Occurrence of Pythium and Phytopythium species isolated from citrus trees infected with gummosis disease in tunisia

A survey was conducted, during 2012/2013, to identify the causal agents of citrus gummosis in Tunisia. The obtained isolates were identified by molecular and pathological criteria. Three species of Pythium (P. aphanidermatum, P. ultimum and P. dissotocum) and two species of Phytopythium (P. vexans and P. mercuriale) were recovered. Pathogenic tests, using twigs inoculation method for five citrus varieties under laboratory conditions, showed necrotic lesions similar to those observed in natural infected citrus trees by gummosis. In greenhouse experiments, the pathogenicity of the species to citrus was demonstrated by soil and trunk inoculations. Results showed necrotic lesions in the trunk of the variety “Clementine”. P. ultimum is the most virulent, while P. mercuriale was the least virulent in trunk inoculation. Symptom of necrotic roots has been also observed. This is the first report of Pythium and Phytopythium species within the citrus area in Tunisia.     

Morphological and molecular identification of some closely related Pythium species in Egypt

Twelve isolates of Pythium species (P. aphanidermatum, P. deliense, P. ultimum var. ultimum and P. ultimum var. sporangiiferum) from different hosts were compared from morphological, pathological and molecular viewpoints. Minimum, optimum and maximum temperatures of P. aphanidermatum and P. deliense were similar while those of P. ultimum var. ultimum and P. ultimum var. sporangiiferum were also similar. All tested isolates were highly virulent against cucumber seedlings with 100% damping-off. RAPD data using three different primers revealed that strains of P. ultimum var. ultimum and P. ultimum var. sporangiiferum are distinct from each other. This data can be used to separate those species from P. aphanidermatum and P. deliense. In contrast, RAPD data cannot be used to separate P. aphanidermatum and P. deliense. Sequence analysis of the ribosomal DNA internal transcribed spacers (ITS) was used to establish phylogenetic relationships among the tested isolates.     

Occurrence of Pythium spp. and Phytophthora spp. in Norwegian Greenhouses and their Pathogenicity on Cucumber Seedlings

Samples of tomato, lettuce and cucumber submitted for diagnosis to the Plant Protection Centre at the Norwegian Crop Research Institute and samples of soil, water and cucumber collected from greenhouses employing hydroponic cultures were examined for the occurrence of Pythium spp. and Phytophthora spp. Two species of Phytophthora and 16 species of Pythium were identified. Phytophthora cryptogea was found on tomato and lettuce. Phytophthora nicotianae was found on tomato fruit. Phytophthora was not found on cucumbers. Pythium irregulare and Pythium group F were the two most commonly found Pythium species in hydroponically cultivated cucumbers. A pathogenicity test with 56 isolates was performed on cucumber seedlings. The most aggressive species were Pythium aphanidermatum, P. irregulare, Pythium paroecandrum and Pythium ultimum.     

Antagonist Microorganisms with the Ability to Control <Emphasis Type="Italic">Pythium</Emphasis> Damping-off of Tomato Seeds in Rockwool

A total of 237 microorganisms were isolated from five different greenhouse tomato growing media. Of those, 40 microorganisms reduced the in vitro mycelial growth of both Pythium aphanidermatum and Pythium ultimum. The ability of these microorganisms to control damping-off was then tested in rockwool. As a result, Pseudomonas corrugata strains 1 and 3, Pseudomonas fluorescens subgroup F and G strains 1, 2, 3, 4 and 5, Pseudomonas marginalis, Pseudomonas putida subgroup B strain 1, Pseudomonas syringae strain 1 and Pseudomonas viridiflava significantly reduced damping-off caused by P. ultimum or P. aphanidermatum. Pseudomonas marginalis was the only microorganism that significantly reduced damping-off caused by both pathogens.     

Biochemical characterisation of Pythium spp. involved in cavity spot of carrots in France

The pathogenicity and growth rate in vivo were assessed on 27 isolates of Pythium spp. recovered from cavity spot lesions on carrots grown in various parts of northwest France. Polyacrylamide gel electrophoresis of isoesterases was used to identify the Pythium spp. involved. Slow-growing isolates were more aggressive than fast-growing ones when inoculated on carrot tap roots. Isoesterase patterns identified the slow-growing isolates as P. violae and P. sulcatum; P. ultimum and P. intermedium were identified among the less aggressive fast-growing isolate group, in which some isolates were also classed as P. sylvaticum or P. irregulare, which have similar electrophoretic profiles. The incidence of Pythium spp. associated with the disease in France is discussed in regard to cavity spot in other countries.     

Chemical,physical and biological control of carrot seedling diseases

In naturally infested soil containingPythium ultimum, P. acanthicum andPhytophthora megasperma, onlyP. ultimum was associated with root rot and damped-off seedlings. Damping-off was promoted by low soil temperatures and by flooding. Seedling stands were markedly reduced when seed was pre-incubated in soil at 12°C but not at 25°C or 35°C. Dusting carrot seed with metalaxyl significantly increased seedling stands in the field at rates from 1.5–6 g kg⁻¹ seed and in both flooded and unflooded, naturally infested soil at 3.15 g kg⁻¹. In greenhouse experiments using artifically infested soil,P. ultimum andP. paroecandrum caused damping-off of carrot seedlings andRhizoctonia solani reduced root and shoot weights.R. solani caused damping-off in nutrient-enriched soil.P. acanthicum andP. megasperma were not pathogenic to seedlings, although both fungi colonized roots. Soil populations of allPythium spp., particularlyP. ultimum, increased during growth of seedlings and population growth ofP. megasperma was promoted by periodic flooding. Infestation of soil withP. acanthicum did not reduce damping-off of carrot seedlings byP. ultimum orP. paroecandrum, but significantly increased root and shoot weights and decreased root colonization byR. solani P. acanthicum has potential as a biocontrol agent againstR. solani.     

Compost-Induced Suppression of Pythium Damping-Off Is Mediated by Fatty-Acid-Metabolizing Seed-Colonizing Microbial Communities

Leaf composts were studied for their suppressive effects on Pythium ultimum sporangium germination, cottonseed colonization, and the severity of Pythium damping-off of cotton. A focus of the work was to assess the role of fatty-acid-metabolizing microbial communities in disease suppression. Suppressiveness was expressed within the first few hours of seed germination as revealed by reduced P. ultimum sporangium germination, reduced seed colonization, and reduced damping-off in transplant experiments. These reductions were not observed when cottonseeds were sown in a conducive leaf compost. Microbial consortia recovered from the surface of cottonseeds during the first few hours of germination in suppressive compost (suppressive consortia) induced significant levels of damping-off suppression, whereas no suppression was induced by microbial consortia recovered from cottonseeds germinated in conducive compost (conducive consortia). Suppressive consortia rapidly metabolized linoleic acid, whereas conducive consortia did not. Furthermore, populations of fatty-acid-metabolizing bacteria and actinobacteria were higher in suppressive consortia than in conducive consortia. Individual bacterial isolates varied in their ability to metabolize linoleic acid and protect seedlings from damping-off. Results indicate that communities of compost-inhabiting microorganisms colonizing cottonseeds within the first few hours after sowing in a Pythium-suppressive compost play a major role in the suppression of P. ultimum sporangium germination, seed colonization, and damping-off. Results further indicate that fatty acid metabolism by these seed-colonizing bacterial consortia can explain the Pythium suppression observed.     

Isolation of Pythium Acanthicum,P. oligandrum,and P. periplocum from Soil and Evaluation of Their Mycoparasitic Activity and Biocontrol Efficacy Against Selected Phytopathogenic Pythium Species

Mycoparasitic Pythium species with spiny oogonia were surveyed in 50 Palestinian agricultural fields subject to different cropping practices using the Sclerotia Bait Technique (SBT) and the Surface-Soil-Dilution-Plate method (SSDP) with the selective VP3 medium. The mycoparasitic Pythium species were obtained from 21 (42%) soils using the SSDP method and from 37 (74%) soils using SBT. Pythium acanthicum and P. oligandrum were isolated by both methods, whereas P. periplocum was isolated only by the SBT. Using a newly modified dual plate culture method (MDPCM), the three mycoparasites showed varying antagonistic performance against several Pythium host species under a range of in vitro conditions. However, P. periplocum and P. oligandrum were found to be active biocontrol agents against P. ultimum, the damping-off organism of cucumber. This pathogen was antagonized, on thin films of water agar, by the three mycoparasites, and was moderately susceptible to P. periplocum while slightly susceptible to P. acanthicum and P. oligandrum. In direct application method in which antagonistic mycoparasites were incorporated into peat/sand mixture artificially infested with P. ultimum under growthroom conditions, Pythium oligandrum and P. periplocum (at 500 CFUg⁻¹) significantly improved seedling emergence and protected seedlings from damping-off. In the seed coating method, biocontrol by two types of seed dressing (homogenate- or oospore coated seeds), was comparable to that achieved by direct application. This revised version was published online in June 2006 with corrections to the Cover Date.     

Epicoccum Nigrum as biocontrol agent of Pythium damping-off and root-rot of cotton seedlings

Efficacy of Epicoccum nigrum and its exudate was tested in control the pre- and post-emergence damping-off and root-rot of Egyptian cotton (cv. Giza 83) in vitro and in vivo. Different isolates of Epicoccum nigrum reduced the radial growth of both Pythium debaryanum and P. ultimum significantly, by production considerable inhibition zones. In liquid cultures E. nigrum exudate showed a high fungicidal effect resulting in a significant reduction of the mycelial dry weight of the two investigated Pythium spp. Also E. nigrum exudate inhibited cellulase and pectinase activity by P. debaryanum and P. ultimum. Soaking of cotton seeds in E. nigrum exudate for different intervals resulted in significant reduction of root-rot severity of seedlings as well as the contamination of seeds and seedlings by fungi during and after germination. These treatments also stimulated germination of cotton seeds and enhanced the seedlings vigour significantly. In pot experiments, the use of E. nigrum as a soil mixture or seed dressing significantly alleviated the hazard effect of P. debaryanum. Pythium ultimum seemed to be weak or non pathogen to the used cotton cultivar (Giza 83). Application of E. nigrum or its exudate not only involved in protection of cotton seedlings against Pythium damping-off and root-rot but also enhanced their vigour and growth characteristics. The main conclusion of this study is that E. nigrum could be used successfully as environmentally safe and economic biological control agent to protect cotton (cv. Giza 83) from damping-off and root-rot diseases caused by P. debaryanum.     

Untersuchungen zum Vorkommen von Pythium spp. im Boden

Investigations on the occurrence of Pythium spp. in soil: I. The isolation of Pythium spp., their distinction to macroscopically characteristics and their determination The aim of the present paper consisted in detection of Pythium spp. directly in the soil. This was possible by using a selective medium and by crumbling smallest particles of agar-covered soil on its surface. On the basis of simple morphologically criteria (growth patterns) this method allows to decide concerning the presence of high and less pathogen or apathogen Pythium spp. in a soil sample within 48 hours. About 700 isolates have been cultivated from hyphal tips, determinated and about 230 tested for pathogenicity to sugar beet seedlings in vitro. Most of the Pythia pathogen to sugar beet belong to P. ultimum Trow followed by P. paroecandrum Drechsler and P. debaryanum sensu Drechsler non Hesse. The taxonomically characteristics are demonstrated by figures of the three species.     

The genus Pythium in Taiwan, China (1) — a synoptic review

The genus Pythium, with slightly over 280 described species, has been classified traditionally with other filamentous, coenocytic, sporangia-producing fungi as “Phycomyetes”. However, with recent advances in chemical, ultrastructural and molecular studies, Pythium spp. are now considered as “fungus-like organisms” or “pseudo-fungi” and are placed in the Kingdom Chromista or Kingdom Straminopila, distinct from the true fungi of the Kingdom Fungi or Kingdom Mycota. They are widely distributed throughout the world as soil saprophytes or plant pathogens. Because of the warm moist maritime climate, Taiwan, China, is especially rich in Pythium species. To date, 48 species of Pythium have been reported from Taiwan, China, with the dominant species being Py. vexans, Py. spinosum, Py. splendens, Py. aphanidermatum, Py. dissotocum and Py. acanthicum. There is no definite geographical distribution of Pythium spp. in Taiwan, China. Twenty nine species of Pythium have proven to be plant pathogens attacking a wide variety of woody and herbaceous plants primarily causing pre- and post-emergence seedling damping-off, root rot, stem rot and rotting of fruits, tubers and ginger rhizomes, resulting in serious economic losses. The most important plant pathogenic species include Py. aphanidermatum and Py. Myriotylum, which are most active during the hot and wet summer months; whereas Py. splendens, Py. spinosum, Py. ultimum and Py. irregulare cause the greatest damage in the cool winter. Most Pythium spp. are non-specific pathogens, infecting mainly juvenile or succulent tissues. This review attempts to assess the taxonomic position of the genus Pythium and provide details of the historical development of the study of Pythium as pathogens in Taiwan, China, causing diseases of sugarcane, trees, vegetables, fruits, specialty crops and flowering plants, as well as measures to control these diseases. Of special note is the introduction of the S-H mixture which, when used as soil amendment, effectively controls many soil-borne Pythium diseases during the early stages of plant growth. The diversity of Pythium species in Taiwan, China, is discussed in comparison with the situation in the mainland of China and suggestions are made to fully utilize Pythium spp. as agents for biological control, in industry and medicine.     

Characterization of two morphological groups of isolates ofPythium ultimum var.ultimum in a vegetable field

Comparisons were made between two morphological groups ofPythium ultimum var.ultimum strains isolated in a vegetable field in Japan. The groups were distinguished as having smaller or larger sexual organs by the sizes of their antheridia and oogonia. Morphological study indicated that the two groups comprised a single taxon,P. ultimum var.ultimum, by the current taxonomical keys. The smaller group grew faster in the lower temperature range of 4–15°C, whereas the larger group grew faster in the higher temperature range of 25–37°C. Random amplified polymorphic DNA (RAPD) and isozyme analyses revealed genetic dissimilarity between the two groups. Cluster analysis of the isozyme banding patterns with four otherPythium spp. demonstrated that the genetic dissimilarity between the two groups was equivalent to species level. In the field survey, the smaller group was frequently detected in February, May and September but not in July, while the larger group was detected mainly in July and September. The two groups were not distinguishable by their pathogenicity to spinach seedlings.     

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.     

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.     

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.     

Identification of Fusarium species causing basal rot of onion in East Azarbaijan province,Iran and evaluation of their virulence on onion bulbs and seedlings

One of the economically important diseases of onion is the basal rot caused by various Fusarium species. Identification of the pathogenic species prevalent in a region is indispensable for designing management strategies, especially to develop resistant cultivars. Eighty Fusarium isolates are obtained from red onion bulbs on infected fields of East Azarbaijan province. Inoculating the onion bulbs with 38 selective isolates indicated that 17 isolates were pathogenic on onion. According to the morphological and molecular characteristics, these isolates were identified as F. oxysporum, F. solani, F. proliferatum and F. redolens. This is the first report of F. redolens on onion in Iran. On the other hand, the virulence of each pathogenic isolate was evaluated on onion bulbs and seedlings. F. oxysporum which causes severe rot and damping-off was considered as a highly virulent species in both conditions. While, F. proliferatum was considered as the most destructive on onion bulbs. Rot ability of F. solani was not considerable, and only the 4S isolate caused pre- and post-emergence damping-off more than 50%. Finally, F. redolens with less pathogenicity on onion bulbs was identified as the most virulent isolate on onion seedlings, which was explanatory of its importance on farm.     

Biological Control of Phoma and Pythium Damping-Off of Sugar-Beet with Pythium oligandrum

Flooding freshly harvested oospores in sterile distilled water (SDW) for several days enhanced germination in 3 out of 4 isolates of Phythium oligandrum. Treatment of SDW-flooded oospores with myo-inositol increased germinability during the first 20 days of storage at 15°C. Seed dressing with oospores of P. oligandrum controlled pre- and post-emergence damping-off of sugar-beet caused by soil-borne P. ultimum and seed-borne Phoma betae. For some isolates, flooded oospores in SDW and treatment with myo-inositol increased efficacy of the seed dressing. However, no significant control of damping-off caused by Rhizoctonia solani was observed. On corn-meal agar, P. oligandrum coiled around and penetrated hyphae of P. ultimum and R. solani, but did not interfere with Ph. betae.     

Isolation and Identification of Pythiaceous Fungi from Irrigation Water and their Pathogenicity to Antirrhinum, Tomato and Chamaecyparis lawsoniana

Pythiaceous fungi were isolated from irrigation water using a variety of natural and artificial baits. Isolates were also obtained by plating water samples directly on the surface of selective agar media. The selective medium of Ocana and Tsaq (1966) PlOVP, was modified by substituting rifampicin and ampicillin (10 and 500 μg cm^?3 respectively) for vancomycin to suppress bacterial growth from water samples. The pythiaceous fungi were identified as Pythiitm dissotocitm, P. middletonii, P. mamillatum, P. rostratum, Pythium“group 1”, “group 2” and “group 3” and Phytophtbom gona-podyides. All isolates of P. gonapodyides were the A1 strain and produced oospores when paired with an A2 isolate of P. drechsleri. Isolates were tested for their pathogenicity to Antirrhinum, tomato and Chatmaecyparis lawsoniana cv. Ellwoodii. Pythium middletonii and Pythium“group 1” caused severe pre-emergence damping-off of Antirrhinum seedlings, P. mamillatuni, P. rostratum and Pythium“group 3” were less pathogenic to the same host while P. dissotocum, Pythium“group 2” and Phytophthora gonapodyides were non-pathogenic. Only isolates of Pythium“group 1” were pathogenic to tomato seedlings. None of the fungi was pathogenic to rooted cuttings of Chamaecyparis lawsoniana cv. Ellwoodii.     

<Emphasis Type="Italic">Pythium montanum</Emphasis> sp. nov., a new species from a spruce stand in the Bavarian Alps

During a study on soilborne Pythiaceae in the Bavarian Alps, 3 isolates of a papillate Pythium species with aplerotic oospores were found in rhizosphere soil from a declining spruce stand. The isolates showed a unique combination of sporangial and gametangial characters, and could not be assigned to any known species of the genus. They were characterised by a relatively low growth rate, internally proliferating sporangia and extremely variable, monoclinous, sessile or shortly stalked antheridia. Direct germination of sporangia frequently occurred in water culture. Internal transcribed spacer (ITS) regions of the ribosomal DNA showed very low levels of similarity to those of all known species of Pythium. This paper describes the isolates as Pythium montanum sp. nov., gives details on its morphology and provides preliminary information on its pathogenicity. Additional molecular evidence for its status as a new species is also provided. Phylogenetic analyses with a set of Pythium spp. showed that P. montanum groups in a clade together with P. vexans, a species whose taxonomical status still remains unclear, and that is considered a ‘border species’ between Pythium and Phytophthora.     

Effects of Fungal Root Pathogens on the Population Dynamics of Biocontrol Strains of Fluorescent Pseudomonads in the Wheat Rhizosphere

The influences of Gaeumannomyces graminis var. tritici (which causes take-all of wheat), Rhizoctonia solani AG-8 (which causes rhizoctonia root rot of wheat), Pythium irregulare, P. aristosporum, and P. ultimum var. sporangiiferum (which cause pythium root rot of wheat) on the population dynamics of Pseudomonas fluorescens 2-79 and Q72a-80 (bicontrol strains active against take-all and pythium root rot of wheat, respectively) in the wheat rhizosphere were examined. Root infection by either G. graminis var. tritici or R. solani resulted in populations of both bacterial strains that were equal to or significantly larger than their respective populations maintained on roots in the absence of these pathogens. In contrast, the population of strain 2-79 was significantly smaller on roots in the presence of any of the three Pythium species than on noninfected roots and was often below the limits of detection (50 CFU/cm of root) on Pythium-infected roots after 40 days of plant growth. In the presence of either P. aristosporum or P. ultimum var. sporangiiferum, the decline in the population of Q72a-80 was similar to that observed on noninfected roots; however, the population of this strain declined more rapidly on roots infected by P. irregulare than on noninfected roots. Application of metalaxyl (which is selectively inhibitory to Pythium spp.) to soil naturally infestated with Pythium spp. resulted in significantly larger rhizosphere populations of the introduced bacteria over time than on plants grown in the same soil without metalaxyl. It is apparent that root infections by fungal pathogens may either enhance or depress the population of fluorescent pseudomonads introduced for their control, with different strains of pseudomonads reacting differentially to different genera and species of the root pathogens.