Flooding events and rising water temperatures increase the significance of the reed pathogen <Emphasis Type="Italic">Pythium phragmitis</Emphasis> as a contributing factor in the decline of <Emphasis Type="Italic">Phragmites australis</Emphasis>

Pythium species are economically significant soilborne plant pathogens with worldwide distribution, causing seedling damping-off or root rot diseases. Pythium phragmitis is a newly described pathogen of common reed (Phragmites australis), widespread in the reed-belt of Lake Constance, Germany. It is highly aggressive towards reed leaves and seedlings, but obviously does not affect roots. In the context of ‘reed decline’ phenomena, P. phragmitis infection of reed inundated during flooding events may be of particular significance. We could show that flooding itself is not necessarily detrimental for reed plants. In the presence of the pathogen, however, most submerged leaves and plants were killed within several weeks. Clipped plants did not show regrowth in the Pythium infested treatments. Significant losses in assimilating leaf area of reeds could, thus, be the result of Pythium infection rather than of flooding alone. Therefore, we suggest that the combination of extended flooding and the presence of P. phragmitis might considerably contribute to ‘reed decline’ at Lake Constance. In parallel, we could show that pathogenicity and spread of this species are considerably favoured by rising temperatures. Since an increase in average water temperature has been found for Lake Constance, we propose that P. phragmitis could be an important factor in the dieback of reed stands likely to be promoted by predicted climate change phenomena.     

Oomycetes associated with Prunus serotina persist in soil after tree harvest

Soil-borne pathogens can shape forest communities by lowering seedling survivorship. Many soil pathogens can persist long-term as survival spores, but how long pathogens outlive tree hosts in gap soils and whether they continue to affect seedling survival is uncertain. We studied the presence of oomycetes and evaluated seedling performance in soils near live Prunus serotina trees, and 0.5 and 1.5-y-old stumps. We isolated five species of oomycetes from soils, two of which were pathogenic (Pythium intermedium and Pythium irregulare) to Prunus serotina. There was a non-significant ~10.5% increase in conspecific seedling survival in stumps versus live trees, and pathogens were present in soils of all stump ages. The continued presence of pathogens of Prunus serotina in gap soils demonstrates the potential for impacts on conspecific regeneration after tree death, though the slight improvement in survival suggests that these effects may weaken with time.     

Land use is a determinant of plant pathogen alpha‐ but not beta‐diversity

Little is known about the diversity patterns of plant pathogens and how they change with land use at a broad scale. We employed DNA metabarcoding to describe the diversity and composition of putative plant pathogen communities in three substrates (soil, roots, and leaves) across five major land uses at a national scale. Almost all plant pathogen communities (fungi, oomycetes, and bacteria) showed strong responses to land use and substrate type. Land use category could explain up to 24% of the variance in composition between communities. Alpha‐diversity (richness) of plant pathogens was consistently lower in natural forests than in agricultural systems. In planted forests, there was also generally low pathogen alpha‐diversity in soil and roots, but alpha‐diversity in leaves was high compared with most other land uses. In contrast to alpha‐diversity, differences in within‐land use beta‐diversity of plant pathogens (the predictability of plant pathogen communities within land use) were subtle. Our results show that large‐scale patterns and distributions of putative plant pathogens can be determined using metabarcoding, allowing some of the first landscape level insights into these critically important communities.     

Virulence of oomycete pathogens from Phragmites australis‐invaded and noninvaded soils to seedlings of wetland plant species

Soil pathogens affect plant community structure and function through negative plant–soil feedbacks that may contribute to the invasiveness of non‐native plant species. Our understanding of these pathogen‐induced soil feedbacks has relied largely on observations of the collective impact of the soil biota on plant populations, with few observations of accompanying changes in populations of specific soil pathogens and their impacts on invasive and noninvasive species. As a result, the roles of specific soil pathogens in plant invasions remain unknown. In this study, we examine the diversity and virulence of soil oomycete pathogens in freshwater wetland soils invaded by non‐native Phragmites australis (European common reed) to better understand the potential for soil pathogen communities to impact a range of native and non‐native species and influence invasiveness. We isolated oomycetes from four sites over a 2‐year period, collecting nearly 500 isolates belonging to 36 different species. These sites were dominated by species of Pythium, many of which decreased seedling survival of a range of native and invasive plants. Despite any clear host specialization, many of the Pythium species were differentially virulent to the native and non‐native plant species tested. Isolates from invaded and noninvaded soils were equally virulent to given individual plant species, and no apparent differences in susceptibility were observed between the collective groups of native and non‐native plant species.     

Only a Few Fungal Species Dominate Highly Diverse Mycofloras Associated with the Common Reed

Plants are naturally colonized by many fungal species that produce effects ranging from beneficial to pathogenic. However, how many of these fungi are linked with a single host plant has not been determined. Furthermore, the composition of plant-associated fungal communities has not been rigorously determined. We investigated these essential issues by employing the perennial wetland reed Phragmites australis as a model. DNA extracted from roots, rhizomes, stems, and leaves was used for amplification and cloning of internal transcribed spacer rRNA gene fragments originating from reed-associated fungi. A total of 1,991 clones from 15 clone libraries were differentiated by restriction fragment length polymorphism analyses into 345 operational taxonomical units (OTUs). Nonparametric estimators for total richness (Chao1 and ACE) and also a parametric log normal model predicted a total of about 750 OTUs if the libraries were infinite. Sixty-two percent of the OTUs sequenced were novel at a threshold of 3%. Several of these OTUs represented undocumented fungal species, which also included higher taxonomic levels. In spite of the high diversity of the OTUs, the mycofloras of vegetative organs were dominated by just a few typical fungi, which suggested that competition and niche differentiation influence the composition of plant-associated fungal communities. This suggestion was independently supported by the results of nested PCR assays specifically monitoring two OTUs over 3 years, which revealed significant preferences for host habitat and host organ.     

Only a few fungal species dominate highly diverse mycofloras associated with the common reed

Plants are naturally colonized by many fungal species that produce effects ranging from beneficial to pathogenic. However, how many of these fungi are linked with a single host plant has not been determined. Furthermore, the composition of plant-associated fungal communities has not been rigorously determined. We investigated these essential issues by employing the perennial wetland reed Phragmites australis as a model. DNA extracted from roots, rhizomes, stems, and leaves was used for amplification and cloning of internal transcribed spacer rRNA gene fragments originating from reed-associated fungi. A total of 1,991 clones from 15 clone libraries were differentiated by restriction fragment length polymorphism analyses into 345 operational taxonomical units (OTUs). Nonparametric estimators for total richness (Chao1 and ACE) and also a parametric log normal model predicted a total of about 750 OTUs if the libraries were infinite. Sixty-two percent of the OTUs sequenced were novel at a threshold of 3%. Several of these OTUs represented undocumented fungal species, which also included higher taxonomic levels. In spite of the high diversity of the OTUs, the mycofloras of vegetative organs were dominated by just a few typical fungi, which suggested that competition and niche differentiation influence the composition of plant-associated fungal communities. This suggestion was independently supported by the results of nested PCR assays specifically monitoring two OTUs over 3 years, which revealed significant preferences for host habitat and host organ.     

Diversity, host, and habitat specificity of oomycete communities in declining reed stands (Phragmites australis) of a large freshwater lake

We studied the diversity of oomycetes in a declining reed belt (Phragmites australis) of Lake Constance, Germany, using conventional baiting with specific reed and standard oak baits, accompanied by molecular techniques. Apart from an Aphanomyces sp. and a Phytophthora sp., baiting from reed rhizosphere samples from flooded, as well as drier, littoral sites revealed only Pythium spp. A total of 67 oomycete isolates was classified according to PCR-RFLP banding patterns and ITS sequencing, and 18 different sequence types could be separated. The majority of these seemed previously unknown species, as indicated by the degree of similarity to those deposited in nucleotide databases. Species communities in both flooded and drier habitats or both reed and oak baits clearly differed from one another, and only few species occurred in both dry and flooded sites, or in both oak and reed baits. A frequently occurring group of related Pythium species appeared to be specifically associated with reed, and these were the only species that proved pathogenic towards this host in vitro. Our study proved that unexplored natural ecosystems harbour diverse communities of oomycete species with specific habitat and host preferences within close-by, but ecologically contrasting, sites. Among the species isolated, those associated with the predominating plant might accumulate and thus may be reed pathogens of considerable importance.     

Morphological and molecular taxonomy of Pythium longisporangium sp. nov. isolated from the Burgundian region of France

During the course of an investigation on the Pythiaceous oomycetes occurring in the Burgundian vineyards, some species of Pythium possessing mainly hypogynous antheridia were found. These had been classified as oomycetes belonging to the "Pythium rostratum" group for a long time. Three of these isolates, having similar structures and growth, are very closely related to a recently described species, Pythium bifurcatum Paul. A close look at these, however, underlines some fundamental differences with the latter. Not all of them produce zoospores but have very large sporangia. The type specimen is F-1200 (B 76a) which is a medium-slow growing saprophyte. The sequence of the ITS region of the rDNA also shows a very close relationship with P. bifurcatum. On the basis of morphological and molecular analysis, we now describe this species as Pythium longisporangium sp. nov. Morphological features of this new species, the sequences of the ITS region of its nuclear ribosomal DNA, and its comparison with related species are discussed.     

Do colonization by dark septate endophytes and elevated temperature affect pathogenicity of oomycetes?

Phialocephala subalpina is one of the most frequent dark septate root endophytes in tree roots but its function in forest ecosystems is largely unknown. A full-factorial infection experiment was performed, using six P.?subalpina isolates, two pathogenic oomycetes (Phytophthora plurivora [syn. Phytophthora citricola s.l.] and Elongisporangium undulatum [syn. Pythium undulatum]) and two temperature regimes (17.9 and 21.6?°C) to examine the ability of P.?subalpina to protect Norway spruce seedlings against root pathogens. Seedling survival, disease intensity and seedling growth were affected by P.?subalpina genotype, temperature and pathogen species. Some P.?subalpina isolates effectively reduced mortality and disease intensity caused by the two pathogens. Elevated temperature adversely affected seedling growth but did not aggravate the effect of the pathogens. Elongisporangium undulatum but not P.?plurivora significantly reduced plant growth. Colonization density of P.?subalpina measured by quantitative PCR was not affected by temperature or the presence of the pathogens. In conclusion, P.?subalpina confers an indirect benefit to its host and might therefore be tolerated in natural ecosystems, despite negative effects on plant health and plant growth.     

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.     

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.     

Zoospore homing and infection events: effects of the biocontrol bacterium Burkholderia cepacia AMMDR1 on two oomycete pathogens of pea (Pisum sativum L.)

Burkholderia cepacia AMMDR1 is a biocontrol agent that protects pea and sweet corn seeds from Pythium damping-off in field experiments. The goal of this work was to understand the effect of B. cepacia AMMDR1 on Pythium aphanidermatum and Aphanomyces euteiches zoospore homing events and on infection of pea seeds or roots. In vitro, B. cepacia AMMDR1 caused zoospore lysis, prevented cyst germination, and inhibited germ tube growth of both oomycetes. B. cepacia AMMDR1 also reduced the attractiveness of seed exudates to Pythium zoospores to nondetectable levels. However, when present at high levels on seeds, B. cepacia AMMDR1 had little net effect on zoospore attraction, probably because it also enhanced seed exudation. Seed-applied B. cepacia AMMDR1 dramatically reduced the incidence of infection by Pythium zoospores in situ compared with an antibiosis-deficient Tn5 mutant strain. This mutant strain also decreased Pythium infection incidence to some extent, but only when the pathogen inoculum potential was low. B. cepacia AMMDR1 did not affect attraction of Aphanomyces zoospores or Aphanomyces root rot incidence. These results suggest that B. cepacia AMMDR1 controls P. aphanidermatum largely through antibiosis, but competition for zoospore-attracting compounds can contribute to the effect. Differences in suppression of Aphanomyces and Pythium are discussed in relation to differences in the ecology of the two pathogens.     

Microbial community responses associated with the development of oomycete plant pathogens on tomato roots in soilless growing systems

AIMS: To determine the spread of different oomycete pathogens in hydroponic, soilless tomato growing systems and their impact on established microbial communities, as baseline studies prior to future introduction of microbial inoculants for disease suppression. METHODS AND RESULTS: The oomycete pathogens, Pythium group F, Pythium aphanidermatum and Phytophthora cryptogea, were introduced into small-scale recirculating tomato growing systems containing rockwool 6 weeks after set-up when roots were well-established. Two weeks later, half of the systems were switched over to run-to-waste. Pythium aphanidermatum spreads the fastest, Pythium group F the slowest and Ph. cryptogea was intermediate in its spread. The switch to run-to-waste had no effect on pathogen recovery. Microbial communities, monitored by dilution plating, were well-established at the first sampling, 6 weeks after set-up and although differences in community levels were found between experiments, changes during any one experiment were small, generally less than 1 log10 CFU g(-1) for bacteria. Pathogen introduction increased microbial community levels in roots but the switch to run-to-waste had no effect. Analysis of bacterial communities through amplification of a fragment of the 16S rRNA gene and DGGE profiling showed that different communities were established within each pathogen experiment and that different communities were established on roots, rockwool and in nutrient solutions. However, no significant changes in microbial profiles were found over time in any experiment. CONCLUSIONS: In these systems, the microbial communities were well-established 6 weeks after set-up and were resistant to biological and physical perturbation. SIGNIFICANCE AND IMPACT OF THE STUDY: The implication for microbial inoculation of such systems for disease suppression is that the micro-organisms would either have to be introduced very early during the set-up of the system or be able to replace an established but variable community.     

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.     

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.     

Dolichopodid Biodiversity and Site Quality Assessment of Reed Marshes And Grasslands in Belgium (Diptera: Dolichopodidae)

The conservation value of grassland and reed marsh habitats in Belgium was determined on the basis of dolichopodid communities (Diptera: Dolichopodidae). Four grassland and one reed marsh sites within the 'Bourgoyen-Ossemeersen' Nature Reserve (B.O.) (Ghent) were sampled with white water traps from March 1993 until March 1994. Subsequently, these soil faunas were compared with those of reed marsh habitats in 'Het Meetjeslandse Krekengebied' (M.K.) sampled in 1990. Comparisons of species communities were carried out by means of multivariate analysis techniques. The DCA and CCA on the B.O. sites produced a clear separation between the grassland and the reed marsh communities. Light intensity and soil humidity were among the most important factors determining species distributions. The comparison between the B.O. and M.K. communities revealed that sampling sites were clustered according to their geographical location rather than to habitat type. Overall species richness and diversity was not significantly different between grasslands and reed marshes. However, conservation quality estimates of the sites using the Site Conservation Quality Index (SCQI) indicated that reed marshes comprise a considerably larger number of rare and Red Data Book species. Moreover, besides typical reed marsh-inhabiting species, reed marshes also house species with other ecological affinities sometimes in rather high abundance. As a result, reed marshes are considered highly valuable for the survival of several wetland dolichopodid species.     

Pythium polare, a new heterothallic oomycete causing brown discolouration of Sanionia uncinata in the Arctic and Antarctic

Pythium polare sp. nov. is a new heterothallic oomycete species isolated from fresh water and moss from various locations in both the Arctic and Antarctic. This water mould is able to infect stems and leaves of Sanionia moss (Sanionia uncinata). Pythium polare causes brown discolouration in in vitro inoculation tests at 5 °C after 5 weeks of inoculation. It is characterized by globose sporangia with various lengths of discharge tubes releasing zoospores and aplerotic oospores with usually one to five antheridia. The sexual structures are only produced in a dual culture of antheridial and oogonial isolates. Phylogenetic analysis, based on ITS sequencing, places all isolated strains of P. polare in a unique new clade, hence it is considered a novel species. Pythium canariense and Pythium violae are the most closely related species of P. polare based both on morphology and the phylogenetic analysis.     

Ethylene insensitivity impairs resistance to soilborne pathogens in tobacco and Arabidopsis thaliana

Transgenic ethylene-insensitive tobacco (Tetr) plants spontaneously develop symptoms of wilting and stem necrosis when grown in nonautoclaved soil. Fusarium oxysporum, F. solani, Thielaviopsis basicola, Rhizopus stolonifer, and two Pythium spp. were isolated from these diseased Tetr plants and demonstrated to be causal agents of the disease symptoms. Pathogenicity of the two Pythium isolates and four additional Pythium spp. was tested on ethylene-insensitive tobacco and Arabidopsis seedlings. In both plant species, ethylene insensitivity enhanced susceptibility to the Pythium spp., as evidenced by both a higher disease index and a higher percentage of diseased plants. Based on the use of a DNA probe specific for Pythium spp., Tetr plants exhibited more pathogen growth in stem and leaf tissue than similarly diseased control plants. These results demonstrate that ethylene signaling is required for resistance to different root pathogens and contributes to limiting growth and systemic spread of the pathogen.     

Effects of winter harvest on water and sediment chemistry in a stand of reed (Phragmites australis)

An investigation of the effects of repeated winter harvest and removal of dry reed culms on water and sediment chemistry was made in a shallow lake dominated by closed, monospecific reed stands. The oxygen concentration and redox potential in the water, as well as temperature, showed regular diurnal fluctuations inside the reed stands. Ammonium and nitrate + nitrite did not exhibit diurnal fluctuations and differences between a harvested and an unharvested reed area were small. Molybdate reactive phosphorus (MRP) concentration was higher in the unharvested area.Because the winter harvest caused an almost doubling of shoot production compared to the unharvested area, net addition of reed litter (leaves, stubble and culms) to the sediment of the harvested area was similar to that of the unharvested area. It is concluded that removal of reed culms in winter has the largest effect on microclimate in spring, and that the rate of mineralization and reducing intensity in the substrate are not immediately affected. Significantly higher summer standing crop of reed after harvest may have caused lower phosphate concentration in the water column and sediment interstitial water.