent with P. insidiosum. DNA sequencing of the Pythium isolate supported the clinical history that the infection was acquired outside of the United States. Despite intensive medical therapy and a second corneal transplant, the ulcer progressed, ultimately requiring enucleation. CONCLUSION: This is the first reported case of culture-proven, contact lens-related Pythium keratitis originating from Israel. Pythium is a fungus-like, aquatic oomycete found in tropical climates. Human pythiosis is uncommon but associated with high morbidity. Case reports describe surgical cure of Pythium keratitis, but this case recurred despite two penetrating keratoplasties and maximal antifungal therapy. In cases of presumed fungal keratitis that do not respond to antifungals, the fungus should be sent for speciation because early surgical intervention is the only means to save the eye in ocular pythiosis.

Identification of Pythium insidiosum by nested PCR in cutaneous lesions of Brazilian horses and rabbits.

Pythium insidiosum is a fungus-like organism present in subtropical and tropical areas, such as Brazil, known to infect humans and various animal species. P. insidiosum is the etiological agent of pythiosis, an emerging and granulomatous disease characterized mainly by cutaneous and subcutaneous lesions in horses, the principal species affected. Accurate diagnosis of pythiosis and identification of its causal agent by microbiological and serological tests can be often difficult and inconclusive principally for horses and humans. The aim of this study was to evaluate the application of the previously described P. insidiosum-specific nested polymerase chain reaction (PCR) assay to directly detect P. insidiosum DNA in clinical and experimental lesions. Universal fungal primers (ITS1 and ITS4) were used during the first-round of PCR to amplify ITS1, 5.8s, and ITS2. A second-round of PCR was conducted with P. insidiosum-specific primers (PI1 and PI2) to amplify a variable region within this ITS1. In this study, a total of 21 equine clinical samples (kunkers) and 28 specimens from experimentally infected rabbits were analyzed by nested PCR. The first-round of PCR generated 800-base pair products, and the second-round produced 105-base pair amplicons for each P. insidiosum-specific sample; no amplicons were generated in negative control samples. Our results suggest that nested PCR is an important and efficient tool for diagnosis of both endemic (horse samples) and experimental (rabbit samples) pythiosis.