<Emphasis Type="Italic">Pythium apinafurcum</Emphasis>: its morphology,molecular phylogeny,and infectivity for plants

During a survey of Pythium species in soils of Japan, Pythium isolates growing at high temperatures were obtained from an uncultivated field soil in Wakayama Prefecture. All six isolates showed similar morphology to each other and had complexly branched secondary hyphae, globose nonproliferating sporangia, and smooth-surfaced oogonia that have one or two oospores per oogonium. The combination of these characteristics differentiated these isolates from other Pythium species reported. Phylogenetic analyses based on sequences of the ribosomal DNA ITS and D1/D2 region of the large subunit showed that all Pythium isolates were clustered in a single clade that was distantly related to other known clades of the genus. We described these isolates as a new Pythium species, Pythium apinafurcum, based on morphology and molecular phylogeny. The P. apinafurcum isolates nonsymptomatically infected the roots of seedlings of bermudagrass, cabbage, and cucumber in a pot inoculation test.     

Morphological and phylogenetic analyses of Pythium species in South Africa

The genus Pythium is important in agriculture, since it contains many plant pathogenic species, as well as species that can promote plant growth and some that have biocontrol potential. In South Africa, very little is known about the diversity of Pythium species within agricultural soil, irrigation and hydroponic systems. Therefore, the aim of the study was to characterise a selection of 85 Pythium isolates collected in South Africa from 1991 through to 2007. The isolates were characterised morphologically as well as through sequence and phylogenetic analyses of the internal transcribed spacer regions (ITS) and the 5.8S gene of the nuclear ribosomal DNA. Phylogenetic analyses showed that the isolates represented ten of the 11 published Pythium clades [Lévesque & De Cock, 2004. Molecular phylogeny and taxonomy of the genus Pythium. Mycological Research 108: 1363–1383]. Characterisation of isolates in clade D and J suggested that the phylogenetic concept of Pythium acanthicum and Pythium perplexum respectively, needs further investigation in order to enable reliable species identification within these clades. Our phylogenetic analyses of Pythium species in clade B also showed that species with globose sporangia group basal within this clade, and are not dispersed within the clade as previously reported. The 85 South African isolates represented 34 known species, of which 20 species have not been reported previously in South Africa. Additionally, three isolates (PPRI 8428, 8300 and 8418) were identified that may each represent putative new species, Pythium sp. WJB-1 to WJB-3.     

Characterization and Identification of Asexual Strains of Pythium Associated with Root Rot of Rose in Japan

This study was conducted to survey the distribution of asexual isolates of Pythium in rose production and to characterize and identify them. Asexual isolates with proliferating globose sporangia belong to group P according to the key of van der Plaats‐Niterink (1981; Monograph of the genus Pythium. Studies in Mycology, Vol. 21, Centraalbueau Voor Schimmelcultures, Baarn, The Netherlands). Group P isolates were recovered from rotted roots of both cutting and miniature roses cultured in rock wool and ebb‐and‐flow culture systems, respectively, throughout the main rose production area of Japan. The typical feature of the P group isolates was that they could grow fast at high temperature, at least 30 mm per 24 h at 35°C. There was no difference between the P group isolates and P. helicoides in morphology and size of sporangia and sporangial germination mode. The symptoms caused by the group P isolates were root rot, followed by leaf blight and plant death in severe cases. In restriction fragment length polymorphism analysis of the rDNA‐ITS region, the banding patterns with five of six enzymes were identical between group P and P. helicoides, the only difference being seen with HhaI. In direct amplification analysis of minisatellite‐region DNA with M13 primer, group P and P. helicoides shared three of five distinct bands. In contrast, P. oedochilum and P. ostracodes showed different banding patterns except for each one band. The results suggest that the group P isolates obtained from rose root rot may be asexual strains of P. helicoides.     

Characterization of Pythium spp. associated with root rot of tobacco seedlings produced using the float tray system in Zimbabwe

The study was undertaken to identify and characterize Pythium isolates associated with root rot disease of tobacco seedlings as a first step towards developing management strategies for the pathogen. A total of 85 Pythium isolates were collected from diseased tobacco seedlings during 2015–2016 tobacco growing season. The isolates were identified to species level using sequencing of the internal transcribed spacer region. Thereafter, a subset of the isolates was tested for sensitivity to the commonly used fungicides, metalaxyl, azoxystrobin and a combination of fenamidone/propamocarbby growing isolates on Potato Dextrose Agar plates amended with the fungicides. The sequence analysis of the ITS‐rDNA identified Pythium myriotylum as the dominant Pythium species associated with the root rot of tobacco seedlings in Zimbabwe. Pythium aphanidermatum and P. insidiosum were also identified albeit at lower frequencies. Phylogenetic analyses of the ITS region of the P. myriotylum isolates showed little sequence diversity giving rise to one distinct clade. The fungicide sensitivity tests showed that metalaxyl provided the best control of P. myriotylum in vitro, as compared to other fungicides. To the best of our knowledge, this is the first comprehensive study to determine and characterize Pythium species associated with root rot of tobacco in the float seedling production system in Zimbabwe.     

Six new species of Pythiogeton in Taiwan,with an account of the molecular phylogeny of this genus

Pythiogeton is a little-studied genus of pythialean Oomycete. The genus is characterized by producing its zoospores outside of the sporangium within an apparently naked protoplasmic mass, which formed from a discharge tube-vesicle complex. A total of nine morphologically distinct Pythiogeton species were identified, of which six were new species (Pythiogeton abundans, Pythiogeton microzoosporum, Pythiogeton oblongilobum, Pythiogeton paucisporum, Pythiogeton proliferatum, and Pythiogeton puliensis). A phylogenetic analysis based on internal transcribed spacer sequences revealed that all isolates of Pythiogeton formed a highly supported clade, nested within the wider clade of Pythium species. Each newly recognized Pythiogeton species that was established on the basis of morphological characters was found to occur in a well-supported subgroup within the Pythiogeton clade, confirming their assignment to new species. Pythiogeton shares a common ancestor with the monophyletic group of Pythium species that have predominantly filamentous sporangia rather than with the separate clade of Pythium species that have predominantly globose or ovoid sporangia. This study confirms that Pythium is an extremely heterogenous and polyphyletic genus containing a number of distinct clades of species, including Pythiogeton, which possess morphologically distinguishable characters. A synoptic key to all the described Pythiogeton species is provided.     

Pythium intermedium,a species complex consisting of three phylogenetic species found in cool-temperate forest ecosystems

Pythium intermedium plays a vital role in the carbon cycle of cool-temperate forests and is widely distributed in Japan's forest soils. In this study, we performed a phylogenetic analysis of the P. intermedium species complex using DNA sequences from multiple loci. The study included 35 isolates from cool-temperate forest soils, seven known P. intermedium isolates, and six known Pythium attrantheridium isolates. We also performed morphological observations and mating tests. Our results showed that all the isolates formed one large clade but were divided into three subclades. Furthermore, we observed many mating reactions between isolates from different subclades, including between P. attrantheridium and P. intermedium. Therefore, we suggest that P. intermedium, P. attrantheridium, and another phylogenetic species belong to one species complex. This is the first report of a species complex within P. intermedium and will be helpful in understanding the evolution of Pythium species in natural ecosystems.     

Oligonucleotide Array for Identification and Detection of Pythium Species

A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies.     

Biocontrol of <Emphasis Type="Italic">Pythium</Emphasis> damping-off in cucumber by arbuscular mycorrhiza-associated bacteria from the genus <Emphasis Type="Italic">Paenibacillus</Emphasis>

The Pythium biocontrol features of 17 Paenibacillus strains, all previously isolated from the rhizosphere, hyphosphere or bulk soil from mycorrhizal and non-mycorrhizal cucumber plants, were examined using a cucumber seedling emergence bioassay. Thirteen strains – four strains of Paenibacillus polymyxa, eight strains of P. macerans and one strain of Paenibacillus sp. – significantly increased the percentage of seedling emergence of seeds inoculated with agar plugs of Pythium aphanidermatum FC42. Overall, the efficacy of Pythium biocontrol did not seem to differ between isolates of Paenibacillus originating from either mycorrhizal or non-mycorrhizal systems. No strains significantly reduced the damping-off incidence caused by the aggressive isolate Pythium sp. B5. Two strains of P. macerans not only reduced the incidence of pre-emergence damping-off by 73%, but they also counteracted the plant growth-depressing effect of P. aphanidermatum FC42, so that 68–82% of the emerged seedlings remained healthy 7 days after sowing. Two strains of P. macerans and one strain of P. polymyxa also significantly increased the percentage of seedling emergence following inoculation with approximately 10⁵ zoospores of P. aphanidermatum FC42. There was no significant difference between the dry weight of three selected bacteria-inoculated and -uninoculated plants in the absence of Pythium; however, the dry weight of bacteria-inoculated plants was significantly higher than that of the uninoculated control plants with bacteria in the presence of P. aphanidermatum FC42.     

Genetic and phenotypic analyses of Pythium isolates from reed suggest the occurrence of a new species,P. phragmiticola,and its involvement in the generation of a natural hybrid

A set of isolates closely related to the reed pathogen Pythium phragmitis was found among Pythium strains obtained from reed (Phragmites australis) soil and plant samples in Germany and Switzerland. These isolates consistently differed from P. phragmitis at 6, 6 and 3 nucleotide positions in the rDNA internal transcribed spacer region, the β-tubulin, and the cytochrome oxidase II gene, respectively. They are formally described here as Pythium phragmiticola sp. nov. An analysis of phenotypic traits like oospore size, growth and aggressiveness to reed showed that P. phragmiticola is almost indiscernible from P. phragmitis, as most features assessed overlapped considerably between species. Phylogenetic analyses provided evidence that P. phragmiticola is the second parent of a previously described Pythium hybrid involving P. phragmitis. The alloploid status of the hybrid, and the parents' reproductive separation was confirmed by molecular evidence and the high ratios of aborted oospores. In natural environments there obviously are two species closely associated with reed, P. phragmitis and P. phragmiticola which are homothallic, but can outcross, resulting in sterile, vegetatively propagated interspecific hybrids. Driving forces leading to the radiation of these two species are unknown, but might be connected with divergent parasitic adaptation to different host tissues or species.     

Morphological and molecular characterisation of Pythium species causing chilli (Capsicum annuum L.) damping-off

Twenty-five Pythium isolates comprising five species viz., Pythium aphanidermatum, P. deliense, P. graminicola, P. heterothallicum and P. ultimum from different geographical locations of Tamil Nadu (Coimbatore, 4; Cuddalore, 6; Dindigul, 1; Dharmapuri, 1; Erode, 1; Madurai, 1; Namakkal, 7; Thanjavur, 1; Theni, 1; Thirunelveli, 1 and Vellore, 1) isolated from chilli crop were analysed with randomly amplified polymorphic DNA (RAPD) markers. Morphological and molecular characteristics of these different species were correlated with the RAPD. Polymerase chain reaction amplification of total genomic DNA with six random primers generated unique banding patterns depending on the primer and the isolate. The isolate I₁₇ produced identical banding patterns, while other isolates produced dissimilar bands within the particular species, indicating the genetic diversity among the isolates within a species. Morphological characters were also different from each other even in isolate I₁₇ which shared identical bands. Cluster analysis showed minimum and maximum per cent similarities among the tested Pythium species which ranged from 49 to 89%, respectively. RAPD markers were better suited for differentiating isolates within a species rather than species.     

Variation in the sensitivity to metalaxyl of Pythium spp. isolated from carrot and other sources

Over a 3-yr period 261 isolates of 17 species of Pythium were tested for sensitivity to metalaxyl at concentrations of 5, 50 or 100 μ/ml. A wide range of responses was observed, from isolates where growth ceased at 5 μg/ml to those where growth at 100 μg/ml was similar to that of the untreated controls. In further tests isolates of 11 different species had ED50's < 1 μg/ml. A lower sensitivity was detected in isolates of six Pythium spp. where values in the range 1–10 μg/ml were obtained. This lower sensitivity was not related to previous known use of metalaxyl. Three isolates of Pythium dissotocum from sites where the fungicide had been used repeatedly had ED50's > 100 μg/ml and were considered resistant. The resistance was stable over a 2-yr period and isolates were cross-resistant to furalaxyl, benalaxyl, ofurace, cyprofuram and oxadixyl. Increasing concentrations of metalaxyl reduced or prevented the production of zoospores by four species of Pythium, although when zoospores were produced, this was followed by the normal processes of encystment and germination. Culturing P. dissotocum on different sub-lethal concentrations of metalaxyl for 18 wk did not induce a high level of resistance to the fungicide.     

Analysis of polyphenols,phytosterols, and bitter principles in <Emphasis Type="Italic">Teucrium</Emphasis> L. species

Total polyphenols, tannins, β-sitosterol, and bitterness values were determined in native and cultivated populations of Teucrium L. species from Croatia (T. arduini L., T. botrys L., T. chamaedrys L., T. flavum L., T. montanum L., T. polium L., and T. scordium L. subsp. scordioides Schreb.). Teucrium species have long been present in folk medicine for diverse medicinal uses, but little is known about polyphenols, phytosterols, and bitter principles. Spectrophotometrically determined contents of total polyphenols (TP) and tannins (T) significantly varied among investigated Teucrium species and were somewhat higher in native populations. The highest TP and T contents were measured in native specimens of T. montanum (TP: 13.68%; T: 3.48%). Scanning densitometry was used for measurements of β-sitosterol levels in plant samples ranged from 0.056% (T. montanum) to 0.129% (T. botrys), and it was not significantly different between native and cultivated specimens of the same plant species. Bitterness values were similar for native and cultivated samples of the same plant species; the highest was measured for T. montanum (15659). The present study suggests that Teucrium species growing in Croatia have potential for cultivation and might be a valuable source of natural bioactive compounds.     

Pythium and Phytopythium species associated with weeds collected in vegetable production fields in Brazil

This study aimed to identify Pythium and Phytopythium species from weeds collected in vegetable fields and test their pathogenicity. Weeds with symptoms of damping-off, root rot or wilt were sampled in the Brazilian states of Ceará, Goiás and Pernambuco, as well as in the Distrito Federal, for isolation and identification of the causal agents. Once isolated, colonies with typical Pythium and Phytopythium characteristics grew in selective V8 medium. Procedures for species identification included morphology and amplification of the ITS and Cox II regions, which were compared with other accessions available at GenBank. The phylogenetic relationships among the isolates and pathogenicity to their original hosts were evaluated. Six Pythium species were identified: P. aphanidermatum, P. oopapillum, P. orthogonon, P. ultimum var. ultimum, P. myriotylum and P. sylvaticum, and two species of Phytopythium, Phy. chamaehyphon and Phy. oedochilum. In the pathogenicity tests, the 10 weed hosts showed symptoms of damping-off or root rot after inoculation, with exception of Portulaca oleraceae in which none of the isolates was pathogenic. Therefore, common weeds in vegetable fields areas can host different Pythium and Phytopythium species and play an important role in the epidemiology of vegetable diseases, in particular on pathogen survival and population increase.     

Effects of Pythium species on the growth of apple and their possible causal role in apple replant disease

Several lines of circumstantial evidence collectively indicated that poor early growth of apple (‘replant disease’) might be associated with the effects of soil-borne pythiaceous fungi. This hypothesis was supported by pathogenicity tests. All isolates tested of P. sylvaticum and certain isolates of seven other Pythium spp. significantly reduced the growth of apple seedlings. The growth reductions caused by certain Pythium isolates were of comparable magnitude to the growth increases occurring after chloropicrin-fumigation of apple orchard soils. The Pythium isolates most virulent to apple were of low virulence to a clonal cherry rootstock. Reappraisal of the nature of the disease as a non-specific soil malaise is consistent with established features of the pathology of Pythium spp. The disease, however, is an ill-defined ‘poor growth phenomenon’ with no diagnostic symptoms and conclusive evidence that Pythium spp. are widely causal is likely to be elusive.     

Effects of Nitrogen, Plant Density, Moisture Stress and Artificial Inoculation with Macrophomina phaseolina on Charcoal Rot Incidence in Grain Sorghum

The occurrence of pythiaceous fungi in pot plant cultures grown in ebb and flow bench systems was investigated monthly from May to December, Phytophthora, Pythium and Saprolegnia (in all 351) were isolated from water samples and identified. Nearly all the isolates of Pythium produced zoospores in water. A pathogenicity test involving 15 isolates of Pythium“group P”, and 7 of Pythium“group F” showed that 73 % were pathogenic on cucumber, 66 % on Gerbera, 59 % on lettuce, 50 % on tomato, and 32 % on cress. Control of Pythium and Phytophthora is important in order to improve the health of plants grown in ebb and flow systems.     

Two new species,Pythium agreste and P. wuhanense,based on morphological characteristics and DNA sequence data

In an investigation of Pythium species in China, two new species, P. agreste and P. wuhanense, were identified based on morphological characteristics and DNA sequence data. Pythium agreste has slightly inflated sporangia, oogonia encompassed by antheridia and antheridial stalks forming a very complicated knot, and plerotic oospores. It differs from the morphologically similar P. volutum which has inflated sporangia, bigger oogonia, and aplerotic oospores; from P. kashmirense which has contiguous inflated sporangia, catenulate oogonia and coiled or bent oogonial stalks; and from P. pectinolyticum which has catenulate oogonia and bigger oogonia and oospores. Pythium wuhanense can be differentiated from morphologically similar P. emineosum and P. paroecandrum by its possession of intercalary cylindrical to elongated sporangia and intercalary oogonia catenulate with sporangia and antheridia. Phylogenetic analyses showed that these two new species were clearly separated from morphologically similar Pythium species, based on the internal transcribed spacer (ITS) region and cytochrome C oxidase subunit I (COI) gene sequences using maximum parsimony and Bayesian methods. The two new species are described and illustrated in detail.     

Phylogenetic relationships among Phytopythium species,and re-evaluation of Phytopythium fagopyri comb. nov., recovered from damped-off buckwheat seedlings in Japan

We examined the phylogenetic relationships among Phytopythium species using the rDNA ITS region, the LSU rDNA region, and the mitochondrial coxI and coxII genes. The genus was resolved into three monophyletic clades (1–3). Clade 1 was the largest clade, composed of 12 known species. Clades 2 contained two known and one new species candidate and clade 3 contained two known species. Three isolates in clade 2 (FP1, HonMa, and a strain designated as P. helicoides CBS293.35) formed a monophyletic group with high bootstrap support. This monophyletic group was distinct from P. helicoides sensu stricto. All three isolates came from damped-off buckwheat seedlings. The isolates were morphologically identical with one another and were characterized by globose, sub-globose, or pyriform sporangia with apical papillae; internally or internally nested proliferating sporangia; simple sympodia; coiling antheridial stalks; and wavy, sessile, or clavate antheridial cells. The isolates grew at temperatures between 15 °C and 40 °C, and the optimum temperature was 30 °C, with a radial growth rate of 20 mm/24 h. The phylogenetic and morphological analyses indicated that these isolates belong to a distinct species, which was previously under the genus Pythium, named here Phytopythium fagopyri comb. nov.     

Molecular characterization of plant growth promoting rhizobacteria that enhance peroxidase and phenylalanine ammonia-lyase activities in chile (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) and tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.)

Pythium and Phytophthora species are associated with damping-off diseases in vegetable nurseries and reduce seedling stand and yield. In this study, bacterial isolates were selected on the basis of in vitro antagonism potential to inhibit mycelial growth of damping-off pathogens along with plant growth properties for field assessment in wet and winter seasons. We demonstrate efficacy of bacterial isolates to protect chile and tomato plants under natural vegetable nursery and artificially created pathogen-infested (Pythium and Phytophthora spp.) nursery conditions. After 21 days of sowing, chile and tomato plants were harvested and analysed for peroxidase and phenylalanine ammonia-lyase activities. Pseudomonas sp. strains FQP PB-3, FQA PB-3 and GRP₃ were most effective in increasing shoot length (P > 0.05%) in both artificial and natural field sites. For example, Pseudomonas sp. FQA PB-3 treatment increased shoot length by 40% in the artificial Pythium 4746 infested nursery site in chile plants in the wet season. The bacterial treatments significantly increased the activity of peroxidase and phenylalanine ammonia-lyase in chile and tomato plant tissues, which are well known as indicators of an active lignification process. Thus, we conclude that treatment with potential bacterial plant growth promoting agents help plants against pathogen invasion by modulating plant peroxidase and phenylalanine ammonia-lyase activities.