Potential anti-Pythium insidiosum therapeutics identified through screening of agricultural fungicides.

Pythiosis is a life-threatening infectious disease caused by the oomycete Pythium insidiosum. Clinical manifestations of pythiosis include an eye, blood vessel, skin, or gastrointestinal tract infection. Pythiosis has been increasingly reported worldwide, with an overall mortality rate of 28%. Radical surgery is required to save patients' lives due to the limited efficacy of antimicrobial drugs. Effective medical treatments are urgently needed for pythiosis. This study aims to find anti-P. insidiosum agents by screening 17 agricultural fungicides that inhibit plant-pathogenic oomycetes and validating their efficacy and safety. Cyazofamid outperformed other fungicides as it can potently inhibit genetically diverse P. insidiosum isolates while exhibiting minimal cellular toxicities. The calculated therapeutic scores determined that the concentration of cyazofamid causing significant cellular toxicities was eight times greater than the concentration of the drug effectively inhibiting P. insidiosum. Furthermore, other studies showed that cyazofamid exhibits low-to-moderate toxicities in animals. The mechanism of cyazofamid action is likely the inhibition of cytochrome b, an essential component in ATP synthesis. Molecular docking and dynamic analyses depicted a stable binding of cyazofamid to the Qi site of the P. insidiosum's cytochrome b orthologous protein. In conclusion, our search for an effective anti-P. insidiosum drug indicated that cyazofamid is a promising candidate for treating pythiosis. With its high efficacy and low toxicity, cyazofamid is a potential chemical for treating pythiosis, reducing the need for radical surgeries, and improving recovery rates. Our findings could pave the way for the development of new and effective treatments for pythiosis.IMPORTANCEPythiosis is a severe infection caused by Pythium insidiosum. The disease is prevalent in tropical/subtropical regions. This infectious condition is challenging to treat with antifungal drugs and often requires surgical removal of the infected tissue. Pythiosis can be fatal if not treated promptly. There is a need for a new treatment that effectively inhibits P. insidiosum. This study screened 17 agricultural fungicides that target plant-pathogenic oomycetes and found that cyazofamid was the most potent in inhibiting P. insidiosum. Cyazofamid showed low toxicity to mammalian cells and high affinity to the P. insidiosum's cytochrome b, which is involved in energy production. Cyazofamid could be a promising candidate for the treatment of pythiosis, as it could reduce the need for surgery and improve the survival rate of patients. This study provides valuable insights into the biology and drug susceptibility of P. insidiosum and opens new avenues for developing effective therapies for pythiosis.

Altering in vivo membrane sterol composition affects the activity of the cyclic lipopeptides tolaasin and sessilin against Pythium.

Cyclic lipopeptides (CLiPs) are secondary metabolites produced by a variety of bacteria. These compounds show a broad range of antimicrobial activities; therefore, they are studied for their potential applications in agriculture and medicine. It is generally assumed that the primary target of the CLiPs is the cellular membrane, where they can permeabilize the lipid bilayer. Model membrane systems are commonly used to investigate the effect of lipid composition on the permeabilizing activity of CLiPs, but these systems do not represent the full complexity of true biological membranes. Here, we introduce a novel method that uses sterol-auxotrophic oomycetes to investigate how the activity of membrane-active compounds is influenced by alterations in membrane sterol composition. More specifically, we investigated how ergosterol, cholesterol, beta-sitosterol and stigmasterol affect the activity of the structurally related Pseudomonas-derived CLiPs tolaasin and sessilin against the oomycete Pythium myriotylum. Both compounds were effective against oomycetes, although tolaasin was considerably more active. Interestingly, tolaasin and sessilin effects were similarly reduced by the presence of sterols, with cholesterol showing the highest reduction of activity.

Insights into the Host Specificity of a New Oomycete Root Pathogen, Pythium brassicum P1: Whole Genome Sequencing and Comparative Analysis Reveals Contracted Regulation of Metabolism, Protein Families, and Distinct Pathogenicity Repertoire.

Pythium brassicum P1 Stanghellini, Mohammadi, Förster, and Adaskaveg is an oomycete root pathogen that has recently been characterized. It only attacks plant species belonging to Brassicaceae family, causing root necrosis, stunting, and yield loss. Since P. brassicum P1 is limited in its host range, this prompted us to sequence its whole genome and compare it to those of broad host range Pythium spp. such as P. aphanidermatum and P. ultimum var. ultimum. A genomic DNA library was constructed with a total of 374 million reads. The sequencing data were assembled using SOAPdenovo2, yielding a total genome size of 50.3 Mb contained in 5434 scaffolds, N50 of 30.2 Kb, 61.2% G+C content, and 13,232 putative protein-coding genes. Pythium brassicum P1 had 175 species-specific gene families, which is slightly below the normal average. Like P. ultimum, P. brassicum P1 genome did not encode any classical RxLR effectors or cutinases, suggesting a significant difference in virulence mechanisms compared to other oomycetes. Pythium brassicum P1 had a much smaller proportions of the YxSL sequence motif in both secreted and non-secreted proteins, relative to other Pythium species. Similarly, P. brassicum P1 had the fewest Crinkler (CRN) effectors of all the Pythium species. There were 633 proteins predicted to be secreted in the P. brassicum P1 genome, which is, again, slightly below average among Pythium genomes. Pythium brassicum P1 had only one cadherin gene with calcium ion-binding LDRE and DxND motifs, compared to Pythium ultimum having four copies. Pythium brassicum P1 had a reduced number of proteins falling under carbohydrate binding module and hydrolytic enzymes. Pythium brassicum P1 had a reduced complement of cellulase and pectinase genes in contrast to P. ultimum and was deficient in xylan degrading enzymes. The contraction in ABC transporter families in P. brassicum P1 is suggested to be the result of a lack of diversity in nutrient uptake and therefore host range.

Long-Read Genome Sequence of the Sugar Beet Rhizosphere Mycoparasite Pythium oligandrum.

Pythium oligandrum is a soil born free living oomycete able to parasitize fungi and oomycetes prey, including important plant and animals pathogens. Pythium oligandrum can colonize endophytically the root tissues of diverse plants where it induces plant defenses. Here we report the first long-read genome sequencing of a P. oligandrum strain sequenced by PacBio technology. Sequencing of genomic DNA loaded onto six SMRT cells permitted the acquisition of 913,728 total reads resulting in 112X genome coverage. The assembly and polishing of the genome sequence yielded180 contigs (N50 = 1.3 Mb; L50 = 12). The size of the genome assembly is 41.9 Mb with a longest contig of 2.7 Mb and 15,007 predicted protein-coding genes among which 95.25% were supported by RNAseq data, thus constituting a new Pythium genome reference. This data will facilitate genomic comparisons of Pythium species that are commensal, beneficial or pathogenic on plant, or parasitic on fungi and oomycete to identify key genetic determinants underpinning their diverse lifestyles. In addition comparison with plant pathogenic or zoopathogenic species will illuminate genomic adaptations for pathogenesis toward widely diverse hosts.

Structure and ecological function of the soil microbiome affecting plant-soil feedbacks in the presence of a soil-borne pathogen.

Interactions between plants and soil microbes are important for plant growth and resistance. Through plant-soil-feedbacks, growth of a plant is influenced by the previous plant that was growing in the same soil. We performed a plant-soil feedback study with 37 grass, forb and legume species, to condition the soil and then tested the effects of plant-induced changes in soil microbiomes on the growth of the commercially important cut-flower Chrysanthemum in presence and absence of a pathogen. We analysed the fungal and bacterial communities in these soils using next-generation sequencing and examined their relationship with plant growth in inoculated soils with or without the root pathogen, Pythium ultimum. We show that a large part of the soil microbiome is plant species-specific while a smaller part is conserved at the plant family level. We further identified clusters of plant species creating plant growth promoting microbiomes that suppress concomitantly plant pathogens. Especially soil inocula with higher relative abundances of arbuscular mycorrhizal fungi caused positive effects on the Chrysanthemum growth when exposed to the pathogen. We conclude that plants differ greatly in how they influence the soil microbiome and that plant growth and protection against pathogens is associated with a complex soil microbial community.

The application of safe for humans and the environment Polyversum antifungal agent containing living cells of Pythium oligandrum for biotransformation of prochiral ketones.

This report presents the whole-cell biotransformation of benzofuranyl-methyl ketone derivatives with the application of Polyversum antifungal agent containing Pythium oligandrum microorganism. Stereochemistry of the reduction of prochiral substrates was modified by the bioconversion conditions (concentration of reagents, a source of the carbon atom, biotransformation medium). In optimized conditions enantioselective process was noted. Secondary alcohols with excellent enantiomeric purity and high yields were obtained. The enantiomeric excess and conversion degree of 1-(benzofuran-2-yl)ethanol, 1-(7-ethylbenzofuran-2-yl)ethanol and 1-(3,7-dimethylbenzofuran-2-yl)ethanol were 99%/98.1%, 94%/94.4% and 99%/72.6%, respectively. In the presence of P. oligandrum, one of the enantiotopic hydrides of the dihydropyridine ring coenzyme is selectively transferred to a re side of the prochiral carbonyl group to give products with S configuration. This study demonstrates an inexpensive, eco-friendly approach in synthesis of optically pure benzofuran derivatives and can be an interesting alternative to organocatalysis. Furthermore, this method can be used in biotechnology processes due to its good chemical performance and a high degree of product isolation.

Genome-wide sequencing and metabolic annotation of Pythium irregulare CBS 494.86: understanding Eicosapentaenoic acid production.

BACKGROUND: Pythium irregulare is an oleaginous Oomycete able to accumulate large amounts of lipids, including Eicosapentaenoic acid (EPA). EPA is an important and expensive dietary supplement with a promising and very competitive market, which is dependent on fish-oil extraction. This has prompted several research groups to study biotechnological routes to obtain specific fatty acids rather than a mixture of various lipids. Moreover, microorganisms can use low cost carbon sources for lipid production, thus reducing production costs. Previous studies have highlighted the production of EPA by P. irregulare, exploiting diverse low cost carbon sources that are produced in large amounts, such as vinasse, glycerol, and food wastewater. However, there is still a lack of knowledge about its biosynthetic pathways, because no functional annotation of any Pythium sp. exists yet. The goal of this work was to identify key genes and pathways related to EPA biosynthesis, in P. irregulare CBS 494.86, by sequencing and performing an unprecedented annotation of its genome, considering the possibility of using wastewater as a carbon source. RESULTS: Genome sequencing provided 17,727 candidate genes, with 3809 of them associated with enzyme code and 945 with membrane transporter proteins. The functional annotation was compared with curated information of oleaginous organisms, understanding amino acids and fatty acids production, and consumption of carbon and nitrogen sources, present in the wastewater. The main features include the presence of genes related to the consumption of several sugars and candidate genes of unsaturated fatty acids production. CONCLUSIONS: The whole metabolic genome presented, which is an unprecedented reconstruction of P. irregulare CBS 494.86, shows its potential to produce value-added products, in special EPA, for food and pharmaceutical industries, moreover it infers metabolic capabilities of the microorganism by incorporating information obtained from literature and genomic data, supplying information of great importance to future work.

The cytochrome bc1 complex inhibitor Ametoctradin has an unusual binding mode.

Ametoctradin is an agricultural fungicide that selectively inhibits the cytochrome bc1 complex of oomycetes. Previous spectrophotometric studies using the purified cytochrome bc1 complex from Pythium sp. showed that Ametoctradin binds to the Qo-site of the enzyme. However, as modeling studies suggested a binding mode like that of the substrate ubiquinol, the possibility for a dual Qo- and Qi-site binding mode was left open. In this work, binding studies and enzyme assays with mitochondrial membrane preparations from Pythium sp. and an S. cerevisiae strain with a modified Qi-site were used to investigate further the binding mode of Ametoctradin. The results obtained argue that the compound could bind to both the Qo- and Qi-sites of the cytochrome bc1 complex and that its position or binding pose in the Qi-site differs from that of Cyazofamid and Amisulbrom, the two Qi-site-targeting, anti-oomycetes compounds. Furthermore, the data support the argument that Ametoctradin prefers binding to the reduced cytochrome bc1 complex. Thus, Ametoctradin has an unusual binding mode and further studies with this compound may offer the opportunity to better understand the catalytic cycle of the cytochrome bc1 complex.

Probing the Phylogenomics and Putative Pathogenicity Genes of Pythium insidiosum by Oomycete Genome Analyses.

Pythium insidiosum is a human-pathogenic oomycete. Many patients infected with it lose organs or die. Toward the goal of developing improved treatment options, we want to understand how Py. insidiosum has evolved to become a successful human pathogen. Our approach here involved the use of comparative genomic and other analyses to identify genes with possible functions in the pathogenicity of Py. insidiosum. We generated an Oomycete Gene Table and used it to explore the genome contents and phylogenomic relationships of Py. insidiosum and 19 other oomycetes. Initial sequence analyses showed that Py. insidiosum is closely related to Pythium species that are not pathogenic to humans. Our analyses also indicated that the organism harbours secreted and adhesin-like proteins, which are absent from related species. Putative virulence proteins were identified by comparison to a set of known virulence genes. Among them is the urease Ure1, which is absent from humans and thus a potential diagnostic and therapeutic target. We used mass spectrometric data to successfully validate the expression of 30% of 14,962 predicted proteins and identify 15 body temperature (37 °C)-dependent proteins of Py. insidiosum. This work begins to unravel the determinants of pathogenicity of Py. insidiosum.

Protein profile of Brazilian Pythium insidiosum isolates.

Pythium insidiosum is an important aquatic Oomycota that causes pythiosis in mammals, especially horses, dogs, and humans; these inhabit marshy environments in tropical and subtropical areas. The aim of this study was to determine the protein profile, as well as identify likely immunodominant proteins, of Brazilian P. insidiosum isolates from southern Brazil, an important equine pythiosis endemic area. P. insidiosum isolates (horses, n = 20 and dogs, n = 02) were analyzed by SDS-PAGE and Western blot techniques. Horse, cattle, dog, and rabbit sera of both diseased and healthy animals were used to identify P. insidiosum proteins. SDS-PAGE protein profile detected antigens of molecular weights ranging from 100 to 20 KDa. Dog isolates revealed a protein profile similar to that of horse isolates. Anti-P. insidiosum antibodies in the sera of the four species could recognize proteins of different molecular weights (∼74 KDa to ∼24 KDa), and proteins ∼50-55 KDa and ∼34 KDa were shown to be immunodominant. Furthermore, ∼74 KDa, ∼60 KDa, ∼30 KDa and ∼24 KDa proteins were poorly recognized by host species antibodies. The Brazilian P. insidiosum isolates analyzed showed a similar protein profile; however, further studies are essential for the identification and characterization of proteins expressed by P. insidiosum, and an evaluation of the immunological profile of hosts susceptible to this Oomycota is necessary.

Eudicot plant-specific sphingolipids determine host selectivity of microbial NLP cytolysins.

Necrosis and ethylene-inducing peptide 1-like (NLP) proteins constitute a superfamily of proteins produced by plant pathogenic bacteria, fungi, and oomycetes. Many NLPs are cytotoxins that facilitate microbial infection of eudicot, but not of monocot plants. Here, we report glycosylinositol phosphorylceramide (GIPC) sphingolipids as NLP toxin receptors. Plant mutants with altered GIPC composition were more resistant to NLP toxins. Binding studies and x-ray crystallography showed that NLPs form complexes with terminal monomeric hexose moieties of GIPCs that result in conformational changes within the toxin. Insensitivity to NLP cytolysins of monocot plants may be explained by the length of the GIPC head group and the architecture of the NLP sugar-binding site. We unveil early steps in NLP cytolysin action that determine plant clade-specific toxin selectivity.

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.

Improved eicosapentaenoic acid production in Pythium splendens RBB-5 based on metabolic regulation analysis.

Eicosapentaenoic acid (EPA) is an essential polyunsaturated fatty acid for human beings. At present, the production of commercially available long-chain polyunsaturated fatty acids, mainly from wild-caught ocean fish, is struggling to meet the increasing demand for EPA. Production of EPA by microorganisms may be an alternative, effective and economical method. The oleaginous fungus Pythium splendens RBB-5 is a potential source of EPA, and thanks to the simple culture conditions required, high yields can be achieved in a facile manner. In the study, lipid metabolomics was performed in an attempt to enhance EPA biosynthesis in Pythium splendens. Synthetic, metabolic regulation and gene expression analyses were conducted to clarify the mechanism of EPA biosynthesis, and guide optimization of EPA production. The results showed that the Δ6 desaturase pathway is the main EPA biosynthetic route in this organism, and ∆6, ∆12 and Δ17 desaturases are the rate-limiting enzymes. All the three desaturase genes were separately introduced into the parent strain to increase the flow of fatty acids into the Δ6 desaturase pathway. Enhanced expression of these key enzymes, in combination with improved regulation of metabolism, resulted in a maximum yield of 1.43 g/L in the D12 transgenic strain, which represents a tenfold increase over the parent strain before optimization. This is the higher EPA production yield yet reported for a microbial system. Our findings may allow the production of EPA at an industrial scale, and the strategy employed could be used to increase the production of EPA or other lipids in oleaginous microorganisms.

Ag nanoparticles generated using bio-reduction and -coating cause microbial killing without cell lysis.

Cost-effective "green" methods of producing Ag nanoparticles (NPs) are being examined because of the potential of these NPs as antimicrobials. Ag NPs were generated from Ag ions using extracellular metabolites from a soil-borne Pythium species. The NPs were variable in size, but had one dimension less than 50 nm and were biocoated; aggregation and coating changed with acetone precipitation. They had dose-dependent lethal effects on a soil pseudomonad, Pseudomonas chlororaphis O6, and were about 30-fold more effective than Ag(+) ions. A role of reactive oxygen species in cell death was demonstrated by use of fluorescent dyes responsive to superoxide anion and peroxide accumulation. Also mutants of the pseudomonad, defective in enzymes that protect against oxidative stress, were more sensitive than the wild type strain; mutant sensitivity differed between exposure to Ag NPs and Ag(+) ions demonstrating a nano-effect. Imaging of bacterial cells treated with the biocoated Ag NPs revealed no cell lysis, but there were changes in surface properties and cell height. These findings support that biocoating the NPs results in limited Ag release and yet they retained potent antimicrobial activity.

Metabolic engineering of long chain-polyunsaturated fatty acid biosynthetic pathway in oleaginous fungus for dihomo-gamma linolenic acid production.

Microbial lipids are promising alternative sources of long chain-polyunsaturated fatty acids (LC-PUFAs) for food, feed, nutraceutical and pharmaceutical sectors. Dihomo-γ-linolenic acid (C20:3Δ(8,11,14); DGLA) is an important LC-PUFAs with anti-inflammatory and anti-proliferative effects. To generate a DGLA-producing strain, fatty acid reconstitution in Aspergillus oryzae was performed by metabolic engineering through co-expression of codon-optimized Pythium Δ(6)-desaturase and Δ(6)-elongase, which had high conversion rates of substrates to respective products as compared to the native enzymes. The Δ(6)-desaturated and Δ(6)-elongated products, γ-linolenic acid (C18:3Δ(6,9,12); GLA) and DGLA, were accumulated in phospholipids rather than triacylglycerol. Interestingly, the manipulation of lipid quality in the oleaginous fungus did not affect growth and lipid phenotypes. This strategy might expand to development of the oleaginous fungal strain for producing other tailor-made oils with industrial applications.

Geographic variation in the elicitin-like glycoprotein, ELI025, of Pythium insidiosum isolated from human and animal subjects.

Oomycetes are fungus-like in appearance, but form a distinct clade within the eukaryotes. While most pathogenic oomycetes infect plants, the understudied oomycete Pythium insidiosum infects humans and animals, and causes a life-threatening infectious disease, called pythiosis. Phylogenetic analyses divide P. insidiosum into 3 groups, according to geographic origins: Clade-I (Americas), Clade-II (Asia and Australia), and Clade-III (Thailand). Surgical removal of the infected organ is the inevitable treatment for patients with pythiosis, but it is often too late or unsuccessful, and many patients die from advanced infection. Understanding P. insidiosum's basic biology could lead to improved infection control. Elicitins, a unique group of proteins found only in oomycetes, are involved in sterol acquisition and stimulation of host responses. Recently, we identified glycosylated and non-glycosylated forms of the elicitin-like protein, ELI025, which is secreted by P. insidiosum, and detected during P. insidiosum infection. In this study, we investigated geographic variation of ELI025 in 24 P. insidiosum strains isolated from humans, animals, and the environment. Genotypes of ELI025, based on 2 sets of PCR primers, correlated well with rDNA-based phylogenetic grouping. Unlike strains in Clade-I and -II, Clade-III strains secreted no glycosylated ELI025. Sera from 17 pythiosis patients yielded a broad range of antibody responses against ELI025, and ∼30% lacked reactivity against the protein. Selective production or secretion of glycosylated ELI025 by different P. insidiosum strains might contribute to the variable host antibody responses. In conclusion, ELI025 was secreted by all P. insidiosum strains isolated from different hosts and geographic origins, but the protein had different biochemical, and immunological characteristics. These finding contribute to the better understanding of the biology and evolution of P. insidiosum, and could lead to appropriate clinical application of the ELI025 protein for diagnosis or treatment of pythiosis.

The Immunoreactive Exo-1,3-ß-Glucanase from the Pathogenic Oomycete Pythium insidiosum Is Temperature Regulated and Exhibits Glycoside Hydrolase Activity.

The oomycete organism, Pythium insidiosum, is the etiologic agent of the life-threatening infectious disease called "pythiosis". Diagnosis and treatment of pythiosis is difficult and challenging. Novel methods for early diagnosis and effective treatment are urgently needed. Recently, we reported a 74-kDa immunodominant protein of P. insidiosum, which could be a diagnostic target, vaccine candidate, and virulence factor. The protein was identified as a putative exo-1,3-ß-glucanase (Exo1). This study reports on genetic, immunological, and biochemical characteristics of Exo1. The full-length exo1 coding sequence (2,229 bases) was cloned. Phylogenetic analysis showed that exo1 is grouped with glucanase-encoding genes of other oomycetes, and is far different from glucanase-encoding genes of fungi. exo1 was up-regulated upon exposure to body temperature, and its gene product is predicted to contain BglC and X8 domains, which are involved in carbohydrate transport, binding, and metabolism. Based on its sequence, Exo1 belongs to the Glycoside Hydrolase family 5 (GH5). Exo1, expressed in E. coli, exhibited ß-glucanase and cellulase activities. Exo1 is a major intracellular immunoreactive protein that can trigger host immune responses during infection. Since GH5 enzyme-encoding genes are not present in human genomes, Exo1 could be a useful target for drug and vaccine development against this pathogen.

A biochemical screening approach to putatively differentiate mammalian pathogenic Oomycota species in the clinical laboratory.

The report of four novel mammalian pathogenic species of the genus Lagenidium prompted us to study the use of biochemical assays to differentiate the Oomycota mammalian pathogens Pythium insidiosum and Lagenidium spp. We investigated the reaction of 23 Lagenidium and eight Pythium species in various biochemical assays. Because the morphological features of the Oomycota species are similar to those of species in the Entomophthoramycota and Mucormycota, five fungal species with coenocytic hyphae were also included. We found that mammalian and plant isolates of Pythium spp. all hydrolysed sucrose, but Lagenidium species and the fungal strains did not. In addition, both Pythium spp. and Lagenidium spp. were found to be maltose-positive, whereas fungal strains did not hydrolyse this sugar. The fungal species and thermo-sensitive Lagenidium giganteum and Lagenidium humanum were urease-negative, but the mammalian Lagenidium spp. and Pythium spp. hydrolysed urea within 24  h. These findings suggest these assays can be used for the presumptive differentiation of mammalian Oomycota species in the laboratory.

Detection of the oomycete Pythium insidiosum by real-time PCR targeting the gene coding for exo-1,3-ß-glucanase.

Pythiosis is a life-threatening infectious disease caused by Pythium insidiosum. Early and accurate diagnosis is the key to prompt treatment and an improved prognosis for patients with pythiosis. An alternative to microbiological and immunological approaches for facilitating diagnosis of pythiosis is the PCR-based assay. Until recently, the ribosomal DNA (rDNA) region was the only target available for PCR-based detection of P. insidiosum. Failure to detect P. insidiosum by PCR amplification using the rDNA-specific primers has been reported. PinsEXO1, encoding an exo-1,3-ß-glucanase, is an alternative, novel and efficient target for identification of P. insidiosum by conventional PCR. In this study, we aimed to develop a real-time (RT)-PCR approach targeting PinsEXO1 and compare its performance with conventional PCR for the detection of P. insidiosum. Both conventional and RT-PCR assays were positive for all 35 P. insidiosum strains tested, whilst all 58 control fungi were negative. The turnaround time for conventional PCR was 10 h, whilst that for RT-PCR was 7.5 h. The lowest amounts of genomic DNA template required for successful amplification by conventional and RT-PCR were 1 and 1 × 10(-4) ng, respectively. In conclusion, the RT-PCR assay retained 100% sensitivity and 100% specificity for detection of P. insidiosum. It showed a substantially improved analytical sensitivity and turnaround time that could improve diagnosis of pythiosis. The assay could also facilitate quantitative DNA analysis and epidemiological studies of P. insidiosum.

Pesticidal activity of metal oxide nanoparticles on plant pathogenic isolates of Pythium.

CuO and ZnO nanoparticles (NPs) have antimicrobial effects that could lead to formulations as pesticides for agriculture or medicine. The responses of two soil-borne plant pathogenic Pythium isolates to the NPs were studied to determine the potential of these metal oxide NPs as pesticides. Growth of the P. ultimum isolate was more sensitive to CuO NPs than the P. aphanidermatum isolate. Growth in liquid medium with CuO NPs eliminated culturability whereas exposure to ZnO NPs resulted in stasis with growth resuming on transfer to medium lacking NPs. The citrate in the medium used for the growth assays was involved in enhanced release of the toxic metals from the NPs. Both CuO and ZnO NPs affected processes involved in Fe uptake. The NPs reduced levels of Fe-chelating siderophore-like metabolites produced by Pythium hyphae. CuO NPs inhibited, but ZnO NPs increased, ferric reductase activity detected at the mycelial surface. These findings illustrate that the toxicity of the metal oxide NPs towards Pythium was influenced by the medium, especially by the presence of a metal chelator. Environmental factors are likely to alter the pesticide potential of the metal oxide NPs when formulated for agricultural use in soils.