Pythium Species Research Articles

First report of wilt disease caused by Pythium myriotylum on peanut plants in Korea.

Peanut (Arachis hypogaea L.) has long been cultivated worldwide as an important crop for oil and protein production. Among the various diseases in peanut plants, wilt diseases caused by soil-borne pathogens such as Ralstonia solanacearum and Verticillium dahliae are especially destructive and substantially diminish both quantity and quality in peanut production (Kokalis-Burelle et al., 1997; Thiessen et al., 2012). In July 2022, wilt symptoms were observed in 1 to 3% of the area of peanut fields in Yeoju-si, Korea (37°23´04.0˝N; 127°33´43.0˝E). The xylem in the stems of the wilted plants was dark brown at the soil-surface, which is a representative symptom of vascular wilt pathogens (Yadeta et al. 2013). To isolate the causative pathogens, the stems exhibiting dark lesions were disinfected with 1% NaOCl for 1 min, rinsed with sterile distilled water, and placed on potato dextrose agar medium. The plates were incubated at 25℃ for 2 days, and white hyphae that grew out from the tissues were subcultured twice on V8 juice agar (V8A) medium. Among the 3 isolates, morphological characteristics of the representative strain YJ1-2 were observed under a microscope. The sporangia were terminal intercalary, filamentous, inflated lobulate, and ranging from 37.4 to 73.6 μm in diameter. The antheridia were diclinous, with clavate, elongate, and crook-necked shapes. The oogonia were mostly globose, with an average of 27.1 μm (range from 20.2 to 35.2 μm, n = 50) in diameter, and mated with one to several antheridia. Both plerotic or aplerotic oospores were observed. Overall, the morphological characteristics of the sporangia, antheridia, oogonia, and oospores indicated that YJ1-2 belongs to the genus Pythium. To genetically characterize YJ1-2, genomic DNA was extracted using cetyltrimethylammonium bromide buffer, and the internal transcribed spacer (ITS) region and cytochrome c oxidase subunit I (cox1) gene were amplified by PCR using primer sets ITS4/ITS5 and OomCoxI-Levlo/ OomCoxI-Levup, respectively (White et al., 1990; Robideau et al. 2011), sequenced, and identified using BLASTN (NCBI, National Center for Biotechnology Information). The ITS sequence (NCBI Acc. No. OR125595) of YJ1-2 has 99% similarity with that of P. myriotylum isolate PY39 (NCBI Acc. No. KX671096). A neighbor-joining phylogenetic tree was constructed from aligned cox1 sequence (NCBI Acc. No. OR224334) of the 10 Pythium species strains including YJ1-2 by CLUSTALW method was used as an outgroup. The YJ1-2 was most closely related to P. myriotylum isolate PM30 (NCBI Acc. No. MT823167). To substantiate the pathogenicity of YJ1-2, the crown roots of peanut plants grown in pots for 4 weeks were wounded using a sterile tweezer, and the mycelial plugs of YJ1-2 cultured for 5 days on V8A were inoculated on the wounds. The inoculated plants were cultivated in a growth chamber at 30℃ and 70% relative humidity with a 12-h photoperiod. The infected peanut plants exhibited wilt symptoms 11 days after inoculation, consistent with the initial observation, while uninoculated plants remained healthy. To satisfy Koch's postulates, white mycelia were re-isolated from the stems of inoculated plants and axenically cultured in V8A. The morphologies and ITS sequences of the re-isolates were consistent with those of YJ1-2. P. myriotylum has been reported as a causal pathogen of peanut pod rot in the United States and China. However, to the best of our knowledge, this is the first report of wilt disease in peanut plants caused by P. myriotylum in Korea. To prevent the incidence of wilt disease, we will continue our investigations to develop control strategies, including the selection of appropriate agrochemicals.

Biocontrol potential of endophytic Pseudomonas strain IALR1619 against two Pythium species in cucumber and hydroponic lettuce.

The use of fungicides to manage disease has led to multiple environmental externalities, including resistance development, pollution, and non-target mortality. Growers have limited options as legacy chemistry is withdrawn from the market. Moreover, fungicides are generally labeled for traditional soil-based production, and not for liquid culture systems. Biocontrol agents for disease management are a more sustainable and environmentally friendly alternative to conventional agroprotectants. Pythium ultimum is a soil borne oomycete plant pathogen with a broad taxonomic host range exceeding 300 plants. Cucumber seedlings exposed to P. ultimum 1 day after a protective inoculation with bacterial endophyte accession IALR1619 (Pseudomonas sp.) recorded 59% survival; with the control assessed at 18%. When the pathogen was added 5 days post endophyte inoculation, 74% of the seedlings treated survived, compared to 36% of the control, indicating a longer-term effect of IALR1619. Under hydroponic conditions, IALR1619 treated leaf type lettuce cv. 'Cristabel' and Romaine cv. 'Red Rosie' showed 29% and 42% higher shoot fresh weight compared to their controls, respectively. Similar results with less growth decline were observed for a repeat experiment with IALR1619. Additionally, an experiment on hydroponic lettuce in pots with perlite was carried out with a mixture of P. ultimum and P. dissotocum after IALR1619 inoculation. The endophyte treated 'Cristabel' showed fresh weight gain, but the second cultivar 'Pensacola' yielded no increase. In summary, the endophyte IALR1619 provided short term as well as medium-term protection against Pythium blight in cucumber seedlings and may be used as an alternative to conventional fungicides in a greenhouse setting. This study also demonstrated the potential of ALR1619 as a biocontrol agent against Pythium blight in hydroponic lettuce.

Picarbutrazox Effectiveness Added to a Seed Treatment Mixture for Management of Oomycetes that Impact Soybean in Ohio.

None of the current oomycota fungicides are effective towards all species of Phytophthora, Phytopythium, Globisporangium, and Pythium that affect soybean seed and seedlings in Ohio. Picarbutrazox is a new oomyceticide with a novel mode of action towards oomycete pathogens. Our objectives were to evaluate picarbutrazox to determine (i) baseline sensitivity (EC50) to 189 isolates of 29 species, (ii) the efficacy with a base seed treatment with three cultivars with different levels of resistance in 14 field environments; and (iii) if the rhizosphere microbiome was affected by the addition of the seed treatment on a moderately susceptible cultivar. The mycelial growth of all isolates was inhibited beginning at 0.001 μg, and the EC50 ranged from 0.0013 to 0.0483 μg of active ingredient (a.i.)/ml. The effect of seed treatment was significantly different for plant population and yield in eight of 14 and six of 12 environments, respectively. The addition of picarbutrazox at 1 and 2.5 g of a.i./100 kg seed to the base seed treatment compared to the base alone was associated with higher plant populations and yield in three and one environments, respectively. There was limited impact of the seed treatment mefenoxam 7.5 g of a.i. plus picarbutrazox 1 g of a.i./100 kg seed on the oomycetes detected in the rhizosphere of soybean seedlings collected at the V1 growth stage. Picarbutrazox has efficacy towards a wider range of oomycetes that cause disease on soybean, and this will be another oomyceticide tool to combat early season damping-off in areas where environmental conditions highly favor disease development.

First Report of Root Rot Caused by Pythium dissotocum on Tobacco in China.

Tobacco (Nicotiana tabacum L.) is an important economic crop that is widely grown around the world. Its annual production in China is estimated at 2.2 million tons (Berbeć and Matyka 2020). Since 2022, a root rot disease was sporadically observed on tobacco seedlings on cultivar Yunyan 87 in cultivated tobacco fields in the Hunan province of China. A disease incidence of about 10% occurred across 48 ha of tobacco fields. The affected tobacco plants had slow and stunted growth with yellowing leaves. The roots turned grayish brown, decayed, and died. Diseased roots were collected from six fields and cut into small pieces (5 mm ×5 mm) from the edge of the rotted portions, and then sterilized with 70% ethanol for 10 s, 0.1% HgCl2 for 1 min, and washed in sterilized water three times. All the sterilized tissue were placed on potato dextrose agar (PDA) medium and cultured at 26 ℃ in the dark. About 3 days later, colonies with similar morphology were removed and sub-cultured on fresh PDA. A total of six strains were obtained from six tobacco samples. Strains were white and had radial growth on PDA. Hyphae were aseptate and the sporangia were filamentous. The oogonia were subglobose, smooth, 16.04 ± 0.25 µm (n=50) in diameter, and developed on unbranched stalks. The antheridia were barrel shaped and clavate. Oospores were globose, aplerotic or nearly plerotic, measuring 6.62 ± 0.33 µm (n=50). These morphological characteristics were consistent with the description of Pythium spp. (van der Plaats-Niterink 1981). For molecular identification, the internal transcribed spacer (ITS) region of rDNA and cytochrome c oxidase subunit I (Cox I) of a representative isolate, GF-3, were amplified and sequenced (GenBank accession nos. OR228424 for ITS and OR237556 for Cox I) using universal primers ITS1/ITS4 (White et al. 1990) and FM58/FM66, respectively (Villa et al. 2006). BLASTn analysis revealed that the ITS and Cox I sequences were 99.76 % (838/840 bp) and 99.85% (671/672 bp) identical to the corresponding sequences of P. dissotocum strain CBS 166.68 (AY598634.2) and UM982 (MT981147.1), respectively. A neighbor-joining phylogenetic tree based on the Cox I sequence showed that GF-3 grouped in the P. dissotocum branch. Based on morphological and molecular characteristics, GF-3 was identified to be P. dissotocum. For pathogenicity testing, four- to five-leaf-old healthy potted tobacco seedlings of the Yunyan 87 cultivar were inoculated with a zoospore suspension (1 × 105 zoospores/ml), which was induced on V8-juice medium. The zoospore suspension was introduced into the soil around plant roots and 10 mL of inoculum was used for each plant. In the control group, plants were inoculated with sterilized water. All of the treated plants were kept in humid chambers at 26°C under a 12 h/12 h photoperiod. The pathogenicity assays were performed twice, with each treatment having three replicated plants. After 5 days, tobacco seedlings inoculated with P. dissotocum showed symptoms resembling that observed in the field. However, the control plants remained healthy. Pythium dissotocum was re-isolated from the infected plants and identified by morphological and molecular methods, thus confirming Koch's postulates. Pythium dissotocum has been reported causing root rot in other plants, including hydroponic lettuce (McGehee et al. 2018) and spinach (Huo et al. 2020). Also, many Pythium species have recently been recovered from float-bed tobacco transplant production greenhouses (Zhang et al. 2022). However, to our knowledge, this is the first report of root rot on tobacco caused by P. dissotocum in China. Since this disease could greatly affect tobacco seedling establishment in the field, appropriate management strategies need to be developed to reduce further losses in tobacco planting fields.

Identification of Some Fungi Causing Okra Root Rot Disease from Different Regions of Anbar and Waist and Testing Pathogenicity

The study was conducted in the Department of Plant Protection - College of Agriculture - University of Anbar for the year 2022, as the results of collecting samples from the regions of Anbar and Waist governorates showed the spread of okra root rot disease in most of the study areas, and the results of phenotypic and molecular diagnosis showed the presence of the fungal species Pythium Aphanidermatum, Fusarium proliferatum, Epicoccum nigrum, Itersonilia perplexans, Fusarium chlamydosporum, Rhizoctonia solani, which were deposited in the International Genbank OQ572440, OQ540926, OQ566939, OQ540927, OQ540928, OQ540929 (NCBI) seeds, respectively. Okra, as the tested isolates achieved a significant decrease in the germination of okra seeds on the culture medium (W.A. Water agar) compared with the control treatment that was not contaminated with any of the tested isolates, in which the infection rate was 0.0%.

Oomycete composition in Proteaceae orchards and natural stands on three continents

The Proteaceae, a diverse family of woody flowering plants in the Southern Hemisphere, contains many species known to be susceptible to Phytophthora cinnamomi, both in the natural environment and in cut-flower orchards. Very little is known about the prevalence of P. cinnamomi and other oomycetes across these landscapes. To address this knowledge gap, we used a double ITS1 and RPS10 gene metabarcoding approach and traditional isolation protocols to investigate oomycetes in orchards and natural stands of Proteaceae across South Africa, South Africa (eastern and western), Australia, and Europe. The RPS10 primers amplified more samples, including various Pythium species, while the ITS primers detected more Phytophthora phylotypes. Both datasets showed that geographic regions influenced oomycete species richness and community composition, while they did not show any variation between orchards and natural vegetation. RPS10 metabarcoding detected the largest number of species and provided greater statistical confidence than ITS1 when considering oomycete species composition. Metabarcoding also showed that orchards had a higher abundance of P. cinnamomi compared to native stands, although this was not found when isolating through baiting of roots and rhizosphere soil. Direct isolation and metabarcoding are complementary, with metabarcoding serving as an early detection tool. However, it cannot distinguish living viable propagules from residual DNA of dead propagules, limiting its use for diagnostic purposes related to Phytophthora management and control. These results, along with those of other recent studies, show that metabarcoding offers an effective tool to describe the dynamics of soil oomycetes in different ecosystems.

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, South Carolina, 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 (four isolates) and P. aphanidermatum (three isolates), and three species of Globisporangium, G. ultimum (one isolate), G. irregulare (one isolate), and G. spinosum (four isolates), 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.

First report of Phytopythium vexans causing root rot on Macadamia integrifolia in China.

Soil-borne plant-pathogenic Phytopythium spp. can cause root rot and damping off on important plant species, resulting in serious economic loss. A survey in October 2021 identified soil-borne diseases occurring on Macadamia integrifolia in Yunnan Province, China. Microbes were isolated from necrotic roots of 23 trees with root rot symptoms by growing on cornmeal-based oomycete-selective 3P (Haas 1964) and P5APR (Jeffers and Martin, 1986) media at 24ºC in the dark for 7 days. Of the 56 single-hyphal isolates obtained, 18 were morphologically similar to Phytopythium vexans (van der Plaats-Niterink 1981; de Cock et al. 2015). Isolates LC04 and LC051 were selected for molecular analyses. The internal transcribed spacer (ITS) region and the cytochrome c oxidase subunit II (CoxII) gene were PCR-amplified using universal primers ITS1/ITS4 (White et al. 1990) and oomycete-specific primers Cox2-F/Cox2-RC4 (Choi et al. 2015), respectively. The PCR products were sequenced with the amplification primers and sequences were lodged in Genbank (Accession no. OM346742, OM415989 for ITS, OM453644, OM453643 for CoxII for isolates LC04 and LC051, respectively). The top BLAST hit in the Genbank nr database for all four sequences was Phytopythium vexans (>99% identity). A maximum-likelihood phylogenetic tree was constructed with analogous concatenated ITS and CoxII sequences from either type or voucher specimens of 13 Phytopythium species in the same phylogenetic clade as P. vexans (Table 1; Bala et. al 2010). Isolates LC04 and LC051 grouped most closely to P. vexans, with LC051 basal and sister to LC04 and P. vexans voucher specimen CBS119.80 with 100% support (Fig. 1). Millet seed inoculated with agar pieces colonized by P. vexans LC04 and LC51 was used to fulfill Koch's postulates (Li et al. 2015) in a completely randomized experimental design. Four 6-month-old M. integrifolia var. Keaau (660) seedlings were transplanted into pasteurized commercial potting mix containing 0.5% (w/w) inoculum. Plants were grown in free draining pots and watered once a day. At 14 days post-inoculation, roots were discolored compared to control plants inoculated with millet seed mixed with agar plugs lacking P. vexans (Fig. 2). By 30 days post-inoculation, infected roots were discolored with obvious decay and reduction in root system size. Control plants were symptomless. P. vexans was successfully re-isolated from two lesioned roots from each plant. The infection experiment was done twice, demonstrating that P. vexans LC04 and LC51 caused root disease on M. integrifolia. P. vexans causes root rot, damping-off, crown rot, stem rot or patch canker on economically important trees in many parts of the world, including seven plant species in China (Farr and Rossman 2022). This is the first report of pathogenic P. vexans on M. integrifolia in China. Reports of pathogenic P. vexans on multiple hosts in several parts of the world suggest it should be considered a quarantine risk and included in risk mitigation or pest management plans that include other species of Phytopythium, or species of Pythium or Phytophthora, to which P. vexans has many similarities (de Cock et al. 2015).

Fitopatógenos do gênero Pythium: polífagos e destrutivos em ambientes úmidos

Pythium is one of the microorganisms of greatest agricultural interest in the world. Most of the species of this genus are natural inhabitants of the soil with a high capacity for surviving even in the absence of hosts, either as saprophytes or by means of resistance structures. Pythium species can cause plant diseases, such as seedling damping off and leaf blights, as well as root, stem, branch and fruit rots. Several environmental factors differentially modulate the behavior of Pythium species, especially humidity, temperature and soil pH. Most species of this genus are polyphagous and produce resistance structures, what makes difficult to manage the diseases they cause. In this sense, management should be based mainly on the adoption of preventive measures, in order to create a less favorable environment for spread, establishment, multiplication and infection. The genus Pythium was created by Pringsheim in 1858. Since then, much has been added in terms of knowledge about the etiology, epidemiology and management of the diseases caused by this pathogen. With the development and improvement of molecular biology techniques, there was an astonishing advance in understanding the biology, physiology and genetic variability of the genus and its species, which contributed to the phylogenetic studies involving Pythium spp. The demand for new research in these areas of study will always be in vogue, as organisms are subject to constant evolutionary processes. This review presents the state of the art on the advances in studies involving the genus Pythium with a view to fostering new lines of research aimed at the ultimate goal of controlling the diseases caused by this pathogenic oomycete.

Gene editing with an oxathiapiprolin resistance selection marker reveals that PuLLP, a loricrin-like protein, is required for oospore development in Pythium ultimum

Oomycetes, such as Pythium species, contain numerous devastating plant pathogens that inflict substantial economic losses worldwide. Although CRISPR/Cas9-based genome editing is available, the selection markers available for genetic transformation in these species are limited. In this study, a mutated version of the Phytophthora capsici oxysterol-binding protein-related protein 1 (PcMuORP1), known to confer oxathiapiprolin resistance, was introduced into the CRISPR/Cas9 system for in situ complementation in Pythium ultimum. We targeted PuLLP, which encodes a loricrin-like protein, and showed significant downregulation when the Puf RNA-binding protein-encoding gene PuM90 was knocked out. The PuLLP knockout mutants could not produce oospores, indicating a similar biological function as PuM90. The reintroduction of PuLLP into the knockout mutant using PcMuORP1 as a selection marker restored oospore production. Further comparisons with the conventional selection marker NPTII indicated that PcMuORP1 could be applied at a lower concentration and cost, resulting in a higher screening efficiency. Successive subculturing in the absence of selective pressure showed that PcMuORP1 had little long-term effect on the fitness of transformants. Hence, it could be reused as an alternative selection marker. This study demonstrates the successful implementation of the PcMuORP1 gene as a selection marker in the genetic transformation of Py. ultimum and reveals the loricrin-like protein PuLLP as a sexual reproduction-related factor downstream of the Puf RNA-binding protein PuM90. Overall, these results will help accelerate the functional genomic investigation of oomycetes.

Identification and Molecular Characterization of Rhizopus delemar from Eastern Ghats of State of India and its Biotechnological Applications

The humid climatic condition of the Koraput district of the eastern ghat of Odisha, India favors the growth of a diverse range of microbial and fungal strains, which not only lay scathing consequences on the environment but also prove to be beneficial in many industrial and biotechnological sectors. Hence, proper exploration of their roles and impacts is the necessity for an hour. With this goal, the present study reports on the identification and molecular characterization of the fungal strain collected from Koraput district of the eastern ghat of Odisha. The fungal strain was cultured, isolated, and identified using Internal transcribed spacer (ITS) 18S rRNA-specific primers. The estimation of fungal biomass was carried out by taking the dry weight of fungal mycelia after 48, 72, 96, and 120 hours of inoculation, which showed an increase in biomass (in w/w) exponentially. The fungal isolate (CAR02) was identified as Rhizopus delemar with GenBank accession number OQ683833. The SDS PAGE protein analysis showed more intensified and concentrated multiple fungal protein bands ranging from 13 to 97 kDa. ELISA (Enzyme- Linked Immunosorbent Assay) was used to estimate the antigenicity of the isolated R. delemar proteins from soil samples of Koraput. The proteins of Pythium species with an absorbance value of 1.5 shared 50-60% of similar antigenic sequences with the R. delemar proteins. Hence, the findings of our investigation suggests that the obtained antigenic sequences could further be used to isolate its epitopic specific regions that are responsible for the rot infections, against which antibodies could be developed. Further study is required to explore the rot causing fungal diversity in the soil of eastern ghats which will be helpful in designing novel disease diagnostic strategies in agriculture sectors. Furthermore, this identified fungus can also be used to synthesize various enzymes that will aid in the production of commercially significant by-products.

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 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 ITS barcoding. Twenty-two species of Pythium, Phytopythium and Globisporangium were identified, with Globisporangium irregulare sensu lato being the most common. To determine the origin of inoculum during the 2011 cropping season, eleven 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. 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.

Utilizing a new target gene, Ppinihp1, identified from genome sequences to detect Phytophthora pini

Phytophthora pini is a globally distributed plant pathogen of the Phytophthora genus. This fungus-like organism was recently associated with foliage blight and shoot dieback disease of Rhododendron pulchrum Sweet, which threaten the health of natural ecosystems and horticultural crops in China. Early and accurate detection of P. pini is critical for effective disease prevention and management. In this study, a new target gene Ppinihp1, which is a hypothetical protein, identified from the whole-genome sequence of P. pini was used to establish a conventional polymerase chain reaction (PCR) assay for the detection of this pathogen. PCR results were proved to be positive for P. pini, but negative for other Phytophthora, Pythium, fungal isolates and Bursaphelenchus species. The test was sensitive enough to detect 100 pg of P. pini purified genomic DNA in a 50 μL reaction mixture. P. pini was also detected in artificially inoculated R. pulchrum samples. Finally, this novel Ppinihp1-PCR-assay was able to detect P. pini in 15 of 40 soil samples collected from R. pulchrum fields in Jiangsu, China. This method established the specificity, sensitivity, accuracy and convenience for P. pini detection, offering a dependable tool for the rapid screening of Phytophthora root and stem rot in R. pulchrum.

Metagenomic identification of a novel oomycete cultured from a pyogranulomatous mass on the tail of a domestic cat.

We report here a transiently culturable oomycete pathogen isolated from a pyogranulomatous tail mass in a cat. The organism was morphologically and genetically distinct from Lagenidium and Pythium species. Following next-generation sequencing (NGS) and assembly of contigs, initial phylogenetic analysis using fragments of the cox1 mitochondrial gene identified this specimen as Paralagenidium sp. after nucleotide alignments with sequences obtained from the Barcode of Life Data System (BOLD). However, further analysis of a concatenation of 13 different mitochondrial genes showed that this organism is unique and different from all known oomycetes. A negative PCR result using primers targeting known oomycete pathogens may not be enough to rule out oomycosis in a suspected case. Additionally, the use of a single gene to classify oomycetes may produce misleading results. The advent of metagenomic sequencing and NGS provides a unique opportunity to further explore the diversity of oomycetes as plant and animal pathogens beyond the current capabilities of global barcoding projects that are based on partial genomic sequences.

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.

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.

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Changes in Production Practices Used for Disease Management in Blueberry Nurseries in Georgia, USA, Over a 15-year Period

Surveys of blueberry [rabbiteye blueberry (Vaccinium virgatum) and southern highbush blueberry (Vaccinium corymbosum interspecific hybrids)] nurseries in the State of Georgia, USA, were conducted in 2007 and 2022 to determine the prevalence of and associations among propagation practices, especially related to disease management. As indicated by the reduction in surveyed nurseries in 2022 (7) compared with 2007 (18), the Georgia blueberry nursery industry has consolidated. However, cultural disease management practices have generally improved in these remaining nurseries. In 2007, in nurseries where cuttings were grown in containers, 77.8% reused containers and 66.7% did not sterilize them before use. The growing medium [pine (Pinus sp.) bark] was reused for subsequent production cycles in 29.4% of nurseries, although such reuse of media tended to be associated with production in beds as opposed to containers (P = 0.08). Nurseries used well water in 88.2% and pond water in 11.8% of cases. Cuttings were grown on benches (vs. the ground) in slightly fewer than half of the nurseries. In contrast, all nurseries surveyed in 2022 grew their cuttings in containers, used well water, and had increased bench use, albeit only slightly. Although all nurseries reused containers, only 28.5% did not sterilize containers before use, and only 14.3% of nurseries reported reusing media. Most nurseries surveyed in 2007 (83.3%) were on a routine, calendar-based fungicide program using a.i. targeted primarily against aboveground diseases (blights and leaf spots) and secondarily against soil-borne water molds (Phytophthora and Pythium species of the Oomycetes class). In contrast, 42.8% of those surveyed in 2022 were on a 2- to 3-week spray schedule, 42.8% used fungicides on an as-needed basis, and 14.4% were on a monthly schedule, indicating that fungicide scheduling varied dramatically among the remaining nurseries.

RPA-CRISPR/Cas12a mediated isothermal amplification for visual detection of Phytophthora sojae.

Phytophthora sojae is among the most devastating pathogens of soybean (Glycine max) and severely impacts soybean production in several countries. The resulting disease can be difficult to diagnose and other Phytophthora species can also infect soybean. Accurate diagnosis is important for management of the disease caused by P. sojae. In this study, recombinase polymerase amplification (RPA) in combination with the CRISPR/Cas12a system were used for detection of P. sojae. The assay was highly specific to P. sojae. The test results were positive for 29 isolates of P. sojae, but negative for 64 isolates of 29 Phytophthora species, 7 Phytopythium and Pythium species, 32 fungal species, and 2 Bursaphelenchus species. The method was highly sensitive, detecting as little as 10 pg.µL-1 of P. sojae genomic DNA at 37°C in 20 min. The test results were visible under UV light and readout coming from fluorophores. In addition, P. sojae was detected from natural inoculated hypocotyls of soybean seedlings using this novel assay. The rapidity and accuracy of the method were verified using 30 soybean rhizosphere samples. In conclusion, the RPA-CRISPR/Cas12a detection assay developed here is sensitive, efficient, and convenient, and has potential for further development as a kit for monitoring root rot of soybean in the field.

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.