Pythium Dissotocum Research Articles

Effect of Treatment of Beech Seeds with Copper Nanoparticles on Seed Coat Mycoflora

Seeds harbor a complex fungal community, of which some members may be pathogenic. For this reason, seeds are often treated with fungicides before sowing. Plant protection is constantly looking for environmentally friendly technological solutions and nanotechnology can provide a solution that is a green and environmentally friendly alternative to fungicide treatment. The aim of this study was to conduct a quantitative and qualitative analysis of the composition of fungal communities inhabiting the seed coats of healthy and damaged European beech seeds and to assess the effect of copper nanoparticles (CuNPs) on the composition of these communities. The seeds were soaked in a solution of CuNPs at a concentration of 50 ppm for 30 min. There were four groups of seeds: control—not treated with CuNPs, seeds examined 1 day after CuNP (CuNP_1day) treatment, seeds examined 3 weeks after CuNP (CuNP_3weeks) treatment, and damaged seeds—not treated. A community analysis was conducted based on the ITS1 region using Illumina sequencing. In total, we found 70 taxa of fungi and Oomycota. The community on the control seeds numbered 38 taxa, on damaged seeds—67, on seeds treated with CuNPs after 1 day—40, and after 3 weeks—15, respectively. In terms of biological diversity and species composition, the community on damaged seeds differed significantly from that on CuNP_3weeks seeds and both of these treatments differed from the remaining two. On the damaged seeds, the dominant species were as follows: Botritis cinerea Pers., Globisporangium intermedium (de Bary) Uzuhashi, Tojo & Kakish., and Pythium dissotocum Drechsler. The communities of the other seed groups were dominated by Fusicoccum quercus Oudem. and Apiognomonia errabunda (Roberge ex Desm.) Höhn., which proved resistant to CuNPs. Taxa belonging to the Oomycota, fungi of the genera Fusarium, Mucor, and Penicillium, were sensitive to CuNPs and did not occur on CuNP_3weeks seeds. The significant reduction in the number of taxa and the most favorable structure of trophic groups being found on the CuNP_3weeks seeds suggest that NPs could potentially replace traditional fungicides for seed treatment.

First Report of Pythium dissotocum Causing Root Rot on Nandina in Tennessee and the United States.

Nandina (Nandina domestica) is a popular landscape plant found extensively beyond the southeastern and southwestern parts of the United States (Wilson et al. 2021). In April 2024, one-year-old nandina 'Fire Power' plants grown in containers at a commercial nursery in Warren County, Tennessee, exhibited severe leaf yellowing, stunted growth, and brown to black root rot lesions. Disease severity affected 65% of the root area, with an incidence of 80% of 450 plants. Symptomatic eight plants' root tissues were surface-sterilized with 70% ethanol and washed twice with distilled water. Small sections of root tissues were placed in Petri dishes containing V8 agar (V8A) and incubated at 24°C in an 8-hour photoperiod cycle. White, radial mycelial growth were observed within 3-4 days of incubation. The aseptate hyphae ranged from 1.81 to 7.21 μm in diameter. The filamentous sporangia formed dendroid-like branches. Oogonia were subglobose, measured 15.06 ± 0.65 μm (n=50) in diameter. Oospores were globose and measured 4.58 ± 0.44 μm (n=50). The isolates (FBG7211 and FBG7216) were identified as Pythium dissotocum based on morphological characterization (Van der Plaats-Niterink 1981). For molecular identification, total DNA was extracted using the DNeasy PowerLyzer Microbial Kit (Qiagen, MA) from 7-day-old cultures of the isolates FBG7211 and FBG7216 grown on V8A. The primer pairs ITS1/ITS4 (White et al. 1990), OomCoxI-Levup/Fm85mod (Robideau et al. 2011), Cox2F/Cox2RC4 (Choi et al. 2015) and Oom-Btub-up415/Oom-Btub-lo1401 (Bilodeau et al., 2007) were used to amplify and sequence isolates using four genetic markers, including the ribosomal internal transcribed spacer (ITS), the mitochondrial cytochrome c oxidase subunit I (COX1), cytochrome c oxidase subunit II (COX2), and nuclear beta-tubulin (BT), respectively. The 100% coverage of sequences (GenBank accession nos. PQ329213 and PQ284875 for ITS; PQ349316 and PQ349317 for COX1; PQ349318 and PQ349319 for COX2; PQ349314 and PQ349315 for BT) were 100%, 99.43%, 100%, and 99.56% Identical to P. dissotocum sequences of ITS, COX1, COX2, and BT genetic markers in the GenBank using core nt BLAST search (ITS: MT348554; COX1: MZ027313; COX2: MG719859; BT: KJ595479). Pathogenicity tests were performed on 1-year-old nandina 'Fire Power' seedlings grown in 1-gal containers to fulfill Koch's postulates. Each seedling was drench-inoculated with a 150 mL/plant of 2 x 105 zoospore suspension of the isolates FBG7211 and FBG7216 (five plants per isolate) (Fu and Kim 2024). Control plants were drenched with sterile distilled water. The plants were maintained in a greenhouse at 21 to 23°C and 70% relative humidity with a 16-h photoperiod and were irrigated twice daily for 2 min using an overhead irrigation system. Fourteen days after inoculation, dark brown necrotic lesions developed on the root of all inoculated plants, similar to those observed in the original samples. In contrast, no symptoms were observed in the control plants. Isolates resembling the morphological characteristics of P. dissotocum were recovered from inoculated plants, and their identity was confirmed as P. dissotocum using sequencing. P. dissotocum has been reported to cause root rot in nursery plants (Weiland et al. 2013). However, to our knowledge, this is the first report of root rot on nandina caused by P. dissotocum in Tennessee and the United States. This finding is crucial to develop an efficient disease management strategy of Pythium root rot on nandina.

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.

First Report of Root Rot on Hydroponically Grown Romaine Lettuce Caused by Pythium dissotocum in Korea.

The romaine lettuce (Lactuca sativa L.) is one of the most frequently consumed vegetables in Korea. In January 2023, the romaine lettuce cultured within an indoor hydroponic farm in South Korea displayed severe disease, with an incidence of approximately 13.7% of 300 plants. The diseased plants showed symptoms of stunted growth, lower leaf yellowing, and brown or black-colored soft and mushy root rot, in which the outer layer of root was sloughed off, leaving a thread-like appearance. These symptoms were similar to those of Pythium root rot previously reported to occur on lettuce (McGehee et al., 2018; Stanghellini and Kronland, 1986). Samples of romaine lettuce with rot symptoms were collected from the hydroponic farm. The infected roots were rinsed three times with sterilized distilled water (SDW), dried on sterilized filter paper, and sliced into segments (about 0.5 cm in length), which were placed into Petri dishes (9 cm in diameter) containing V8 juice agar (V8A: 8% V8 juice and 1.5% agar powder) and cultured at 25°C for 2 days. The emerging hyphae were transferred to new Petri dishes containing V8A. After four rounds of sub-culturing, a total of 11 strains were isolated and all of them exhibited the same morphology. Strain KNU2301TP was purified via isolation of a single zoospore and stored at -80°C. The mycelia were non-pigmented. The hyphae obtained from a three-day-old culture grown in V8A were aseptate and the diameter of major hyphae was up to 7 µm. The filamentous sporangia were not to slightly inflated and formed dendroid-like branches. Vesicles containing zoospores were formed on the filamentous sporangia. The encysted zoospores were spherical with a diameter of 8.1 to10 µm (average 8.9 ± 0.6 µm; n = 50). These morphological characteristics of KNU2301TP were similar to those of a previously reported oomycete, Pythium dissotocum (Van der Plaats-Niterink 1981). The genomic DNA was extracted from mycelia cultured on V8A by a previously described extraction method (Chi et al. 2009). Sequences of internal transcribed spacer (ITS) region and cytochrome c oxidase subunit II (cox2) gene were amplified with paired primers ITS1/ITS4 and cox2_F/R, respectively (Callaghan et al., 2022; Hudspeth et al. 2000; White et al. 1990). The resulting sequences were deposited in GenBank (GenBank Accession Nos.; ITS: OQ683867; cox2: OQ700848). The sequences of ITS and cox2 gene of strain KNU2301TP were 99.61% and 100% identical to the sequences of ITS (MG719858) and cox2 gene (MG719859) of P. dissotocum strain YNP-3, respectively. Based on the result of the morphological characterization and sequence analysis, the strain KNU2301TP was identified as P. dissotocum. For Koch's postulates, 15 lettuce seedlings (eighteen-day-old) were inoculated by immersing the roots in a spore suspension (1 × 105 zoospores/ mL) and incubated at 25°C under 16/8 h light/dark cycles for 8 days. Fifteen plants of same age were treated with SDW in the same manner as a control The symptoms resembling those originally found at the farm were developed on the inoculated plants, but not on controls. The strain reisolated from the inoculated plants by same method mentioned above was confirmed as strain KNU2301TP by analysis of morphology and ITS and cox2 sequences. To our knowledge, this is the first report of root rot on hydroponically grown lettuce caused by P. dissotocum in Korea. Root rot on lettuce caused by P. dissotocum has been previously reported in USA, Canada, and Finland (McGehee et al. 2018; Stanghellini and Rasmussen 1994). Since lettuce is an important and popular leafy vegetable around the world, further work would focus on developing efficient strategies to manage this Pythium root rot disease.

Morphological and molecular Characterization of Pythium s.l. species from Khyber Pakthunkhwa province, Pakistan, with some new records and description of Globisporangium ghaffarianum sp. nov.

Pythium s.l. is an ecologically important taxon of the phylum Oomycota that lives in terrestrial and aquatic ecosystems as saprobes. Many species are facultative parasites of plants, causing losses worldwide. Thirty-six putative isolates of the genus from the District Bajaur were characterised morphologically and molecularly during the present studies. Globisporangium ghaffarianum sp. nov. is introduced as a novel taxon. Besides this, G. orthogonon, G. paroecandrum, G. selbyi, G. schmitthenneri, Pythium kashmirense, P. aristosporum, and Phytopythium helicoides are new records for Pakistan. Globisporangium ultimum var. ultimum, Pythium dissotocum, P. catenulatum, P. acanthicum, P. oligandrum, Phytopythium sindhum, Ph. vexans, and Ph. litorale are new records for the province.

Diversity and Pathogenicity of Pythium Species Associated with Reduced Yields of Processing Tomatoes (Solanum lycopersicum) in Victoria, Australia.

Yield decline associated with poor crop establishment, stunting, wilting, and diminished root systems was reported in processing tomato crops in Victoria, Australia. During surveys between 2016 and 2018 Pythium species were isolated by soil baiting and by culturing from the diseased roots and collars of plants exhibiting these symptoms. Eleven species of Pythium were identified based on cultural characteristics and phylogenetic analysis with ITS, Cox-1, and Cox-2 gene sequences. None of the 11 Pythium species had been reported previously from processing or fresh tomatoes in Australia. Pythium dissotocum was the most abundant and widespread species isolated during surveys in each of two growing seasons. In pathogenicity tests, these Pythium species ranged from nonpathogenic to highly aggressive. P. aphanidermatum, P. ultimum, and P. irregulare were consistently the most aggressive species, causing serious damage or death at the pregermination, postgermination, and later stages of plant growth. Five processing tomato cultivars varied significantly in their susceptibility to Pythium disease. These results suggest that Pythium species could be contributing to yield loss in processing tomatoes in Victoria both in the crop establishment phase and through the season.

Peronosporales Species Associated with Strawberry Crown Rot in the Czech Republic.

The symptoms of crown rot on strawberry plants are considered typical for the pathogen Phytophthora cactorum, which causes high losses of this crop. However, an unknown number of related species of pathogens of Peronosporales cause symptoms quite similar to those caused by P. cactorum. To determine their spectrum and importance, strawberry plants were sampled from 41 farms in the Czech Republic. The cultures were isolated from the symptomatic plants using the baiting method, with subsequent cultivation on a semiselective medium. Isolates were identified to the species level using nuclear ribosomal internal transcribed spacer (ITS) barcoding after preliminary morphological determination. In total, 175 isolates of 24 species of Phytophthora, Phytopythium, Pythium, and Globisporangium were detected. The most represented was Phytophthora cactorum, with 113 (65%) isolates, which was recorded in 61% of farms, and the Pythium dissotocum complex with 20 (11%) isolates, which was recorded in 27% of farms. Other species were represented in units of percent. Large differences between farms in the species spectra were ascertained. The differences between species in cardinal growth temperatures and different management of the farms are discussed as a main reason for such a diversification. Regarding the dissimilar sensitivity of various species of Peronosporales against fungicides, the proper determination of the cause of disease is of crucial significance in plant protection.

Characterization and Pathogenicity of Pythium-Like Species Associated with Root and Collar Rot of Kiwifruit in Turkey.

During the period of June to October in 2018, a widespread decline was observed on kiwifruit vines in the vineyards located in the Altınordu, Fatsa, and Perşembe districts of Ordu province in Turkey. The symptoms were associated with reddish-brown rots expanding from the root to the collar with sparse off-color foliage. Based on the percentage of the total infected samples across 18 vineyards, the most common oomycete species were Globisporangium intermedium (37.1%), Phytopythium vexans (34.3%), Globisporangium sylvaticum (14.3%), Globisporangium heterothallicum (11.4%), and Pythium dissotocum (2.9%). The morphological identification of isolates was confirmed based on partial DNA sequences containing the nuclear rDNA internal transcribed spacer region gene and the mitochondrial cytochrome c oxidase subunit II gene. The optimum growth temperature and the optimum pH values of the five species ranged from 22.98 to 28.25°C and 5.67 to 8.51, respectively. Pathogenicity tests on the seedlings of kiwifruit cv. Hayward revealed significant differences in virulence among isolates. Phytopythium vexans and Globisporangium sylvaticum isolates caused severe root and collar rot resulting in seedling death, while Globisporangium heterothallicum and Globisporangium intermedium isolates had relatively lower virulence. All Globisporangium spp. and Phytopythium vexans isolates significantly decreased plant growth parameters (plant height, shoot and root dry weights, and root length); however, Pythium dissotocum caused very mild symptoms and did not affect these parameters of growth. To our knowledge, this is the first study reporting Globisporangium sylvaticum, Globisporangium heterothallicum, and Globisporangium intermedium causing root and collar rot on kiwifruit not only in Turkey but also in the world.

First Report of Root Rot Caused by Pythium dissotocum on Hydroponically Grown Collard Greens (Brassica oleracea var. acephala).

Collards (Brassica oleracea var. acephala) are grown throughout the United States. Hydroponic greens are more common now due to technological advances lowering the cost and increasing the efficacy of production. In January 2021, a 325 m2 indoor hydroponic farm opened to provide fresh produce for a school in Los Angeles County, CA. Three week old collard seedlings were purchased from a local nursery, rinsed of their rooting media, and transplanted into deep water culture beds (1.2 m x 2.5 m x 0.3 m). Two weeks later, symptoms including plant stunting, chlorosis, leaf curling and wilting, and brown necrotic roots appeared. By and by 80-100% of usable plants were lost to disease. Symptomatic roots were plated on corn meal agar (CMA) amended with 2 ml of 25% lactic acid and CMA amended with pimaricin, ampicillin, rifampicin, and pentachloronitrobenzene (PARP) (Kannwischer et al. 1978). After 2 days a single colony type emerged on PARP but no growth occurred on acidified CMA. Representative isolates were transferred to CMA and to filtered (0.02 µm) soil extract solution with boiled grass blades (Martin 1992), both of which were incubated at 22 C and ambient light conditions. On CMA, isolates produced coenocytic mycelium with minimal aerial hyphae. After 24 h in soil extract, isolates developed filamentous sporangia, elongated discharge tubes with slightly inflated tips, and zoospores. Oospores were not observed. Pathogenicity was confirmed by soaking the roots of five day old collard seedlings in beakers containing zoospores (1 x 102 zoospores/ml) in filtered soil extract. Four isolates were tested on 15 seedlings each. After 24 h at 22 C in ambient light conditions, plants were transferred to new beakers with roots placed on filter paper at the bottom and saturated with sterile distilled water. Three days after this transfer, leaves on all plants turned chlorotic and roots developed brown lesions from which morphologically identical colonies were isolated. Control plants, soaked in filtered soil extract, developed no root or foliar symptoms. To molecularly identify the collard isolates, DNA was extracted from mycelial original and re-isolated isolates and was amplified by PCR using mitochondrial primers for the cytochrome oxidase I (COI) gene (Robideau et al. 2011) and the cytochrome oxidase II (COX2) gene (Martin 2000). The only species that matched both loci from the original and re-isolated isolates with a high percent identity was Pythium dissotocum. The COI locus from the original isolate (MZ027311) matched P. dissotocum with 99% identity and with 332/334 base pairs matching the isolate with Sequence ID MT981134.1. From the re-isolated isolate (MZ027313), the COIequence perfectly matched 657/657 base pairs of P. dissotocum (Sequence ID MT981147.1). The COX2 locus from the original isolate (MZ027312) matched P. dissotocum (Sequence ID MG719859.1) with a 99% identity and 517/518 matching base pairs and the re-isolated isolate (MZ027314) perfectly matched P. dissotocum (Sequence ID MG719859.1) with 515/515 matching base pairs. Based on these molecular and morphological data, the isolates were identified as Pythium dissotocum. To our knowledge, this is the first report of P. dissotocum causing root rot on collards. At this same facility, P. dissotocum was also confirmed as the cause of declining bean (Phaseolus vulgaris) plants. As hydroponics will be necessary to feed a growing population - especially in urban areas -- and because leafy greens are a main crop of the hydroponics industry, we anticipate this issue may become common. Hydroponic systems are highly conducive to the persistence of Oomycetes and a record of infection and plan of action will be necessary to preserve crop health.

Methylobacterium sp. 2A Is a Plant Growth-Promoting Rhizobacteria That Has the Potential to Improve Potato Crop Yield Under Adverse Conditions.

A Gram-negative pink-pigmented bacillus (named 2A) was isolated from Solanum tuberosum L. cv. Desirée plants that were strikingly more developed, presented increased root hair density, and higher biomass than other potato lines of the same age. The 16S ribosomal DNA sequence, used for comparative gene sequence analysis, indicated that strain 2A belongs to the genus Methylobacterium. Nucleotide identity between Methylobacterium sp. 2A sequenced genome and the rest of the species that belong to the genus suggested that this species has not been described so far. In vitro, potato plants inoculated with Methylobacterium sp. 2A had a better performance when grown under 50 mM NaCl or when infected with Phytophthora infestans. We inoculated Methylobacterium sp. 2A in Arabidopsis thaliana roots and exposed these plants to salt stress (75 mM NaCl). Methylobacterium sp. 2A-inoculated plants, grown in control or salt stress conditions, displayed a higher density of lateral roots (p < 0.05) compared to noninoculated plants. Moreover, under salt stress, they presented a higher number of leaves and larger rosette diameter. In dual confrontation assays, Methylobacterium sp. 2A displayed biocontrol activity against P. infestans, Botrytis cinerea, and Fusarium graminearum, but not against Rhizoctonia solani, and Pythium dissotocum. In addition, we observed that Methylobacterium sp. 2A diminished the size of necrotic lesions and reduced chlorosis when greenhouse potato plants were infected with P. infestans. Methylobacterium sp. 2A produces indole acetic acid, solubilizes mineral phosphate and is able to grow in a N2 free medium. Whole-genome sequencing revealed metabolic pathways associated with its plant growth promoter capacity. Our results suggest that Methylobacterium sp. 2A is a plant growth-promoting rhizobacteria (PGPR) that can alleviate salt stress, and restricts P. infestans infection in potato plants, emerging as a potential strategy to improve crop management.

Pathogens and Molds Affecting Production and Quality of Cannabis sativa L.

Plant pathogens infecting marijuana (Cannabis sativa L.) plants reduce growth of the crop by affecting the roots, crown, and foliage. In addition, fungi (molds) that colonize the inflorescences (buds) during development or after harvest, and which colonize internal tissues as endophytes, can reduce product quality. The pathogens and molds that affect C. sativa grown hydroponically indoors (in environmentally controlled growth rooms and greenhouses) and field-grown plants were studied over multiple years of sampling. A PCR-based assay using primers for the internal transcribed spacer region (ITS) of ribosomal DNA confirmed identity of the cultures. Root-infecting pathogens included Fusarium oxysporum, Fusarium solani, Fusarium brachygibbosum, Pythium dissotocum, Pythium myriotylum, and Pythium aphanidermatum, which caused root browning, discoloration of the crown and pith tissues, stunting and yellowing of plants, and in some instances, plant death. On the foliage, powdery mildew, caused by Golovinomyces cichoracearum, was the major pathogen observed. On inflorescences, Penicillium bud rot (caused by Penicillium olsonii and Penicillium copticola), Botrytis bud rot (Botrytis cinerea), and Fusarium bud rot (F. solani, F. oxysporum) were present to varying extents. Endophytic fungi present in crown, stem, and petiole tissues included soil-colonizing and cellulolytic fungi, such as species of Chaetomium, Trametes, Trichoderma, Penicillium, and Fusarium. Analysis of air samples in indoor growing environments revealed that species of Penicillium, Cladosporium, Aspergillus, Fusarium, Beauveria, and Trichoderma were present. The latter two species were the result of the application of biocontrol products for control of insects and diseases, respectively. Fungal communities present in unpasteurized coconut (coco) fiber growing medium are potential sources of mold contamination on cannabis plants. Swabs taken from greenhouse-grown and indoor buds pre- and post-harvest revealed the presence of Cladosporium and up to five species of Penicillium, as well as low levels of Alternaria species. Mechanical trimming of buds caused an increase in the frequency of Penicillium species, presumably by providing entry points through wounds or spreading endophytes from pith tissues. Aerial distribution of pathogen inoculum and mold spores and dissemination through vegetative propagation are important methods of spread, and entry through wound sites on roots, stems, and bud tissues facilitates pathogen establishment on cannabis plants.

Efficacy of biofungicides against root rot and damping-off of microgreens caused by Pythium spp.

Pythium spp. are the causal agents of Pythium root rot and damping-off on microgreens. The objective of this project was to assess the efficacy of biofungicides on Pythium root rot and damping-off caused by Pythium aphanidermatum and Pythium dissotocum on microgreens in greenhouses. In the first experiment, arugula (Eruca sativa Mill.), kale (Brassica oleracea var. sabellica L.), radish (Raphanus raphanistrum subsp. sativus L.), and mustard (Brassica juncea L. Czern) microgreens were treated with Companion® (Bacillus subtilis GB03), Triathlon BA® (Bacillus amyloliquefaciens D747), or RootShield Plus® (Trichoderma harzianum KRL-AG2 and Trichoderma virens G-41) in a hydroponic nutrient film technique system. Two days after treatment, the plants were inoculated with 3 × 105 zoospores of Pythium spp. After seven days, we measured root necrosis, damping-off incidence and severity, and plant biomass. All plants infected with Pythium spp. were smaller by 28% or more compared with non-inoculated plants. Overall disease was low, but biomass was lower in all treatments inoculated with Pythium spp. Arugula infected with Pythium spp. and treated with Triathlon BA® resulted in 8% lower disease incidence compared with the positive control, yet Triathlon BA® resulted in the highest root necrosis. On a separate experiment, arugula and mustard were grown in propagation trays, irrigated manually, and treated with the biofungicides mentioned above or Cease® (Bacillus subtilis QST 713). Arugula and mustard plants inoculated with Pythium spp. had 74.4% reduction of shoot dry weight. Arugula and mustard treated with Cease®, with and without Pythium spp., resulted in ≥59% more biomass compared with the untreated inoculated control. In the tray experiment, all the infected plants treated with biofungicides had more biomass than plants with no biofungicides. Results from this experiment suggest that microbial biofungicides can be introduced in nutrient solutions in nutrient film technique or applied in the irrigation to prevent Pythium root rot and damping-off in brassica microgreens. However, biofungicides can reduce plant biomass and growers may need to extend production time to achieve target yields.

Fusarium and Pythium species infecting roots of hydroponically grown marijuana (Cannabis sativa L.) plants

An increase in the cultivation of Cannabis sativa (cannabis or marijuana) plants in Canada is becoming associated with increased incidence and severity of various diseases, many of which have not been previously reported. In this study, hydroponically grown C. sativa plants were sampled over a 3-year period (2014–2017) to determine the prevalence of root pathogens. Following isolation, pathogenicity studies were conducted to establish the extent of disease symptoms caused by the recovered microbes. Root rot was found to be caused by two Pythium species – Pythium dissotocum Drechsler and P. myriotylum Drechsler. As well, two Fusarium species were recovered from diseased plants – Fusarium oxysporum Schlecht. emend. Snyder & Hansen and F. solani (Mart.) Sacc. Upon inoculation onto healthy plants, all isolates of Pythium spp. caused browning and a reduction in root mass, accompanied by stunting. Inoculation of plants with F. oxysporum caused browning of roots and crown rot infection, accompanied by pith and vascular discolouration, and in some cases wilting of plants, while root and crown infection was observed with F. solani. Phylogenetic analysis of internal transcribed spacer (ITS) and elongation factor 1α (EF-1α) sequences revealed that the Fusarium species affecting cannabis plants shared 99–100% sequence homology with isolates causing stem rot and wilt in other hosts, including cumin and tomato, suggesting they were not uniquely adapted to cannabis. The potential for spread of F. oxysporum through the hydroponic system was confirmed by its detection in the recirculating nutrient solution. Furthermore, rooted cuttings obtained from commercial propagators were found to harbour Fusarium root infection that resulted in subsequent stunting, yellowing and occasional death of plants. This demonstrates the potential for long-distance spread of the pathogen. The two Pythium species recovered from cannabis plants have an extremely broad host range and are not unique to this host. An additional species, P. aphanidermatum (Edson) Fitzp., was recovered from diseased plants grown under greenhouse conditions in 2018. The management of these root pathogens on C. sativa will require the evaluation and implementation of sanitization methods, biological control agents, and chemical products adapted from greenhouse vegetable production practices. The use of pathogen-free propagation materials and identification of potential sources of disease resistance should also become a priority.

First Report of Pythium dissotocum Causing Pythium Root Rot on Hydroponically Grown Lettuce in Connecticut

In August 2016, lettuce (Lactuca sativa L.) plants presented root rot, severe wilting, and high incidence of mortality in New Haven County, CT. The plants were grown in a hydroponic deep-water culture system in a commercial greenhouse. Mature plants were stunted, the lower leaves were chlorotic, the roots were necrotic, and some plants collapsed. Lettuce roots were collected from several symptomatic plants, washed three times with sterile deionized water, blotted dry, and plated on PARP-V8 selective medium (0.005 g of pimarcin, 0.25 mg of ampicillin, 0.01 g of rifampicin, 0.10 g of pentachloronitrobenzene, 200 ml of clarified V8 juice, and 800 ml of distilled water) (Moorman et al. 2002). The plates were incubated in the dark at 21°C. Mycelium resembling the morphology of Pythium species was observed in all plates after 3 days. The isolate was identified as Pythium dissotocum based on the morphology of sexual and asexual structures following van der Plaats-Niterink’s key (van der Plaats-Niterink 1981). Colonies grown on V8-juice agar had filamentous sporangia with a dendroid structure, the oogonia were 23 μm in diameter and subglobose, the antheridia developed on unbranched stalks and were sessile, and the oospores were aplerotic. DNA was extracted from mycelial mats using a DNeasy Plant Mini Kit (Qiagen, U.S.A.), and the internal transcribed spacer (ITS) region was amplified using universal primers ITS1 and ITS4 (White et al. 1990). BLAST nucleotide analyses against publicly available Pythium sequences in GenBank confirmed that the isolates obtained were P. dissotocum with 97% query coverage and 99% identity match to GenBank accession number KM061701.1. The nucleotide sequence has been assigned the GenBank accession number MG993546. To confirm Koch’s postulates, 14-day-old lettuce seedlings, cultivar Rex (n = 25), were inoculated with 1 × 10⁵ zoospores of the isolate of P. dissotocum produced on V8 broth. After 7 days, symptoms appeared as reduced growth, necrotic roots, and chlorotic leaves. Control plants were symptomless and healthy. Roots were collected from each infected lettuce plant, and four root fragments were plated on PARP-V8 selective medium. Mycelial growth resembling Pythium was observed in all plates from inoculated plants, and no growth was observed from roots of noninoculated plants (n = 25, 1 plate per plant). The putative P. dissotocum was reisolated from the plates; DNA was extracted from mycelial mats using the same methods described above and was confirmed to be Pythium using primers ITS1 and ITS4. The amplicon was the same size as for the P. dissotocum recovered from the original plants. This is the first report of P. dissotocum being detected on lettuce in Connecticut. P. dissotocum reduces crop yield and was first reported in 1986 as a root pathogen on hydroponic lettuce in Arizona (Stanghellini and Kronland 1986). There are currently no registered synthetic-chemical fungicides in the United States for the suppression of Pythium species in hydroponic lettuce (Utkhede et al. 2009). Prevention of this disease is recommended by controlling temperature and salt levels in the nutrient solution, sanitizing surfaces, and applying preventative biological fungicides registered for Pythium root rot.

English

Common beans (Phaseolus vulgaris L.) is the main leguminous crop grown primarily by small-holder farmers in the East and South African countries. Pythium root rot disease is the major production constraints which results in yield losses of 70% to most commercial bean cultivars in eastern Africa. Study focused on ascertaining preliminary information on bean cultivation practices in Tanzania, morphological and molecular characterization and identification of Pythium species from infected beans plants and determining the relationship between soil pH and the occurrence and distribution of the Pythium spp. Soil samples and infected bean plants were collected by aseptic pathogenic isolation and DNA extraction. Universal primers (ITS1 and ITS4) were used for amplification and followed by sequencing. About 63.0% of farmers practiced sole beans cropping, 31.0% mixed cropping and 6.0% intercropping. Corn, banana, cassava, Irish potatoes and coffee were either mixed or intercropped with beans. Also, 52.4% of farmers use farm saved seeds and 92.9% do not use fertilizer in their bean fields. Eleven species of the Pythium spp. were identified: Pythium aphanidermatum, Pythium splendens, Pythium ultimum, Pythium attrantheridium, Pythium graminicola, Pythium oligandrum, Pythium dissotocum, Pythium irregurale, Pythium camurandrum, Pythium paroecandrum and Pythium acanthophoron. Phylogenetic analysis showed diversity and homogenity among the Pythium spp. across the collection area. A high incidence and wide distribution of Pythium species were recorded in soils in the 5.03 to 5.95 pH range. Key words: Incidence, internal transcribed spacer (ITS), leguminous, molecular characterization of pathogen.

The Concentration of 2-Propenyl Glucosinolate in Biofumigant Crops Influences Their Anti-Fungal Activity (In-Vitro) against Soil-Borne Pathogens

This study investigated the biofumigation potential of nine Brassica species/cultivars by determining the levels of 2-propenyl glucosinolate in their roots and shoots, and their in-vitro suppression of four major soil-borne pathogens of vegetable crops. Hydrolysis of 2-propenyl GSL produces volatile isothiocyanate (ITC) compounds which are known to have anti-fungal activity. HPLC results showed that 2-propenyl GSL only occurred in root and shoot residue of flowering plants of four Brassica cultivars developed for green manuring (Caliente 199?, Mustclean?, Nemfix? and BQ Mulch?) and in the standard (mustard seed meal) treatment Fumafert?. Levels of 2-propenyl GSL varied several fold within the four Brassica cultivars, with 77-88% of the total concentrations recorded in the shoot tissues. In in vitro assays, the level of fungal suppression by volatiles emitted by hydrated shoot and root residues related to their content of 2-propenyl GSL, and the dose of residue applied to five soilborne test pathogens (S. minor, Rhizoctonia solani, Fusarium oxysporum, Pythium dissotocum and Rhizoctonia solani). The variation in 2-propenyl GLS levels found in the Brassica green manure crops tested provides scope for selecting cultivars with greater potential for biofumigation, and to control multiple soil-borne disease problems in vegetable farms.

Studies of Aquatic Fungi. XXIV. Aquatic Fungi in the Water of Melting Snow

The work was undertaken to investigate the mycoflora in the water of melting snow. Samples of water were collected in March 1987-1988 for hydrochemical analysis (3 sites) and studies of the fungus content (9 sites). Forty-nine species of fungi were found in this waters. The following fungi unknown from Poland were found: &lt;i&gt;Skirgiella septigena, Monoblepharis macraodra, M. polymorpha, M. fascicutlta, M. insignis, Achlya apiculata, Apodachlya punctata, Pythium dissotocum, Hansenula holstii, H. saturnus, Actiaospora megalospora&lt;i&gt; and &lt;i&gt;Heliscus lugdunensis&lt;/i&gt;.

Sensitivity of Pythium irregulare, P. sylvaticum, and P. ultimum from Forest Nurseries to Mefenoxam and Fosetyl-Al, and Control of Pythium Damping-off.

Mefenoxam and fosetyl-Al are common fungicides used to supplement disease control of Pythium damping-off and root rot in forest nurseries of the western United States. However, it is unknown whether fungicide-resistant Pythium isolates are present or whether new fungicide and biological treatments might also provide supplemental disease control. Isolates of Pythium irregulare, P. sylvaticum, and P. ultimum from three forest nurseries were evaluated for in vitro sensitivity to mefenoxam and fosetyl-Al. A greenhouse study was also conducted to assess efficacy of fungicide and biological treatments in Douglas-fir (Pseudotsuga menziesii) seedlings inoculated with Pythium dissotocum, P. irregulare, and P. 'vipa'. P. irregulare was approximately three times less sensitive to mefenoxam (0.20 μg/ml) than P. sylvaticum (0.06 μg/ml) and P. ultimum (0.06 μg/ml), and two resistant isolates of P. ultimum were identified (≥311 μg/ml). All three Pythium spp. were similarly sensitive to fosetyl-Al (1,256 to 1,508 μg/ml) and no resistant isolates were found. In the disease control efficacy trial, both fosetyl-Al and phosphorous acid consistently provided good protection against damping-off caused by P. dissotocum, P. irregulare, and P. 'vipa'. Other treatments, including mefenoxam, also provided good or intermediate protection but efficacy depended upon which Pythium sp. was used. Growers should consider rotating mefenoxam use with other fungicide chemistries or biological treatments to prevent further development and spread of mefenoxam-resistant isolates.

Components of different signalling pathways regulated by a new orthologue of AtPROPEP1 in tomato following infection by pathogens

Plants express different defence mechanisms in response to pathogens. Understanding the recognition of pathogen‐associated molecular patterns (PAMPs) by specific receptors, and the role of endogenous signals such as AtPep1 that regulate expression of genes in Arabidopsis thaliana, has aided the understanding of the defence mechanisms in different species. The aim of this study was to identify possible orthologous sequences of AtPROPEPs in tomato (Solanum lycopersicum) and characterize its role in resistance to necrotrophic pathogens. The presence of an orthologue of the A. thaliana AtPROPEP1 gene in S. lycopersicum, SlPROPEP, by in silico analysis, is reported here. This has 96% identity with the C‐terminal region of a previously described potato peptide, another possible orthologue of AtPep1. A virus‐induced gene silencing (VIGS) system was employed to investigate the role of the SlPROPEP. Silencing of SlPROPEP in tomato made plants more susceptible to Pythium dissotocum; approximately 30% of SlPROPEP‐silenced plants showed stem constriction compared with 4% in control plants. Furthermore, quantification of P. dissotocum by qPCR revealed that the increase in symptom severity in SlPROPEP‐silenced plants was associated with a 15 times increase in growth of the pathogen compared to control plants. Silencing of SlPROPEP also resulted in decreased expression of genes involved in plant defence against pathogens, such as PR‐1, PR‐5, ERF1, LOX‐D and DEF2. These results suggest that SlPROPEP is involved in tomato resistance to P. dissotocum and probably acts as a pathogen‐associated molecular pattern through signalling pathways mediated by jasmonic acid/ethylene (JA/ET).

First Report of Pythium dissotocum Affecting Cilantro in Hydroponic Systems in Puerto Rico

Pythium dissotocum was detected on cilantro in hydroponic systems in Puerto Rico. Isolates from stem lesions were inoculated in cilantro ‘Lonestar’ and in pea ‘Sugar Baby,’ producing soft rot 21 and 15 days after inoculation, respectively. The pathogen was re-isolated from diseased plants and it was confirmed to be Pythium dissotocum by PCR. Accepted for publication 5 December 2012. Published 14 December 2012.