Role of ectomycorrhizal colonization in enhancement of nutrients for survival of plants collected from mountainous cold stress areas.

BACKGROUND: Ectomycorrhizal (ECM and ECM-like) structures associated with plant root systems are a challenge for scientists. The dispersion pattern of roots within the soil profile and the nutritional conditions are both favourable factors to motivate the plants to make ECM associations. RESULTS: This study discusses the colonization of mycorrhizal associations in Kobresia and Polygonum species including Polygonum viviparum, Kobresia filicina, K. myosuroides, Alnus nitida, Betula pendula, Pinus sylvestris, and Trifolium repens grown naturally in cold stressed soils of Gilgit-Baltistan (high-altitude alpine Deosai plains), Hazara, Swat, Dir, and Bajaur. Sieved soil batches were exposed to +5 °C (control), -10, -20, -30, -40, -50, -125 °C for 5 h, and selected plants were sown to these soils for 10 weeks under favourable conditions for ECM colonization. Ectomycorrhizal associations were examined in the above mentioned plants. Some ECM fungi have dark mycelia that look like the mantle and Hartig net. Examples of these are Kobresia filicina, K. myosuroides, and Polygonum viviparum. Findings of this study revealed that K. myosuroides excelled in ECM root tip length, dry mass, and NH4 concentration at -125 °C. Contrarily, A. nitida demonstrated the lower values, indicated its minimum tolerance. Notably, T. repens boasted the highest nitrogen concentration (18.7 ± 1.31 mg/g), while P. sylvestris led in phosphorus (3.2 ± 0.22 mg/g). The B. pendula showed the highest potassium concentration (9.4 ± 0.66 mg/g), emphasising species-specific nutrient uptake capabilities in extreme cold conditions. The PCA analysis revealed that the parameters, e.g., NH4 in soil mix (NH4), NO3 in soil mix (NO3), phosphorus in soil in species of Polygonum viviparum, Kobresia filicina, K. myosuroides, Alnus nitida, Betula pendula, Pinus sylvestris, and Trifolium repens are most accurately represented in cases of + 5 °C, -10 °C, and -20 °C temperatures. On the other hand, the parameters for ECM root tips (ECM) and Dry Mass (DM) are best described in -40 °C, -50 °C, and - 125 °C temperatures. All parameters have a strong influence on the variability of the system indicated the efficiency of ECM. The heatmap supported the nutrients positively correlated with ECM colonization with the host plants. CONCLUSION: At lower temperatures, hyphae and spores in roots were reduced, while soluble phosphorus concentrations of leaves were increased in cold stress soils. Maximum foliar nutrient concentrations were found in K. myosuroides at the lowest temperature treatments due to efficient functioning and colonization of ECM.

Anaerobacillus alkaliphilus sp. nov., a novel alkaliphilic and moderately halophilic bacterium.

Two alkaliphilic and moderately halophilic bacterial strains B16-10T and Z23-18 characterized by optimal growth at pH 9.0-10.0 and 5 % (w/v) NaCl, were isolated from the rhizosphere soil of the bayonet grass (Bolboschoenus maritimus) in the Kiskunság National Park, Hungary. Cells of both strains stained Gram-positive, were motile straight rods, and formed terminal, ellipsoidal endospores with swollen sporangia. The isolates were facultative anaerobic, catalase positive, oxidase negative. Both strains contained meso-diaminopimelic acid as diagnostic diaminoacid of the peptidoglycan. Menaquinone-7 (MK-7) was the predominant isoprenoid quinone. Anteiso-C15 : 0, C16 : 1ω11c and iso-C14 : 0 were the major cellular fatty acids. The DNA G+C content of both strains was 35.8 mol%. The 16S rRNA gene based phylogenetic analysis revealed that the facultative anaerobic strains B16-10T and Z23-18 showed the highest similarities to the type strains of anaerobic Anaerobacillus isosaccharinicus NB2006T (98.7 and 99.1 %), A. macyae JMM-4T (98.2 and 98.4 %), A. alkalidiazotrophicus MS 6T (97.7 and 98.4 %), A. alkalilacustris Z-0521T (97.5 and 98.3 %) and A. arseniciselenatis DSM 15340T (97.5 and 98.2 %). However, the distinctive phenotypic and genetic results of this study confirmed that strains B16-10T and Z23-18 represent a novel species, for which the name Anaerobacillus alkaliphilus sp. nov. is proposed. The type strain is B16-10T (=DSM 29790T=NCAIM B 02608T).

Biosynthetic and antimicrobial potential of actinobacteria isolated from bulrush rhizospheres habitat in Zhalong Wetland, China.

The wetland ecosystem is known to possess unique vegetation and serves multiple functions within the environment. In this study, bacterial bioprospecting of bulrush rhizospheres in the Zhalong Wetland, China, was performed using comprehensive methods, including strain isolation and phylogenetic analysis, PCR detection of biosynthetic gene clusters, assessment of antimicrobial activity, metabolite profiling and genome analysis. A total of 27 actinobacterial strains were isolated, and their biosynthetic gene clusters (NRPS, PKS-I and PKS-II) were investigated; all of the tested strains had at least one of the three aforementioned biosynthetic gene clusters. Furthermore, fermentation broth extracts produced by these strains showed antimicrobial activities against certain pathogens, and ten of the extracts exhibited broad-spectrum antimicrobial activity. Liquid chromatography-mass spectrometry (LC-MS) analysis indicated chemical diversity of secondary metabolites from these extracts. Among these strains, ZLSD-24 generated the largest amounts and types of secondary metabolites. Subsequent genome analysis showed that 41 secondary metabolite biosynthetic gene clusters were present in the strain ZLSD-24, which was in accordance with the LC-MS data. Taken together, the results of this study reveal that bulrush rhizosphere habitat in the Zhalong wetland is a promising source of novel natural products.

Influences of Different Halophyte Vegetation on Soil Microbial Community at Temperate Salt Marsh.

Salt marshes are transitional zone between terrestrial and aquatic ecosystems, occupied mainly by halophytic vegetation which provides numerous ecological services to coastal ecosystem. Halophyte-associated microbial community plays an important role in the adaptation of plants to adverse condition and also affected habitat characteristics. To explore the relationship between halophytes and soil microbial community, we studied the soil enzyme activities, soil microbial community structure, and functional gene abundance in halophytes- (Carex scabrifolia, Phragmites australis, and Suaeda japonica) covered and un-vegetated (mud flat) soils at Suncheon Bay, South Korea. Higher concentrations of total, Gram-positive, Gram-negative, total bacterial, and actinomycetes PLFAs (phospholipid fatty acids) were observed in the soil underneath the halophytes compared with mud flat soil and were highest in Carex soil. Halophyte-covered soils had different microbial community composition due to higher abundance of Gram-negative bacteria than mud flat soil. Similar to PLFA concentrations, the increased activities of ß-glucosidase, cellulase, phosphatase, and sulfatase enzymes were observed under halophyte soil compared to mud flat soil and Carex exhibited highest activities. The abundance of archaeal 16S rRNA, fungal ITS, and denitrifying genes (nirK, nirS, and nosZ) were not influenced by the halophytes. Abundance bacterial 16S rRNA and dissimilatory (bi)sulfite (dsrA) genes were highest in Carex-covered soil. The abundance of functional genes involved in methane cycle (mcrA and pmoA) was not affected by the halophytes. However, the ratios of mcrA/pmoA and mcrA/dsrA increased in halophyte-covered soils which indicate higher methanogenesis activities. The finding of the study also suggests that halophytes had increased the microbial and enzyme activities, and played a pivotal role in shaping microbial community structure.

Contribution of arbuscular mycorrhizal fungi of sedges to soil aggregation along an altitudinal alpine grassland gradient on the Tibetan Plateau.

The diversity of arbuscular mycorrhizal fungi (AMF) in sedges on the Tibetan Plateau remains largely unexplored, and their contribution to soil aggregation can be important in understanding the ecological function of AMF in alpine ecosystems. Roots of Kobresia pygmaea C.B. Clarke and Carex pseudofoetida Kük. in alpine Kobresia pastures along an elevational transect (4149-5033 m) on Mount Mila were analysed for AMF diversity. A structural equation model was built to explore the contribution of biotic factors to soil aggregation. Sedges harboured abundant AMF communities covering seven families and some operational taxonomic units are habitat specific. The two plant species hosted similar AMF communities at most altitudes. The relative abundance of the two sedges contributed largely to soil macroaggregates, followed by extraradical mycorrhizal hyphae (EMH) and total glomalin-related soil protein (T-GRSP). The influence of plant richness was mainly due to its indirect influence on T-GRSP and EMH. There was a strong positive correlation between GRSP and soil total carbon and nitrogen. Our results indicate that mycorrhization might not be a major trait leading to niche differentiation of the two co-occurring sedge species. However, AMF contribute to soil aggregation and thus may have the potential to greatly influence C and N cycling in alpine grasslands.

Presence and persistence of wastewater pathogen Escherichia coli O157:H7 in hydroponic reactors of treatment wetland species.

Treatment wetlands (TWs) efficiently remove many pollutants including a several log order reduction of pathogens from influent to effluent; however, there is evidence to suggest that pathogen cells are sequestered in a subsurface wetland and may remain viable months after inoculation. Escherichia coli is a common pathogen in domestic and agricultural wastewater and the O157:H7 strain causes most environmental outbreaks in the United States. To assess attachment of E. coli to the TW rhizosphere, direct measurements of E. coli levels were taken. Experiments were performed in chemostats containing either Teflon nylon as an abiotic control or roots of Carex utriculata or Schoenoplectus acutus. Flow of simulated wastewater through the chemostat was set to maintain a 2 hour residence time. The influent was inoculated with E. coli O157:H7 containing DsRed fluorescent protein. Root samples were excised and analyzed via epifluorescent microscopy. E. coli O157:H7 was detected on the root surface at 2 hours after inoculation, and were visible as single cells. Microcolonies began forming at 24 hours post-inoculation and were detected for up to 1 week post-inoculation. Image analysis determined that the number of microcolonies with >100 cells increased 1 week post-inoculation, confirming that E. coli O157:H7 is capable of growth within biofilms surrounding wetland plant roots.

Functional analysis of a tannic-acid-inducible and hypoviral-regulated small heat-shock protein Hsp24 from the chestnut blight fungus Cryphonectria parasitica.

A small heat-shock protein gene, CpHsp24, of Cryphonectria parasitica was selected based on its expression pattern, which showed that it was tannic acid inducible and that its induction was severely hampered by a hypovirus. The predicted protein sequence of CpHsp24 consisted of a hallmark α-crystalline domain flanked by a variable N-terminal and a short C-terminal region. Disruption of CpHsp24 resulted in a slow growth rate under standard growth conditions. The CpHsp24-null mutant showed enhanced sensitivity to heat shock, which was consistent with Northern and Western analyses displaying the heat-shock induction of the CpHsp24 gene and protein, respectively. Virulence tests on the excised bark revealed a severe decrease in the necrotic area of the CpHsp24-null mutant. When the hypovirus was transferred, virus-containing CpHsp24-null progeny displayed severely retarded growth patterns with hypovirulent characteristics of reduced pigmentation and sporulation. Because the tannic-acid-inducible and hypoviral-suppressible expression and the severely impaired virulence are also characteristics of the laccase3 gene (lac3), lac3 expression in the CpHsp24-null mutant was also examined. The resulting lac3 induction was severely affected in the CpHsp24-null mutant, suggesting that CpHsp24 is important for lac3 induction and that CpHsp24 may act as a molecular chaperone for the lac3 protein.

Molecular diversity of arbuscular mycorrhizal fungi associated with two co-occurring perennial plant species on a Tibetan altitudinal gradient.

Plant communities on Mount Segrila on the Tibetan Plateau show distinct changes at different altitudes, but little information is available on belowground communities of arbuscular mycorrhizal fungi (AMF). Root samples of two co-occurring species, Pennisetum centrasiaticum and Kobresia sp., growing in open grasslands at eight altitudes (3,446-4,556 m) were analyzed for diversity of AMF by PCR, cloning, and sequencing. Dominant plants were well colonized by AMF even at higher altitudes where spore density in rhizospheres decreased dramatically. A total of 29 operational taxonomic units (OTUs) of AMF were detected, and some novel sequence types were found. Acaulosporaceae and Glomeraceae were the dominant families. There was no significant difference in OTU richness along elevational gradients in Kobresia sp., but OTU richness in P. centrasiaticum was higher at intermediate elevations. Elevation, host plant species, and soil variables (pH, soil organic matter, and available P and N) were found to have significant effects on the overall AMF community across all elevations. Fungal community composition differed significantly between the two plant species at each elevation, and the similarity was generally higher at the intermediate elevations. No significant difference in compositional similarity was observed for Kobresia sp. with increasing elevation, but the dissimilarity increased significantly for P. centrasiaticum. These results suggest that host identity is an important determinant for the structure of the AMF communities along the elevational gradients in high altitude environments.

Rhizosphere effect of Scirpus triqueter on soil microbial structure during phytoremediation of diesel-contaminated wetland.

Though phytoremediation has been widely used to restore various contaminated sites, it is still unclear how soil microbial communities respond microecologically to plants and pollutants during the process. In this paper, batch microcosms imitating in situ phytoremediation of petroleum-contaminated wetland by Scirpus triqueter were set up to monitor the influence of plant rhizosphere effect on soil microbes. Palmitic acid, one of the main root exudates of S. triqueter, was added to strengthen rhizosphere effect. Abundances of certain microbial subgroups were quantified by phospholipid fatty acid profiles. Results showed that diesel removal extents were significantly higher in the rhizosphere (57.6 +/-4.2-65.5 +/- 6.9%) than those in bulk soil (27.8 +/-6.5-36.3 +/- 3.2%). In addition, abundances of saturated, monounsaturated, and polyunsaturated fatty acids were significantly higher (P < 0.05) in planted soil than those in the bulk soil. When it was less than 15,000 mg diesel kg soil-1, increasing diesel concentration led to higher abundances of fungi, Gram-positive and Gram-negative bacteria. The addition of palmitic acid amplified the rhizosphere effect on soil microbial populations and diesel removal. Principal component analysis revealed that plant rhizosphere effect was the dominant factor affecting microbial structure. These results provided new insights into plant-microbe-pollutant coactions responsible for diesel degradation, and they were valuable to facilitate phytoremediation of diesel contamination in wetland habitats.

Enhanced inhibition of Aspergillus niger on sedge (Lepironia articulata) treated with heat-cured lime oil.

AIMS: This study aimed to examine heat curing effect (30-100°C) on antifungal activities of lime oil and its components (limonene, p-cymene, ß-pinene and α-pinene) at concentrations ranging from 100 to 300 µl ml(-1) against Aspergillus niger in microbiological medium and to optimize heat curing of lime oil for efficient mould control on sedge (Lepironia articulata). METHODS AND RESULTS: Broth dilution method was employed to determine lime oil minimum inhibitory concentration, which was at 90 µl ml(-1) with heat curing at 70°C. Limonene, a main component of lime oil, was an agent responsible for temperature dependencies of lime oil activities observed. Response surface methodology was used to construct the mathematical model describing a time period of zero mould growth on sedge as functions of heat curing temperature and lime oil concentration. Heat curing of 90 µl ml(-1) lime oil at 70°C extended a period of zero mould growth on sedge to 18 weeks under moist conditions. CONCLUSIONS: Heat curing at 70°C best enhanced antifungal activity of lime oil against A. niger both in medium and on sedge. SIGNIFICANCE AND IMPACT OF THE STUDY: Heat curing of lime oil has potential to be used to enhance the antifungal safety of sedge products.

Rhizosphere bacteria of Costularia spp. from ultramafic soils in New Caledonia: diversity, tolerance to extreme edaphic conditions, and role in plant growth and mineral nutrition.

Rhizosphere bacteria were isolated from Costularia spp., pioneer sedges from ultramafic soils in New Caledonia, which is a hotspot of biodiversity in the South Pacific. Genus identification, ability to tolerate edaphic constraints, and plant-growth-promoting (PGP) properties were analysed. We found that 10(5) colony-forming units per gram of root were dominated by Proteobacteria (69%) and comprised 21 genera, including Burkholderia (28%), Curtobacterium (15%), Bradyrhizobium (9%), Sphingomonas (8%), Rhizobium (7%), and Bacillus (5%). High proportions of bacteria tolerated many elements of the extreme edaphic conditions: 82% tolerated 100 µmol·L(-1) chromium, 70% 1 mmol·L(-1) nickel, 63% 10 mmol·L(-1) manganese, 24% 1 mmol·L(-1) cobalt, and 42% an unbalanced calcium/magnesium ratio (1/16). These strains also exhibited multiple PGP properties, including the ability to produce ammonia (65%), indole-3-acetic acid (60%), siderophores (52%), and 1-aminocyclopropane-1-carboxylate (ACC) deaminase (39%); as well as the capacity to solubilize phosphates (19%). The best-performing strains were inoculated with Sorghum sp. grown on ultramafic substrate. Three strains significantly enhanced the shoot biomass by up to 33%. The most successful strains influenced plant nutrition through the mobilization of metals in roots and a reduction of metal transfer to shoots. These results suggest a key role of these bacteria in plant growth, nutrition, and adaptation to the ultramafic constraints.

Mycorrhizal status of Cyperaceae from New Caledonian ultramafic soils: effects of phosphorus availability on arbuscular mycorrhizal colonization of Costularia comosa under field conditions.

Plants from the Cyperaceae family (sedges), usually considered as non-mycorrhizal, constitute almost exclusively the herbaceous stratum of the ultramafic maquis in New Caledonia. These plants are pioneers and are important for the ecological restoration of mined areas. Costularia comosa, one of the most common sedges in this environment, was grown under field conditions on ultramafic soil, fertilized or not with phosphate and/or nitrogen. Results showed that the addition of phosphate to the soil induced a clear increase in mycorrhizal colonization of C. comosa and an increase in arbuscule abundance, reflecting the establishment of a functional mycorrhizal symbiosis. Significant positive correlations were found among mycorrhizal parameters and plant or soil phosphorus concentrations. Nitrogen fertilization did not affect mycorrhizal colonization of C. comosa. The improvement in mycorrhizal colonization by phosphate fertilization did not influence significantly nickel concentrations in the roots and shoots of plants. This study demonstrated that phosphate fertilization of ultramafic soil improved mycorrhizal colonization of C. comosa, with formation of a functional symbiosis under field conditions.

Effect of environmental gradient in coastal vegetation on communities of arbuscular mycorrhizal fungi associated with Ixeris repens (Asteraceae).

The community structure of arbuscular mycorrhizal (AM) fungi associated with Ixeris repens was studied in coastal vegetation near the Tottori sand dunes in Japan. I. repens produces roots from a subterranean stem growing near the soil surface which provides an opportunity to examine the effects of an environmental gradient related to distance from the sea on AM fungal communities at a regular soil depth. Based on partial sequences of the nuclear large subunit ribosomal RNA gene, AM fungi in root samples were divided into 17 phylotypes. Among these, five AM fungal phylotypes in Glomus and Diversispora were dominant near the seaward forefront of the vegetation. Redundancy analysis of the AM fungal community showed significant relationships between the distribution of phylotypes and environmental variables such as distance from the sea, water-soluble sodium in soil, and some coexisting plant species. These results suggest that environmental gradients in the coastal vegetation can be determinants of the AM fungal community.

Nocardioides caricicola sp. nov., an endophytic bacterium isolated from a halophyte, Carex scabrifolia Steud.

A Gram-staining-positive, coccoid to rod-shaped bacterium, designated strain YC6903(T), was isolated from a halophytic plant (Carex scabrifolia Steud.) collected from sand dunes at Namhae Island, Korea, and its taxonomic position was investigated by using a polyphasic approach. Strain YC6903(T) grew optimally at 30 °C and at pH 8.0. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain YC6903(T) belongs to the genus Nocardioides in the family Nocardioidaceae. Strain YC6903(T) was related most closely to Nocardioides pyridinolyticus OS4(T) (97.0 % 16S rRNA gene sequence similarity), Nocardioides dokdonensis FR1436(T) (96.6 %), Nocardioides aquiterrae GW-9(T) (96.6 %) and Nocardioides hankookensis DS-30(T) (96.6 %). The cell-wall peptidoglycan contained LL-diaminopimelic acid and MK-8(H(4)) was the major respiratory quinone. The mean (±SD) level of DNA-DNA relatedness between strain YC6903(T) and N. pyridinolyticus OS4(T) was 53.5±5.5 %. The predominant cellular fatty acid of strain YC6903(T) was iso-C(16 : 0) (28.9 %). The DNA G+C content was 71.7 mol%. Phenotypic, phylogenetic and chemotaxonomic data indicated that strain YC6903(T) represents a novel species of the genus Nocardioides, for which the name Nocardioides caricicola sp. nov. is proposed. The type strain is YC6903(T) (=KACC 13778(T) =DSM 22177(T)).

New insights into the mycorrhizal status of Cyperaceae from ultramafic soils in New Caledonia.

In New Caledonia, a hot spot of biodiversity, plants from the Cyperaceae family are mostly endemic and considered pioneers of the nickel-rich natural serpentine ecosystem. The aim of the study was to highlight the mycorrhizal status of these Cyperaceae and to bring new insights into the role of this symbiosis in plant tolerance to ultramafic soils. Nine Cyperaceae species were studied and presented evidence of root colonization by arbuscular mycorrhizas (AMs), with frequencies ranging from 8% to 57%. The highest level of AM colonization was observed in plants from the endemic dominant genus Costularia. Molecular evidence demonstrated the presence of Glomus sp. inside the roots. In a controlled greenhouse assay, AM inoculation of Costularia comosa grown under ultramafic conditions significantly enhanced plant growth, with an increase in biomass by up to 2.4-fold for shoots and 1.2-fold for roots, and also reduced nickel content in roots by 2.5-fold, as compared with the controls. All these data support our hypotheses (i) that a relationship exists between the mycorrhizal status of Cyperaceae and their habitat, and (ii) that AM have a positive role in plant tolerance to ultramafic soils (mineral nutrition and metal tolerance), suggesting the use of these pioneer plants with AM management as potential tools for nickel mine site rehabilitation in New Caledonia.

Ectomycorrhizal fungi associated with two species of Kobresia in an alpine meadow in the eastern Himalaya.

The diversity of ectomycorrhizal fungi (EMF) on Kobresia filicina and Kobresia capillifolia in an alpine meadow in China's southwestern mountains, one of the word's hotspots of biodiversity, was estimated based on internal transcribed spacer rDNA sequence analysis of root tips. Seventy EMF operational taxonomical units (OTUs) were found in the two plant species. Dauciform roots with EMF were detected in species of Kobresia for the first time. OTU richness of EMF was high in Tomentella/Thelophora and Inocybe, followed by Cortinarius, Sebacina, the Cenococcum geophilum complex, and Russula. Tomentella/Thelophora and Inocybe were general and dominant mycobiont genera of the two sedges. Besides the C. geophilum complex, the ascomycete components Hymenoscyphus and Lachnum were also detected on the two plants. Alpine plants in different geographical regions share similar main genera and/or families of EMF while harboring predominantly different mycobiont species; most of the members detected by us have not been found elsewhere. Significant differences in the profile of EMF occurrences were not found between the two plant species and among the three sampling seasons in our sample size.

Impact of rhizobacteria on growth and chromium accumulation in Scirpus lacustris L. grown under chromium supplementation.

Four chromate tolerant rhizobacterial strains viz., RZB-01, RZB-02, RZB-03 and RZB-04 were isolated from rhizosphere of Scirpus lacustris collected from Cr-contaminated area. These strains characterized at morphological and biochemical levels. The most efficient chromate tolerant strain RZB-03 was inoculated to fresh plant of S. lacustris and grown in 2 microg ml(-1) and 5 microg ml(-1) of Cr+6 supplemented nutrient solution under controlled laboratory condition. The effects of rhizobacterial inoculation on growth and chromium accumulation in S. lacustris were evaluated. The inoculation of rhizobacteria increased biomass by 59 and 104%, while total chlorophyll content by 1.76 and 15.3% and protein content increased by 23 and 138% under 2 microg ml(-1) and 5 microg ml(-1) concentrations of Cr+6, respectively after 14 d as compared to non-inoculated plant. Similarly, the Cr accumulation also increased by 97 and 75% in shoot and 114 and 68% in root of inoculated plants as compared to non inoculated plants at 2 microg ml(-1) and 5 microg ml(-1) Cr+6 concentrations, respectively after 14 d. The chromate tolerant rhizobacteria which play an important role in chromium uptake and growth promotion in plant may be useful in development of microbes assisted phytoremediation system for decontamination of chromium polluted sites.

Silencing of Kex2 significantly diminishes the virulence of Cryphonectria parasitica.

Cryphonectria parasitica is the causal agent of chestnut blight. Infection of this ascomycete with Cryphonectria hypovirus 1 (CHV1) results in reduction of virulence and sporulation of the fungus. The virus affects fungal gene expression and several of the CHV1 downregulated genes encode secreted proteins that contain consensus Kex2 processing signals. Additionally, CHV1 has been shown to colocalize in infected cells primarily with fungal trans-Golgi network vesicles containing the Kex2 protease. We report here the cloning, analysis, and possible role of the C. parasitica Kex2 gene (CpKex2). CpKex2 gene sequence analysis showed high similarity to other ascomycete kexin-like proteins. Southern blot analyses of CpKex2 showed a single copy of this gene in the fungal genome. In order to monitor the expression and evaluate the function of CpKex2, antibodies were raised against expressed protein and Kex2-silenced mutants were generated. Western blots indicate that the Kex2 protein was constitutively expressed. Growth rate of the fungus was not significantly affected in Kex2-silenced strains; however, these strains showed reduced virulence, reduced sexual and asexual sporulation, and reductions in mating and fertility. The reduced virulence was correlated with reduced Kex2 enzymatic activity and reduced relative mRNA transcript levels as measured by real time reverse-transcriptase polymerase chain reaction. These results suggest that secreted proteins processed by Kex2 are important in fungal development and virulence.

Heterotrophic N2-fixation contributes to nitrogen economy of a common wetland sedge, Schoenoplectus californicus.

A survey of the ecological variability within 52 populations of Schoenoplectus californicus (C.A. Mey.) Soják across its distributional range revealed that it is commonly found in nitrogen (N) limited areas, but rarely in phosphorus limited soils. We explored the hypothesis that S. californicus supplements its nitrogen demand by bacterial N2-fixation processes associated with its roots and rhizomes. We estimated N2-fixation of diazotrophs associated with plant rhizomes and roots from several locations throughout the species' range and conducted an experiment growing plants in zero, low, and high N additions. Nitrogenase activity in rhizomes and roots was measured using the acetylene reduction assay. The presence of diazotrophs was verified by the detection of the nifH gene. Nitrogenase activity was restricted to rhizomes and roots and it was two orders of magnitude higher in the latter plant organs (81 and 2032 nmol C2H4 g DW-1 d-1, respectively). Correspondingly, 40x more nifH gene copies were found on roots compared to rhizomes. The proportion of the nifH gene copies in total bacterial DNA was positively correlated with the nitrogenase activity. In the experiment, the contribution of fixed N to the plant N content ranged from 13.8% to 32.5% among clones from different locations. These are relatively high values for a non-cultivated plant and justify future research on the link between N-fixing bacteria and S. californicus production.

Cadophora meredithiae and C. interclivum, new species from roots of sedge and spruce in a western Canada subalpine forest.

Two new species of Cadophora are described based on multigene phylogenetic analyses and phenotypic and ecological characters. The species delimitation was based on concordance of gene genealogies. The cultures of the Cadophora species were isolated from the roots of long-beaked sedge and white spruce from a subalpine forest in western Canada; however, they probably have a broader distribution because their internal transcribed spacer (ITS) sequences have high similarity with a number of GenBank sequences from ecological studies of plant roots. The taxonomy of Cadophora in Leotiomycetes is discussed based on the phylogeny generated in this study. Results from this work will facilitate ecological and evolutionary studies on root-associated fungi.