Ecology of Hymexazol-Insensitive Pythium Species in Field Soils

Soils from 100 irrigated fields (95 under vegetables, 5 under citrus) in different geographical locations in the West Bank (Palestinian Autonomous Territory) were surveyed for hymexazol-insensitive (HIS) Pythium species using the surface soil dilution plate (SSDP) method with the VP3 medium amended with 50 mg/L hymexazol (HMI) (VP3H50), over a period of 12 months. HIS Pythium species were isolated from 37% of the soils surveyed, with mean population levels ranging from 4.3-1422 CFU g(-1) dry weight. Eight HIS Pythium taxa were recovered on the VP3H50 medium, the most abundant of which was P. vexans (found in 29% of field soils surveyed). Seasonal variations in population levels of HIS Pythium species were studied in four fields over a period of 12 months. Significant seasonal variations in HIS population levels were detected in the four fields, with the highest population levels of HIS Pythium spp. encountered in spring and the lowest population levels in winter in three of the fields surveyed. Effects of HMI on linear growth and colony morphology of 149 Pythium ssp. isolates were examined on CMA amended with HMI at five concentrations. Pythium vexans isolates responded differently from those of the other Pythium species. Isolates of this important pathogen were more insensitive to HMI at high concentrations than the other main species tested. A large proportion of the P. ultimum isolates was either insensitive or weakly sensitive to HMI. Furthermore, a few isolates of other Pythium species were insensitive to the fungicide at various concentrations. The colony morphology of P. vexans isolates was not affected by HMI, whereas colonies of the other species showed sparse growth on the HMI amended medium relative to the control. The pathogenicity of P. vexans and P. ultimum isolates to cucumber seedlings was examined in growth chambers. Insensitive isolates of both species were found to be more virulent damping-off pathogens than the sensitive isolates. The present study demonstrates that HMI can not be used effectively in controlling Pythium spp. in soil inhabited with high densities of HIS Pythium spp. pathogens.     

Pythium species associated with cavity spot on carrots in Norway

Carrot roots with cavity spot lesions from eight different counties in Norway were sampled and Pythium species were isolated on selective medium. Pythium spp. were characterised morphologically and by species-specific PCR. Laboratory experiments with inoculations of carrot roots were performed. A total of 130 isolates out of 230 Pythium -like isolates tested with PCR were identified as pathogenic species of Pythium. These were P. intermedium (29%), P. sulcatum (23%), P. sylvaticum (16%), P. violae (15%) and a possible new Pythium species designated P . ' vipa ' (18%). There were some differences between geographical regions and ages of cavities regarding the frequency of the different species isolated. When rating sunken lesions in the laboratory inoculation experiments, P. ' vipa ' was the most aggressive and P. violae the least aggressive species. P. intermedium and P. ' vipa ' caused more discolouration of the infected carrot tissue than the other species. The importance of the different Pythium spp. as agents of cavity spot in Norway is discussed.     

Cavity spot of carrots: Interactions between the host and pathogen, related to the cell wall

This study investigates the structural aspects of cavity spot pathogenesis. Different Pythium spp. isolated from infected carrots, apples and melons were cultured on agar in Petri dishes and used for inoculation of uninfected carrots. Only slow-growing Pythium spp. (< 15 mm day^-1), such as P. violae and P. sulcatum caused cavity spot lesions. It is suggested that slow-growing species are able to penetrate, albeit slowly, into the plant tissue for 3 to 4 days before a hypersensitive reaction develops. Fast-growing species, however, did not cause lesions. Based on ultrastructural observations, we suggest that the following sequence of events occurs between the plant and the pathogen: The fungus infects the walls and grows for several days, during which time small amounts of wall-degrading enzymes are secreted. Phenylalanine ammonia lyase (PAL) activity and phenols increase linearly immediately upon inoculation. There was a lag phase of about 5 days before lignin began to increase linearly for about a month. Dissolution of wall components decreases the solute potential and water potential in the apoplast. Thus, water moves from the symplast into the apoplast, the turgor pressure gradually dissipates, and the cells shrink and eventually die.     

<Emphasis Type="Italic">Pythium</Emphasis> infection activates conserved plant defense responses in mosses

The moss Physcomitrella patens (P. patens) is a useful model to study abiotic stress responses since it is highly tolerant to drought, salt and osmotic stress. However, very little is known about the defense mechanisms activated in this moss after pathogen assault. In this study, we show that P. patens activated multiple and similar responses against Pythium irregulare and Pythium debaryanum, including the reinforcement of the cell wall, induction of the defense genes CHS, LOX and PAL, and accumulation of the signaling molecules jasmonic acid (JA) and its precursor 12-oxo-phytodienoic acid (OPDA). However, theses responses were not sufficient and infection could not be prevented leading to hyphae colonization of moss tissues and plant decay. Pythium infection induced reactive oxygen species production and caused cell death of moss tissues. Taken together, these data indicate that Pythium infection activates in P. patens common responses to those previously characterized in flowering plants. Microscopic analysis also revealed intracellular relocation of chloroplasts in Pythium-infected tissues toward the infection site. In addition, OPDA, JA and its methyl ester methyl jasmonate induced the expression of PAL. Our results show for the first time JA and OPDA accumulation in a moss and suggest that this defense pathway is functional and has been maintained during the evolution of plants. Authors Juan Pablo Oliver and Alexandra Castro contributed equally to this work.     

Diversity of Micromonospora strains isolated from nitrogen fixing nodules and rhizosphere of Pisum sativum analyzed by multilocus sequence analysis

It was recently reported that Micromonospora inhabits the intracellular tissues of nitrogen fixing nodules of the wild legume Lupinus angustifolius. To determine if Micromonospora populations are also present in nitrogen fixing nodules of cultivated legumes such as Pisum sativum, we carried out the isolation of this actinobacterium from P. sativum plants collected in two man-managed fields in the region of Castilla and León (Spain). In this work, we describe the isolation of 93 Micromonospora strains recovered from nitrogen fixing nodules and the rhizosphere of P. sativum. The genomic diversity of the strains was analyzed by amplified ribosomal DNA restriction analysis (ARDRA). Forty-six isolates and 34 reference strains were further analyzed using a multilocus sequence analysis scheme developed to address the phylogeny of the genus Micromonospora and to evaluate the species distribution in the two studied habitats. The MLSA results were evaluated by DNA-DNA hybridization to determine their usefulness for the delineation of Micromonospora at the species level. In most cases, DDH values below 70% were obtained with strains that shared a sequence similarity of 98.5% or less. Thus, MLSA studies clearly supported the established taxonomy of the genus Micromonospora and indicated that genomic species could be delineated as groups of strains that share > 98.5% sequence similarity based on the 5 genes selected. The species diversity of the strains isolated from both the rhizosphere and nodules was very high and in many cases the new strains could not be related to any of the currently described species.     

Pathogenic free-living amoebae in Korea

Acanthamoeba and Naegleria are widely distributed in fresh water, soil and dust throughout the world, and cause meningoencephalitis or keratoconjunctivitis in humans and other mammals. Korean isolates, namely, Naegleria sp. YM-1 and Acanthamoeba sp. YM-2, YM-3, YM-4, YM-5, YM-6 and YM-7, were collected from sewage, water puddles, a storage reservoir, the gills of a fresh water fish, and by corneal washing. These isolates were categorized into three groups based on the mortalities of infected mice namely, highly virulent (YM-4), moderately virulent (YM-2, YM-5 and YM-7) and nonpathogenic (YM-3). In addition, a new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Korean isolate YM-4. The morphologic characters of its cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Based on experimentally infected mouse mortality, Acanthamoeba YM-4 was highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. Moreover, an anti-Acanthamoeba YM-4 monoclonal antibody reacted only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of a 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster based on phylogenic distances. Thus Acanthamoeba YM-4 was identified as a new species, and assigned Acanthamoeba sohi. Up to the year 2002 in Korea, two clinical cases were found to be infected with Acanthamoeba spp. These patients died of meningoencephalitis. In addition, one case of Acanthamoeba pneumonia with an immunodeficient status was reported and Acanthamoeba was detected in several cases of chronic relapsing corneal ulcer, chronic conjunctivitis, and keratitis.     

Reprint of: Saprolegnia strains isolated from river insects and amphipods are broad spectrum pathogens

Saprolegnia species are destructive pathogens to many aquatic organisms and are found in most parts of the world. Reports based on phylogenetic analysis suggest that Saprolegnia strains isolated from aquatic animals such as crustaceans and frogs are close to Saprolegnia strains isolated from infected fish or fish eggs and vice versa. However, it has often been assumed that host specificity occurs for each individual isolate or strain. Here we demonstrate that Saprolegnia spp. can have multiple hosts and are thus capable of infecting different aquatic organisms. Saprolegnia delica, Saprolegnia hypogyna, and 2 strains of Saprolegnia diclina were isolated from aquatic insects and amphipods while S. delica, Saprolegnia ferax, Pythium pachycaule, and a Pythium sp. were isolated from the water of a medium to fast flowing river. The ITS region of the rRNA gene was sequenced for all isolates. In challenge experiments, all four isolates from insects were found to be highly pathogenic to eggs of Atlantic salmon (Salmo salar) and embryos of the African clawed frog (Xenopus laevis). We found that Saprolegnia spp. isolated from salmon eggs were also able to successfully establish infection in nymphs of stonefly (Perla bipunctata) and embryos of X. laevis). These results suggest that Saprolegnia spp. are capable of infecting multiple hosts, which may give them an advantage during seasonal variation in their natural environments.     

新近从宁夏分离的腐霉

到现在为止全世界已发现loo余种腐霉。我国已报道54种。在我国31个省、市自治区中,仅宁夏和西藏2个自治区未见报道过腐霉。最近我们从宁夏回族自治区土壤和植物病根用多种方法进行分离,得到259株,鉴定出10个种。它们是:瓜果腐霉Pythium aphanidermatum (Edson) Fitzpatrick、链状腐霉P. catenulatum Matthews、畸雌腐霉P. irregulare Buisman、寡雄腐霉P. oligandrum Drechsler、侧雄腐霉P.paroecandrum Drechsler,缠器腐霉P. periplocum Drechsler、无序腐霉P. perplexum Kouyeas & Theohari、角柄腐霉P. salpingophocum DrechsIer、终极腐霉P. ultimum Trow和钟器腐霉P. vexans de Bary。它们全是宁夏地区新记录种。其中无序腐霉还是我国新记录种。本文仅描述了无序腐霉,对其余9个种进行了讨论.有些种类除从土壤中分离到以外,还见于一些寄主植物:大葱Allium fistulosum L.、韭葱A. juncea L.、芹菜Apium-graveolens L.、油菜Brassica campestris L.、雪里蒸B. juncea (L.) Czern et Coss var.crispifolia Bailey白菜B. pekinensis Rupr.、辣椒Capsicum frutescens L.、胡萝卜Daucuscarota L. var.saliva DC.、草每Fragaria ananassa Duchesne、番茄esculentum Mill.、菜豆Phaseolus vulgaris L.和萝卜Raphanus sativus L.。以“腐霉名称:寄主名称”的格式列出如后:瓜果腐霉:胡萝卜;链状腐霉:胡萝卜;寡雄腐霉:白菜和辣椒;侧雄腐霉:大葱、芹菜和白菜;缠器腐霉:韭葱;终极腐霉:大葱、韭葱、油菜、雪里蕻、白菜、辣椒、胡萝卜、草墓、番茄、菜豆和萝卜。在我国,这些腐霉从前述寄主上分离出均属首次报道。     

Plantago lanceolata and Plantago rugelii Extracts are Toxic to Meloidogyne incognita but not to Certain Microbes

Extracts from the plants Plantago lanceolata and P. rugelii were evaluated for toxicity to the root-knot nematode Meloidogyne incognita, the beneficial microbes Enterobacter cloacae, Pseudomonas fluorescens and Trichoderma virens, and the plant-pathogenic fungi Fusarium oxysporum f. sp. gladioli, Phytophthora capsici, Pythium ultimum, and Rhizoctonia solani. Wild plants were collected, roots were excised from shoots, and the plant parts were dried and ground to a powder. One set of extracts (10% w/v) was prepared in water and another in methanol. Treatments included extract concentrations of 25%, 50%, 75% and 100%, and water controls. Meloidogyne incognita egg hatch was recorded after 7-day exposure to the treatments, and second-stage juvenile (J2) activity after 48 hours. All extracts were toxic to eggs and J2, with P. lanceolata shoot extract tending to have the most activity against M. incognita. Numbers of active J2 remained the same or decreased in a 24-hour water rinse following the 48-hour extract treatment, indicating that the extracts were lethal. When data from water- and methanol-extracted roots and shoots of both plant species were combined for analysis, J2 tended to be more sensitive than eggs to the toxic compounds at lower concentrations, while the higher concentrations (75% and 100%) were equally toxic to both life stages. The effective concentrations causing 50% reduction (EC₅₀) in egg hatch and in J2 viability were 44.4% and 43.7%, respectively. No extract was toxic to any of the bacteria or fungi in our assays.     

PYTHIUM SYLVATICUM鉴定及其专一性PCR引物

从杭州采集的水稻、棉花和大豆猝倒苗中分离到国内新记录腐霉种Pythium sylvaticum。该腐霉为异宗配合种,菌丝膨大体球形或柠檬形,雄器异丝生,藏卵器光滑,每个藏卵器上1~3个雄器,雄器常在接近藏卵器处形成二叉状分枝,卵孢子不满器。测定了该种4 个菌株的核糖体内转录间隔区(ITS)的序列,根据与59种腐霉ITS序列的比较,设计了P. sylvaticum种专一性引物PSF1和PSR2。实际结果表明:该引物能从11种共14株腐霉DNA中特异性地扩增P. sylvaticum,从而与其它10种腐霉区分。     

A new species of Rhopalosiphum (Hemiptera, Aphididae) on Chusquea tomentosa (Poaceae, Bambusoideae) from Costa Rica

The new species Rhopalosiphum chusqueae Pérez Hidalgo & Villalobos Muller, is described from apterous viviparous females caught on Chusquea tomentosa in Cerro de la Muerte (Costa Rica). The identity of the species is supported both by the morphological features and by a molecular phylogenetic analysis based on a fragment of the mitochondrial DNA containing the 5' region of the cytochrome c oxidase 1 (COI) and on the nuclear gene coding for the Elongation factor-1 alpha (EF1α). The taxonomic position of the new species is discussed. An identification key to the Aphidinae species living on plants of Bambusoideae (Poaceae) is presented.     

ent-Labdane glycosides from the aquatic plant Potamogeton lucens and analytical evaluation of the lipophilic extract constituents of various Potamogeton species

Two new ent-labdane glycosides, one known furano-ent-labdane and a new hydroxylated fatty acid were isolated from the dichloromethane extract of the freshwater aquatic plant Potamogeton lucens. The new compounds were assigned the structures of beta-d-glucopyranosyl-8(17),13-ent-labdadien-16,15-olid-18-oate, 18-beta-d-glucopyranosyloxy-8(17),13-ent-labdadien-16,15-olide and 13(R)-hydroxy-octadeca-(9Z,11E,15Z)-trien-oic acid by spectroscopic means. The algicidal activity of these compounds was tested against Raphidocelis subcapitata. Based on our previous study of Potamogeton pectinatus, other constituents were identified in P. lucens by LC-UV-MS, LC-NMR and GC-MS. The lipophilic extract profiles of both species are presented. Two other species, Potamogeton perfoliatus and P. crispus, were also investigated by analytical comparison of their non-polar extracts. The distribution of ent-labdanes characterized in Potamogeton is summarized.     

Morphological and molecular characteristics of a new species of Pasteuria parasitic on Meloidogyne ardenensis

A species of the hyper-parasitic bacterium Pasteuria was isolated from the root-knot nematode Meloidogyne ardenensis infecting the roots of ash (Fraxinus excelsior). It is morphologically different from some other Pasteuria pathogens of nematodes in that the spores lack a basal ring on the ventral side of the spore and have a unique clumping nature. Transmission electron microscopy (TEM) showed that the clumps of spores are not random aggregates but result from the disintegration of the suicide cells of the thalli. Sporulation within each vegetative mycelium was shown to be asynchronous. In addition to the novel morphological features 16S rRNA sequence analysis showed this to be a new species of Pasteuria which we have called P. hartismeri. Spores of P. hartismeri attach to juveniles of root-knot nematodes infecting a wide range of plants such as mint (Meloidogyne hapla), rye grass (unidentified Meloidogyne sp.) and potato (Meloidogyne fallax).     

Materials forPythium flora of Japan VIII Two species ofPythium:P. pyrilobum andP. oligandrum

Two species ofPythium (P. pyrilobum, P. oligandrum) having spherical sporangia with complex subglobose elements were isolated from the crown of creeping bentgrass [Agrostis palustris (cv. Penncross)] and from vegetable field soil, respectively. They are reported for the first time in Japan.     

Host Specificity of Pathogenic Pythium Species: Implications for Tree Species Diversity

In the Janzen–Connell hypothesis, host-specific natural enemies enhance species diversity and influence the structure of plant communities. This study tests the explicit assumption of host specificity for soil pathogens of the genus Pythium that cause damping-off disease of germinating seeds and seedlings. We isolated Pythium spp. from soil of a tropical forest in Panama. Then, in an inoculation experiment, we determined the pathogenicity of 75 tropical isolates of unknown pathogenicity and seven pathogenic temperate isolates of Pythium on seeds and/or seedlings of eight tropical tree species. Only three tropical isolates, one identified as P. ultimum and two as P. aphanidermatum , were pathogenic. Tropical pathogenic isolates were pathogenic on 4–6 of eight tree species. Temperate isolates were pathogenic on 0–4 of eight species, indicating that some tropical tree species are susceptible to novel isolates of Pythium . No tree species was susceptible to all isolates and two species were not susceptible to any isolate. Collectively, these results indicate that these Pythium isolates vary widely in their pathogenicity, causing differential mortality of potential host species; likewise, the tree species vary in their susceptibility to a given Pythium isolate. These differences in pathogenicity and susceptibility indicate some support for the Janzen–Connell assumption of host specificity. While they are not restricted to a single species, their intermediate level of specificity suggests that Pythium spp. have the potential to have some effect on forest community structure and diversity.     

Insecticidal bacteria isolated from predatory larvae of the antlion species Myrmeleon bore (Neuroptera: Myrmeleontidae)

Various bacterial species were isolated from the crop (digestive organ) of the antlion species Myrmeleon bore and tested for their insecticidal activity against caterpillars by injection. Sixty-eight isolates from the antlion crop were grouped into twenty-four species based on homologies of 16S rRNA gene sequences and biochemical properties. Isolated Bacillus cereus, Bacillus sphaericus, Morganella morganii, Serratia marcescens and a Klebsiella species killed 80% or more cutworms when injected at a dose of 5x10(5)cells per insect. In addition, cutworms killed by these isolates resembled observations made of caterpillars attacked by antlions. A culture-independent analysis showed that the isolated bacterial species are likely to be frequently present in the antlion crop. These results suggest that insecticidal microorganisms associate with antlions, and may promote the death of prey.     

Pythium spp. present in irrigation water in the Poniente region of Almería (south-eastern Spain)

Pythium catenulatum, P. diclinum and P. paroecandrum, as well as asexual forms with filamentous sporangia were isolated from irrigation water in the Poniente region of Almería (south-eastern Spain). The taxonomic and morphological details of these fungal isolates are described. P. catenulatum and P. diclinumare new reports to Spain. This revised version was published online in June 2006 with corrections to the Cover Date.     

Direct and indirect influences of arbuscular mycorrhizal fungi on phosphorus uptake by two root hemiparasitic Pedicularis species: do the fungal partners matter at low colonization levels?

Background and Aims

Because most parasitic plants do not form mycorrhizal associations, the nutritional roles of arbuscular mycorrhizal (AM) fungi in them have hardly been tested. Some facultative root hemiparasitic Pedicularis species form AM associations and hence are ideal for testing both direct and indirect effects of AM fungi on their nutrient acquisition. The aim of this study was to test the influence of AM inoculation on phosphorus (P) uptake by Pedicularis rex and P. tricolor.

Methods

³²P labelling was used in compartmented pots to assess the contribution of the AM pathway and the influence of AM inoculation on P uptake from a host plant into the root hemiparasites. Laboratory isolates of fungal species (Glomus mosseae and G. intraradices) and the host species (Hordeum vulgare ‘Fleet’) to which the two Pedicularis species showed obvious responses in haustorium formation and growth in previous studies were used.

Key Results

The AM colonization of both Pedicularis spp. was low (<15 % root length) and only a very small proportion of total plant P (<1 %) was delivered from the soil via the AM fungus. In a separate experiment, inoculation with AM fungi strongly interfered with P acquisition by both Pedicularis species from their host barley, almost certainly because the numbers of haustoria formed by the parasite were significantly reduced in AM plants.

Conclusions

Roles of AM fungi in nutrient acquisition by root parasitic plants were quantitatively demonstrated for the first time. Evidence was obtained for a novel mechanism of preventing root parasitic plants from overexploiting host resources through AM fungal-induced suppression of the absorptive structures in the parasites.