Pythium and Globisporangium species associated with cucumber rhizosphere causing damping-off and their effects on cucumber seed decay in Oman.

Pythium sensu lato (s.l.) is a pathogenic oomycete. The present study was conducted to isolate and identify Pythium s.l. species associated with the rhizosphere and roots of greenhouse-growing cucumbers showing damping-off symptoms in 10 Omani governorates (provinces). A total of 166 isolates were recovered from 276 rhizosphere soil and root samples and were identified based on the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit I (COX I) gene region. Pythium aphanidermatum, P. myriotylum, Globisporangium spinosum, Globisporangium sp.1 (isolates Kb003/PySyCu-1 and Kb004/PySyCu-2), and Globisporangium sp.2 (isolate Ib002R) were identified. Among these species, P. aphanidermatum was the most abundant species, represented by 143 isolates (86.1%), followed by G. spinosum with 18 isolates (10.8%), Globisporangium sp.1 and P. myriotylum each with 2 isolates (2.4%), and Globisporangium sp.2 with 1 isolate (0.6%). Pathogenicity tests were also conducted for 38 isolates, including P. aphanidermatum (25), P. myriotylum (2), Globisporangium sp.2 (1), G. spinosum (8), and Globisporangium sp.1 (2). Among the tested isolates, only Globisporangium sp.2 isolate was avirulent, and none of the seeds were rotted at the end of the treatment. However, the other species induced the symptoms of seed decay with the incidence ranged from 86.7 to 100%. Phylogenetic analyses were conducted based on 222 ITS and 53 COX I sequences, and confirmed morphological identification. In addition, the genetic diversity of 93 P. aphanidermatum isolates was assessed via the amplified fragment length polymorphism (AFLP) method. The analysis produced 93 genotypes and 449 polymorphic loci. Pythium aphanidermatum populations were found to have moderate levels of genetic diversity (H = 0.2) and a moderate Shannon information index (I = 0.3793). Analysis of molecular variance (FST = 0.1, P = 0.0) revealed a moderate level of genetic differentiation among P. aphanidermatum isolates between Oman governorates. The sensitivity of 15 P. aphanidermatum isolates was evaluated against hymexazol at different concentrations (10, 100, and 1000 ppm). The results revealed that P. aphanidermatum could grow well at concentrations of up to 100 ppm hymexazol. However, hymexazol at 1000 ppm retarded the growth of P. aphanidermatum. This study showed that P. aphanidermatum is the most prevalent species in greenhouses in Oman and exhibited a moderate level of genetic diversity. Most of the isolates exhibited differences in tolerance to hymexazol but showed no resistance.

Wheat rhizosphere dynamics of Trichoderma gamsii A5MH and suppression of a Pythium root rot-Fusarium crown rot disease complex over two consecutive cropping seasons.

AIMS: Determine the wheat rhizosphere competence of Trichoderma gamsii strain A5MH and in planta suppression of the Pythium root and Fusarium crown rot pathogens Globisporangium irregulare and Fusarium pseudograminearum. METHODS AND RESULTS: Wheat was continuously cropped (eight years) at a minimum tillage, low growing season rainfall (GSR ≤ 170 mm) site shown as highly conducive to Pythium root and Fusarium crown rots. Root isolation frequency (RIF) and qPCR were used to determine the rhizosphere dynamics of strain A5MH and the target pathogens at tillering, grain harvest, and in postharvest stubble over the final 2 years. Strain A5MH actively colonized the wheat rhizosphere throughout both growing seasons, had high root abundance at harvest [log 4.5 genome copies (GC) g-1] and persisted in standing stubble for at least 293-d postinoculation. Globisporangium irregulare was most abundant in roots at tillering, whereas F. pseudograminearum was only abundant at harvest and up to 9-fold greater in the drier, second year (GSR 105 mm). Strain A5MH decreased RIF of both pathogens by up to 40%, root abundance of G. irregulare by 100-fold, and F. pseudogaminearum by 700-fold, but was ineffective against crown rot in the second year when pathogen abundance was >log 6.0 GC g-1 root. Strain A5MH increased crop emergence and tillering biomass by up to 40%. CONCLUSIONS: Further trials are required to determine if the A5MH-induced pathogen suppression translates to yield improvements in higher rainfall regions where non-cereal rotations reduce crown rot inoculum.

Plant growth-promoting and biocontrol traits of endophytic Bacillus licheniformis against soft rot causing Pythium myriotylum in ginger plant.

Bacterial endophytes from plants harbor diverse metabolites that play major roles in biocontrol and improve plant growth. In this study, a total of 12 endophytic bacteria were isolated from the ginger rhizome. The strain K3 was highly effective in preventing mycelia growth of Pythium myriotylum (78.5 ± 1.5% inhibition) in dual culture. The cell-free extract (2.5%) of endophyte K3 inhibited 76.3 ± 4.8% mycelia growth, and 92.4 ± 4.2% inhibition was observed at a 5% sample concentration. The secondary metabolites produced by Bacillus licheniformis K3 showed maximum activity against Pseudomonas syringae (24 ± 1 mm zone of inhibition) and Xanthomonas campestris (28 ± 3 mm zone of inhibition). The strain K3 produced 28.3 ± 1.7 IU mL-1 protease, 28.3 ± 1.7 IU mL-1 cellulase, and 2.04 ± 0.13 IU mL-1 chitinase, respectively. The ginger rhizome treated with K3 in the greenhouse registered 53.8 ± 1.4% soft rot incidence, and the streptomycin-treated pot registered 78.3 ± 1.7% disease incidence. The selected endophyte K3 improved ascorbate peroxidase (1.37 ± 0.009 µmole ASC min-1 mg-1 protein), catalase (8.7 ± 0.28 µmole min-1 mg-1 protein), and phenylalanine ammonia-lyase (26.2 ± 0.99 Umg-1) in the greenhouse. In addition, K3 treatment in the field trial improved rhizome yield (730 ± 18.4 g) after 180 days (p < 0.01). The shoot length was 46 ± 8.3 cm in K3-treated plants, and it was about 31% higher than the control treatment (p < 0.01). The lytic enzyme-producing and growth-promoting endophyte is useful in sustainable crop production through the management of biotic stress.

A Pythium myriotylum Small Cysteine-Rich Protein Triggers Immune Responses in Diverse Plant Hosts.

The oomycete Pythium myriotylum is a necrotrophic pathogen that infects many crop species worldwide, including ginger, soybean, tomato, and tobacco. Here, we identified a P. myriotylum small cysteine-rich protein, PmSCR1, that induces cell death in Nicotiana benthamiana by screening small, secreted proteins that were induced during infection of ginger and did not have a predicted function at the time of selection. Orthologs of PmSCR1 were found in other Pythium species, but these did not have cell death-inducing activity in N. benthamiana. PmSCR1 encodes a protein containing an auxiliary activity 17 family domain and triggers multiple immune responses in host plants. The elicitor function of PmSCR1 appears to be independent of enzymatic activity, because the heat inactivation of PmSCR1 protein did not affect PmSCR1-induced cell death or other defense responses. The elicitor function of PmSCR1 was also independent of BAK1 and SOBIR1. Furthermore, a small region of the protein, PmSCR186-211, is sufficient for inducing cell death. A pretreatment using the full-length PmSCR1 protein promoted the resistance of soybean and N. benthamiana to Phytophthora sojae and Phytophthora capsici infection, respectively. These results reveal that PmSCR1 is a novel elicitor from P. myriotylum, which exhibits plant immunity-inducing activity in multiple host plants. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Cutaneous Pythiosis in 2 Dogs, Italy.

We report cutaneous pythiosis in 2 dogs in Italy that had recurrent exposure to the same freshwater habitat. Phylogenetic analysis placed the isolates within Pythium insidiosum complex cluster IV, corresponding to P. periculosum. In Italy, pythiosis should be considered in differential diagnoses by human and veterinary health professionals.

Activity of volatiles induced by microbes and natural plants stifled the growth of Pythium aphanidermatum - the damping off in Tomato.

BACKGROUND: Volatilomes from natural plants and microbes imparts diverse antifungal properties to suppress the growth of plant pathogens and therefore can be a suitable alternative of chemical fungicides. The present experiment was to study effect of volatiles produced by natural plants and microbes on the fungal growth of Pythium aphanidermatum, which is a tomato seedling pathogen. RESULTS: Isolate of P. aphanidermatum, causing damping off in tomato were isolated and incubated at 25 ± 2 °C. The isolate was tested for the anti-oomycetes activities of volatiles in vitro. The volatiles produced by the leaves of Mentha spicata and Cymbopogon citratus showed the maximum inhibitory effect of 45.56 and 24.70 percent, respectively on the mycelial growth of P. aphanidermatum, whereas, the pathogen was not inhibited on exposure to the volatiles of macro-basidiomycetes fungi. The volatiles of T. asperellum showed the maximum inhibitory effect of 69.26 percent against P. aphanidermatum. The study also included the identification of Volatile Organic Compounds (VOCs) involved in the suppression of pathogens by Headspace Gas Chromatography Mass Spectrometry (HS GCMS). The results revealed the production of carvone by the leaves of M. spicata; citronellol and geraniol by C. citratus; isopentyl alcohol and limonene by T. asperellum with increased peak area percentage and these compounds possessed antifungal properties. The vaporous action of isopentyl alcohol completely suppressed the mycelial growth of P. aphanidermatum, which is highly correlated to the T. asperellum extract on pathogenic growth. While the compounds, carvone, and citronellol showed the maximum inhibitory effect of 89.02 and 85.49 percent, respectively when used at 500 ppm and also altered the sporulation behavior of P. aphanidermatum. CONCLUSION: Results showed that volatiles of M. spicata and T. asperellum have anti-oomycetes action on pathogenic growth leading to a distortion of sporulation of P. aphanidermatum. High antifungal properties make VOCs suitable for incorporation as a new integrated plant disease management programs.

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.

Specific interaction of an RNA-binding protein with the 3'-UTR of its target mRNA is critical to oomycete sexual reproduction.

Sexual reproduction is an essential stage of the oomycete life cycle. However, the functions of critical regulators in this biological process remain unclear due to a lack of genome editing technologies and functional genomic studies in oomycetes. The notorious oomycete pathogen Pythium ultimum is responsible for a variety of diseases in a broad range of plant species. In this study, we revealed the mechanism through which PuM90, a stage-specific Puf family RNA-binding protein, regulates oospore formation in P. ultimum. We developed the first CRISPR/Cas9 system-mediated gene knockout and in situ complementation methods for Pythium. PuM90-knockout mutants were significantly defective in oospore formation, with empty oogonia or oospores larger in size with thinner oospore walls compared with the wild type. A tripartite recognition motif (TRM) in the Puf domain of PuM90 could specifically bind to a UGUACAUA motif in the mRNA 3' untranslated region (UTR) of PuFLP, which encodes a flavodoxin-like protein, and thereby repress PuFLP mRNA level to facilitate oospore formation. Phenotypes similar to PuM90-knockout mutants were observed with overexpression of PuFLP, mutation of key amino acids in the TRM of PuM90, or mutation of the 3'-UTR binding site in PuFLP. The results demonstrated that a specific interaction of the RNA-binding protein PuM90 with the 3'-UTR of PuFLP mRNA at the post-transcriptional regulation level is critical for the sexual reproduction of P. ultimum.

Volatile Organic Compounds from Pythium oligandrum Play a Role in Its Parasitism on Plant-Pathogenic Pythium myriotylum.

The oomycete Pythium oligandrum is a soil-inhabiting parasite and predator of both fungi and oomycetes, and uses hydrolytic enzymes extensively to penetrate and hydrolyze its host or prey. Other mechanisms have been studied less, and we investigated the contribution of P. oligandrum-produced volatile organic compounds (VOCs) to parasitism. The growth-inhibiting activity of P. oligandrum VOCs was tested on Pythium myriotylum-a host or prey of P. oligandrum-coupled with electron microscopy, and biochemical and transcriptomic analyses. The P. oligandrum-produced VOCs reduced P. myriotylum growth by 80% and zoospore levels by 60%. Gas chromatography-mass spectrometry (GC-MS) identified 23 VOCs, and methyl heptenone, d-limonene, 2-undecanone, and 1-octanal were potent inhibitors of P. myriotylum growth and led to increased production of reactive oxygen species at a concentration that did not inhibit P. oligandrum growth. Exposure to the P. oligandrum VOCs led to shrinkage of P. myriotylum hyphae and lysis of the cellular membranes and organelles. Transcriptomics of P. myriotylum exposed to the P. oligandrum VOCs at increasing levels of growth inhibition initially showed a strong upregulation of putative detoxification-related genes that was not maintained later. The inhibition of P. myriotylum growth continued immediately after the exposure to the VOCs was discontinued and led to the reduced infection of its plant hosts. The VOCs produced by P. oligandrum could be another factor alongside hydrolytic enzymes contributing to its ecological role as a microbial parasite in particular ecological niches such as in soil, and may also contribute to the biocontrol of diseases using P. oligandrum commercial preparations. IMPORTANCE Microbe-microbe interactions in nature are multifaceted, with multiple mechanisms of action, and are crucial to how plants interact with microbes. Volatile organic compounds (VOCs) have diverse functions, including contributing to parasitism in ecological interactions and potential applications in biocontrol. The microbial parasite P. oligandrum is well known for using hydrolytic enzymes as part of its parasitism. We found that P. oligandrum VOCs reduced the growth of, and caused major damage to, the hyphae of P. myriotylum (a host or prey of P. oligandrum). Transcriptomic analyses of P. myriotylum exposed to the VOCs revealed the upregulation of genes potentially involved in an attempt to detoxify the VOCs. The inhibitory effects of the VOCs had a knock-on effect by reducing the virulence of P. myriotylum toward its plant hosts. The P. oligandrum VOCs could contribute to its ecological role as a microbial parasite. The VOCs analyzed here may also contribute to the biocontrol of diseases using P. oligandrum commercial preparations.

Comparative genomics reveals the presence of simple sequence repeats in genes related to virulence in plant pathogenic Pythium ultimum and Pythium vexans.

In this study, we evaluated the occurrence, relative abundance (RA), and density (RD) of simple sequence repeats (SSRs) in the complete genome and transcriptomic sequences of the plant pathogenic species of Pythium to acquire a better knowledge of their genome structure and evolution. Among the species, P. ultimum had the highest RA and RD of SSRs in the genomic sequences, whereas P. vexans had the highest RA and RD in the transcriptomic sequences. The genomic and transcriptomic sequences of P. aphanidermatum showed the lowest RA and RD of SSRs. Trinucleotide SSRs were the most prevalent class in both genomic and transcriptomic sequences, while dinucleotide SSRs were the least prevalent. The G + C content of the transcriptomic sequences was found to be positively correlated with the number (r = 0.601) and RA (r = 0.710) of SSRs. A motif conservation study revealed the highest number of unique motifs in P. vexans (9.9%). Overall, a low conservation of motifs was observed among the species (25.9%). A gene enrichment study revealed P. vexans and P. ultimum carry SSRs in their genes that are directly connected to virulence, whereas the remaining two species, P. aphanidermatum and P. arrhenomanes, harbour SSRs in genes involved in transcription, translation, and ATP binding. In an effort to enhance the genomic resources, a total of 11,002 primers from the transcribed regions were designed for the pathogenic Pythium species. Furthermore, the unique motifs identified in this work could be employed as molecular probes for species identification.

Infection with Pythium flevoense in a harbour porpoise (Phocoena phocoena) as a novel cause of dermatitis in marine mammals.

The oomycete Pythium flevoense was diagnosed as the cause of dermatitis in a young adult female harbour porpoise (Phocoena phocoena) that had been trapped in a pound net in a temperate saltwater environment. Disease from Pythium sp. infection-pythiosis-is infrequently diagnosed in humans, horses, dogs, cattle, and few other mammalian species. Pythiosis is typically associated with exposure to tropical or subtropical freshwater conditions, and typically caused by Pythium insidiosum. However, until now, pythiosis has been reported in neither marine mammals nor temperate saltwater conditions, and P. flevoense is not known as a cause of pythiosis in mammals. This porpoise developed generalised dermatitis despite treatment and euthanasia was necessary. Histopathological evaluation revealed a chronic active erosive dermatitis, with intralesional hyphae morphologically consistent with a Pythium sp. PCR analysis and sequencing of affected skin matched Pythium flevoense with a 100% similarity to the reference strain. Additional diagnostics excluded other pathogens. Based on this case report, P. flevoense needs to be considered as a mammalian pathogen. Furthermore, harbour porpoises and possibly other marine mammals may be at risk of infection with P. flevoense, and pythiosis should be included in the differential diagnosis of dermatitis in marine mammals.

Soil Suppressiveness Against Pythium ultimum and Rhizoctonia solani in Two Land Management Systems and Eleven Soil Health Treatments.

The soil microbiome is known to be crucial for the control of soil-borne plant diseases. However, there is still little knowledge on how to modify the soil microbiome to induce or increase disease suppressiveness. In the present study, we applied eleven soil health treatments combined with conventional and organic agricultural management in a long-term field experiment. Suppressiveness against Pythium ultimum and Rhizoctonia solani was assessed in bioassays for 2 years. In addition, the microbiome community composition and microbial abundance were determined. We found that while several treatments changed the microbial community composition compared to the control, only a combination treatment of anaerobic soil disinfestation, hair meal, and compost addition resulted in suppressiveness against P. ultimum. Pythium suppressiveness is likely to have been caused by an increased microbial abundance and activity. Moreover, the increased abundance of several bacterial taxa, such as Pseudomonas sp., Chryseobacterium sp., members of the family Chitinophagaceae, and the fungal genus Mortierella sp. and family Trichosporonaceae, was measured. There was no overall difference in suppressiveness between conventional and organic land management. Also, no suppressiveness against R. solani could be detected. Our results indicate that a treatment combining the reduction of microorganisms followed by a recovery phase with high amounts of organic amendments may be more effective in inducing suppressiveness than treatments consisting of only one of these measures.

First Study Case of Microbial Biocontrol Agents Isolated from Aquaponics Through the Mining of High-Throughput Sequencing Data to Control Pythium aphanidermatum on Lettuce.

Aquaponics is defined as a sustainable and integrated system that combines fish aquaculture and hydroponic plant production in the same recirculated water loop. A recent study using high-throughput sequencing (HTS) technologies highlighted that microbial communities from an aquaponic system could control one of the most problematic pathogens in soilless lettuce culture, namely, Pythium aphanidermatum. Therefore, this study aims at isolating the microorganisms responsible for this biocontrol action. Based on the most promising genera identified by HTS, an innovative strategy for isolating and testing original biocontrol agents from aquaponic water was designed to control P. aphanidermatum. Eighty-two bacterial strains and 18 fungal strains were isolated, identified by Sanger sequencing, and screened in vivo to control damping-off of lettuce seeds caused by P. aphanidermatum. Out of these 100 isolates, the eight most efficacious ones were selected and further tested individually to control root rot disease caused by the same pathogen at a later stage of lettuce growth. Strains SHb30 (Sphingobium xenophagum), G2 (Aspergillus flavus), and Chito13 (Mycolicibacterium fortuitum) decreased seed damping-off at a better rate than a propamocarb fungicide and a Pseudomonas chlororaphis registered biocontrol agent did. In root rot bioassays, lettuce mortality was prevented by applying strains G2 and Chito13, which were at least as efficacious as the fungicide or biopesticide controls. Lettuce disease symptoms and mortality were eradicated by strain SHb30 in the first bioassay, but not in the second one. These results show that aquaponic systems are promising sources of original biocontrol agents, and that HTS-guided strategies could represent interesting approaches to identify new biocontrol agents.

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

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

Case Series: Mixed Infectious Keratitis by Pythium insidiosum and Fungal Species.

SIGNIFICANCE: This case series is the first to illustrate mixed infection from Pythium sp. and fungal species in corneal ulcer. PURPOSE: This case series aimed to alert all toward the possibility of both Pythium sp. and fungal species infection in case of nonresponding corneal ulcer treated with either antifungals or antipythium drugs alone. Increased suspicion of mixed infection in case of nonresponding fungal/ Pythium keratitis may facilitate early and prompt management. CASE REPORTS: Six patients presented with signs of either fungal or Pythium keratitis. They underwent ophthalmological examinations, smear examinations, cultures, and polymerase chain reaction (PCR). Therapeutic penetrating keratoplasty was performed in cases where symptoms worsened after treatment with either antifungal or antipythium drugs. The half corneal button (HCB) was shared for histopathological and microbiological examinations. In the first case, smear examination from corneal scraping (CS) revealed Pythium -like filaments, which were confirmed with PCR; however, Aspergillus nidulans grew in culture. In the second case, iodine-potassium iodide (IKI) staining was positive for Pythium ; however, PCR was positive for both Pythium and fungus, which was further confirmed by DNA sequencing. In the third case, IKI staining and HCB were positive for Pythium ; however, PCR was positive for fungus, which was identified as Candida saitoana with DNA sequencing. In the fourth case, Pythium grew in the CS culture; however, Candida sp. grew in the HCB culture. In the fifth case, Cladosporium sp. grew in culture from CS; however, Pythium insidiosum grew from the anterior chamber exudate after therapeutic penetrating keratoplasty. In the sixth case, smear examination revealed septate fungal filaments, and Cladosporium sp. grew in culture; however, HCB on histopathological examination showed features of Pythium keratitis. CONCLUSIONS: In unresponsive cases of Pythium or fungal keratitis, diagnostic modalities such as IKI and PCR should be implemented as a routine practice, in addition to smears and cultures.

Oomicidal activity of polypyrrole nanoparticles against Pythium insidiosum.

This study evaluated in-vitro action of a new molecule, the polypyrrole nanoparticles (Ppy-NP), against Pythium insidiosum isolates using M38-A2/CLSI; the minimal inhibitory (MIC) and minimal oomicidal (MOC) concentrations were also determined. Additionally, changes in the hyphae wall of P. insidiosum CBS 575.85 treated with Ppy-NP were evaluated by scanning electron microscopy (SEM). The MIC100 and MOC for all isolates ranged from 8 to 32 µg mL-1, and the MIC90 and MIC50 were 16 µg mL-1. The SEM showed structural damage to the hyphae of P. insidisoum treated with Ppy-NP, as hyphae surfaces with less turgidity were found, thereby showing scaling and ruptures compared to the control (untreated hyphae). Our findings highlighted the anti-P. insidiosum properties of Ppy-NP proved to be a promising candidate for research using pythiosis experimental models.

Anti-Pythium insidiosum activity of bioactive compounds from medicinal plants.

Pythiosis is a serious disease caused by the aquatic oomycete Pythium insidiosum that mainly affects mammals. Unlike fungal and bacterial resistance induced by the indiscriminate use of drugs, P. insidiosum has low susceptibility to antifungal drugs. In this sense, essential oils and their major components emerge as a promising treatment line for this disease. Given the above, this study sought to verify P. insidiosum (n = 34) susceptibility to the bioactive compounds eugenol, α-terpineol, menthol, and carvacrol and correlate them with the respective essential oils of Eugenia caryophyllata, Melaleuca alternifolia, Mentha piperita, and Origanum vulgare. The essential oils and bioactive compounds were purchased commercially and tested according to the Clinical and Laboratory Standards Institute protocol M38-A2. Our findings showed that eugenol, α-terpineol, and carvacrol had superior anti-P. insidiosum action than their respective essential oils, suggesting that they may be responsible for inhibitory activity against P. insidiosum. Notably, the major compound with the best anti-P. insidiosum activity was α-terpineol; nonetheless, menthol showed less activity than its essential oil. The results imply that essential oils and their major compounds may be important allies in treating pythiosis, expanding the perspectives of developing new drugs with anti-P. insidiosum activity.

Differential Susceptibility of Two Citrullus amarus Pollenizer Watermelons to Five Species of Pythium and Globisporangium.

Pollenizer watermelon (Citrullus amarus) must be planted alongside triploid watermelon cultivars to provide a source of pollen. Early season death of pollenizer watermelon cultivars SP-6 and SP-7 was observed in a research field in Charleston County, SC, in April 2022, 3 to 4 weeks after transplanting. Disease incidence was 15 and 12% for the two cultivars, respectively. Two species of Pythium (P. myriotylum and P. aphanidermatum) and three species of Globisporangium (G. ultimum, G. irregulare, and G. spinosum) were recovered from 13 of 17 plants cultured and identified based on the sequences of the cytochrome c oxidase subunit I gene. Pythium spp. were recovered from both cultivars, while Globisporangium spp. were recovered only from 'SP-7'. In pathogenicity tests, 'SP-7' had lower area under the disease progress curve (AUDPC) values than 'SP-6' with one isolate each of four species and lower AUDPC values than seedless watermelon 'Estrella' with one isolate each of two species. Mean AUDPC values did not differ between 'Estrella' and 'SP-6' with any isolate. AUDPC values were greater with G. ultimum on all three cultivars than with one isolate of G. spinosum and both isolates of P. myriotylum and P. aphanidermatum. Susceptibility of the new cultivar SP-7 to Pythium and Globisporangium spp. differs from susceptibility of 'SP-6'. These results will help growers choose which pollenizer cultivar to plant.

Diversity of Pythium Species Recovered from Float-Bed Tobacco Transplant Production Greenhouses.

Pythium diseases are common in hydroponic crop production and often threaten the greenhouse production of cucumber, tomato, lettuce, and other crops. In tobacco transplant production, where float-bed hydroponic greenhouses are commonly used, Pythium diseases can cause up to 70% seedling loss. However, there have been few comprehensive studies on the composition and diversity of Pythium communities in tobacco greenhouses. In a 2017 survey, 360 Pythium isolates were collected from 41 tobacco greenhouses across four states (VA, MD, GA, and PA). Samples were collected from one to seven sites within each greenhouse. Twelve described Pythium species were identified (P. adhaerens, P. aristosporum, P. attrantheridium, P. catenulatum, P. coloratum, P. dissotocum, P. inflatum, P. irregulare, P. myriotylum, P. pectinolyticum, P. porphyrae, and P. torulosum) among the isolates obtained. Approximately 80% of the surveyed greenhouses harbored Pythium in at least one of four sites (bay water, tobacco seedlings, weeds, and center walkways) within the greenhouse. The structure of Pythium communities was diverse among the surveyed greenhouses: multiple Pythium species coexisted in the same sample, and multiple species were present within the same greenhouse at different sites. This diversity appeared to be influenced by the sampling sites within the surveyed tobacco greenhouses, sample type, and sampling time. Intraspecific variation may also exist among the P. dissotocum populations found in this study. These results uncovered the complexity and diversity of the Pythium communities within float tobacco transplant greenhouses, which could play a role in the variation in Pythium diseases observed in these production systems.

Effect of Infection Timing by Four Pythium spp. on Soybean Damping-Off Symptoms with and Without Cold Stress.

Pythium spp. cause damping-off of soybean, especially when soil conditions at or shortly after planting are cool and wet. Soybean planting dates continue to shift to earlier dates, so germinating seed and seedlings are exposed to periods of cold stress at a time which favors infection by Pythium, and seedling disease occurs. The objective of this study was to assess infection timing and cold stress on soybean seedling disease severity caused by four Pythium spp. prevalent in Iowa, namely P. lutarium, P. oopapillum, P. sylvaticum, and P. torulosum. Each species was used individually to inoculate soybean cultivar 'Sloan' using a rolled towel assay. Two temperature treatments (continuous 18°C [C18]; a 48-h cold stress period at 10°C [CS]) were applied. Soybean seedling age was divided into five growth stages (GS1 to GS5). Root rot severity and root length were assessed at 2, 4, 7, and 10 days after inoculation (DAI). At C18, root rot was greatest when soybean was inoculated with P. lutarium or P. sylvaticum at GS1 (seed imbibes water) and with P. oopapillum or P. torulosum at GS1, GS2 (radicle elongation), and GS3 (hypocotyl emergence). After CS, soybean susceptibility to P. lutarium and P. sylvaticum was reduced compared to C18 for inoculation at all GSs except GS5 (unifoliate leaf emergence). Conversely, root rot by P. oopapillum and P. torulosum was greater after CS compared to C18. Data from this study demonstrate that greater root rot, and consequently more damping-off, is likely if infection occurs at early germination stages before seedling emergence.