Igneous phosphate rock solubilization by biofilm-forming mycorrhizobacteria and hyphobacteria associated with Rhizoglomus irregulare DAOM 197198.

Biofilm formation on abiotic and biotic surfaces was studied with two hyphobacteria, strongly attached to the surface of the arbuscular mycorrhizal fungus (AMF) Rhizoglomus irregulare (Ri) DAOM 197198 and two mycorrhizobacteria, loosely attached to the roots of different mycorrhizal plants. When the sparingly soluble igneous phosphate rock (PR) from Quebec, or when the chemical hydroxyapatite were used as sole phosphorus (P) source, hyphobacteria Rhizobium miluonense Rm3 and Burkholderia anthina Ba8 produced significantly more biofilms than mycorrhizobacteria Rahnella sp. Rs11 and Burkholderia phenazinium Bph12, as indicated by the crystal violet assay or by quantifying biofilm exopolysaccharides. As previously observed with planktonic bacteria, biofilms mobilized P by lowering the pH and releasing gluconic acid. The high efficiency of P mobilization by the hyphobacteria Ba8 was linked to the presence of more viable cells in its biofilm as revealed by the hydrolysis of fluorescein diacetate. Scanning electron microscopy micrographs showed a high adherence of the best P-solubilizer hyphobacteria Ba8 on the surface of Quebec PR. Hydroxyapatite porous structure did not allow a good adherence of Ba8. Ba8 formed an important biofilm on the hyphae of Ri DAOM 197198 with low reactive Quebec PR while no biofilm was observed with the high reactive hydroxyapatite. Results confirm the possible presence of specificity between the Ri DAOM 197198 and the hyphobacteria and suggest that the interaction would be regulated by the availability of P.

Phosphate transporter genes as reliable gene markers for the identification and discrimination of arbuscular mycorrhizal fungi in the genus glomus.

An inorganic phosphate transporter gene sequence (852-bp section) allowed discrimination between 10 Glomus fungal species represented by 25 strains. It was particularly valuable in differentiating between morphologically similar species with nucleotide and amino acid sequence differences higher than 3%. This gene is proposed as a reliable barcode for the Glomeromycetes.

Impact of endochitinase-transformed white spruce on soil fungal biomass and ectendomycorrhizal symbiosis.

The impact of transgenic white spruce [Picea glauca (Moench) Voss] containing the endochitinase gene (ech42) on soil fungal biomass and on the ectendomycorrhizal fungi Wilcoxina spp. was tested using a greenhouse trial. The measured level of endochitinase in roots of transgenic white spruce was up to 10 times higher than that in roots of nontransformed white spruce. The level of endochitinase in root exudates of three of four ech42-transformed lines was significantly greater than that in controls. Analysis soil ergosterol showed that the amount of fungal biomass in soil samples from control white spruce was slightly larger than that in soil samples from ech42-transformed white spruce. Nevertheless, the difference was not statistically significant. The rates of mycorrhizal colonization of transformed lines and controls were similar. Sequencing the internal transcribed spacer rRNA region revealed that the root tips were colonized by the ectendomycorrhizal fungi Wilcoxina spp. and the dark septate endophyte Phialocephala fortinii. Colonization of root tips by Wilcoxina spp. was monitored by real-time PCR to quantify the fungus present during the development of ectendomycorrhizal symbiosis in ech42-transformed and control lines. The numbers of Wilcoxina molecules in the transformed lines and the controls were not significantly different (P > 0.05, as determined by analysis of covariance), indicating that in spite of higher levels of endochitinase expression, mycorrhization was not inhibited. Our results indicate that the higher levels of chitinolytic activity in root exudates and root tissues from ech42-transformed lines did not alter the soil fungal biomass or the development of ectendomycorrhizal symbiosis involving Wilcoxina spp.

Morchella tomentosa: a unique belowground structure and a new clade of morels.

Mechanisms involved in post-fire morel fructification remain unclear. A new undescribed belowground vegetative structure of Morchella tomentosa in a burned boreal forest was investigated north of Fairbanks, Alaska. The name "radiscisclerotium" is proposed to define this peculiar and elaborate below-ground vegetative structure of M. tomentosa. Bayesian and maximum parsimony analyses based on ITS rRNA regions and nLSU gene strongly supported a new clade composed of M. tomentosa within the genus Morchella.

Ectomycorrhizal fungi affect the physiological responses of Picea glauca and Pinus banksiana seedlings exposed to an NaCl gradient.

We tested the effects of ectomycorrhizal (ECM) inoculation on greenhouse-grown white spruce (Picea glauca (Moench) Voss) and jack pine (Pinus banksiana L.) seedlings to be used for revegetation of salt-affected tailing sands resulting from the exploitation of oil sand in northeastern Alberta, Canada. White spruce and jack pine seedlings were inoculated with three ECM fungi selected for their in vitro tolerance to excess Na+ and Cl-: Hebeloma crustuliniforme (Bull) Quel. UAMH 5247, Laccaria bicolor Maire (Orton) UAMH 8232 and a Suillus tomentosus (Kauff.) Sing., Snell and Dick isolate from a salt-affected site. The physiological responses of the seedlings to a gradient of NaCl concentration (0, 50, 100 and 200 mM) were assessed over four weeks by: (1) Na+ accumulation and allocation; (2) chlorophyll a fluorescence; (3) growth, (4) water content; and (5) organic osmolyte accumulation. Jack pine seedlings were more sensitive than white spruce seedlings to increasing Na+ and Cl- concentrations. Both species showed decreasing biomass accumulation, and increasing concentrations of organic osmotica and Na with increasing NaCl concentration. White spruce seedlings inoculated with the S. tomentosus isolate had the best growth response at all NaCl concentrations tested. Although jack pine seedlings inoculated with the L. bicolor or S. tomentosus isolate exhibited the highest growth in the 50 and 100 mM NaCl treatments, both fungi increased the photochemical stress and dehydration of their hosts in the 200 mM NaCl treatment. At the latter concentration, jack pine seedlings inoculated with H. crustuliniforme showed the greatest tolerance to salt stress. Although the different fungi altered the physiological response of the host in different ways, inoculation with salt-stress-tolerant ECM fungi increased growth and reduced the negative effects of excess NaCl. Use of controlled mycorrhization may increase survival of coniferous seedlings used for revegetation of salt-affected sites.

Use of phylogenetical analysis to predict susceptibility of pathogenic Candida spp. to antifungal drugs.

Successful treatment of a Candida infection relies on 1) an accurate identification of the pathogenic fungus and 2) on its susceptibility to antifungal drugs. In the present study we investigated the level of correlation between phylogenetical evolution and susceptibility of pathogenic Candida spp. to antifungal drugs. For this, we compared a phylogenetic tree, assembled with the concatenated sequences (2475-bp) of the ATP2, TEF1, and TUF1 genes from 20 representative Candida species, with published minimal inhibitory concentrations (MIC) of the four principal antifungal drug classes commonly used in the treatment of candidiasis: polyenes, triazoles, nucleoside analogues, and echinocandins. The phylogenetic tree revealed three distinct phylogenetic clusters among Candida species. Species within a given phylogenetic cluster have generally similar susceptibility profiles to antifungal drugs and species within Clusters II and III were less sensitive to antifungal drugs than Cluster I species. These results showed that phylogenetical relationship between clusters and susceptibility to several antifungal drugs could be used to guide therapy when only species identification is available prior to information pertaining to its resistance profile. An extended study comprising a large panel of clinical samples should be conducted to confirm the efficiency of this approach in the treatment of candidiasis.

Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state.

The extent to which the N2-fixing bacterial endophyte Azoarcus sp. strain BH72 in the rhizosphere of Kallar grass can provide fixed nitrogen to the plant was assessed by evaluating inoculated plants grown in the greenhouse and uninoculated plants taken from the natural environment. The inoculum consisted of either wild-type bacteria or nifK- mutant strain BHNKD4. In N2-deficient conditions, plants inoculated with strain BH72 (N2-fixing test plants) grew better and accumulated more nitrogen with a lower delta15N signature after 8 months than did plants inoculated with the mutant strain (non-N2-fixing control plants). Polyadenylated or polymerase chain reaction-amplified BH72 nifH transcripts were retrieved from test but not from control plants. BH72 nifH transcripts were abundant. The inocula could not be reisolated. These results indicate that Azoarcus sp. BH72 can contribute combined N2 to the plant in an unculturable state. Abundant BH72 nifH transcripts were detected also in uninoculated plants taken from the natural environment, from which Azoarcus sp. BH72 also could not be isolated. Quantification of nitrogenase gene transcription indicated a high potential of strain BH72 for biological N2 fixation in association with roots. Phylogenetic analysis of nitrogenase sequences predicted that uncultured grass endophytes including Azoarcus spp. are ecologically dominant and play an important role in N2-fixation in natural grass ecosystems.

Carbon partitioning in a split-root system of arbuscular mycorrhizal plants is fungal and plant species dependent.

• Root carbon (C) partitioning in two host plant species colonized by one of three arbuscular mycorrhizal (AM) fungal species was investigated. • Split-root systems of barley (Hordeum vulgare) and sugar maple (Acer saccharum) were inoculated on one side with one of three AM fungi. Leaves were labelled with 14 CO2 3 wk after inoculation. Plants were harvested 24 h later and the root systems from the mycorrhizal (M) and nonmycorrhizal (NM) sides were analysed separately for 14 C. • Partitioning of 14 C between M and NM sides varied depending on the fungal and host plant species used. Gigaspora rosea showed a strong C-sink capacity with both plant species, Glomus intraradices showed a strong C-sink capacity with barley, and Glomus mosseae did not affect 14 C partitioning. The C-sink strength of the M barley roots inoculated with G. rosea or G. intraradices was linearly correlated with the degree of colonization. • The use of three AM fungal and two plant species allowed us to conclude that C-sink strength of AM fungi depends on both partners involved in the symbiosis.

Glomus intraradices causes differential changes in amino acid and starch concentrations of in vitro strawberry subjected to water stress.

The effect of colonization of tissue-cultured strawberry (Fragaria×ananassa Duch. cv. Kent) plantlets in vitro by the arbuscular mycorrhizal fungus (AMF) Glomus intraradices on plantlet response to poly(ethylene glycol) (PEG)-8000-induced water stress was investigated. The plantlets were inoculated axenically and co-cultured with the AMF for 4 wk, then transferred to 15% PEG-8000 solutions for 4, 8 and 12 h. Relative water content, water potential, osmotic potential, leaf conductance for water vapour diffusion and photosynthetic efficiency as estimated by chlorophyll a fluorescence were all affected by the PEG treatment and its duration but not by the presence of the intraradical phase of the AMF. However, distinct differences in PEG-induced changes in amino acid content were observed between nonmycorrhizal and mycorrhizal plantlets. In the latter, the treatment with PEG caused a substantial decrease in asparagine levels in leaves that was accompanied by a marked increase in asparagine concentration in roots. The opposite was observed in nonmycorrhizal plantlets. Furthermore, concentrations of aspartic acid, serine, threonine, amino-N-butyric acid, alanine and starch increased in roots of mycorrhizal and decreased in nonmycorrhizal plantlets. Our results suggest the presence of a mobile pool of asparagine that can be translocated from leaves to roots or vice versa in response to PEG-induced water stress, depending on the mycorrhizal status of the plantlets. These opposite patterns suggest different strategies of mycorrhizal and nonmycorrhizal plantlets to water stress, which seem to involve different adjustments in nitrogen and carbon metabolism.

14C transfer between the spring ephemeral Erythronium americanum and sugar maple saplings via arbuscular mycorrhizal fungi in natural stands.

We investigated in the field the carbon (C) transfer between sugar maple (Acer saccharum) saplings and the spring ephemeral Erythronium americanum via the mycelium of arbuscular mycorrhizal (AM) fungi. Sugar maple saplings and E. americanum plants were planted together in pots placed in the ground of a maple forest in 1999. Ectomycorrhizal yellow birches (Betula alleghaniensis) were added as control plants. In spring 2000, during leaf expansion of sugar maple saplings, the leaves of E. americanum were labelled with 14CO2. Seven days after labelling, radioactivity was detected in leaves, stem and roots of sugar maples. Specific radioactivity in sugar maples was 13-fold higher than in yellow birches revealing the occurrence of a direct transfer of 14C between the AM plants. The quantity of 14C transferred to sugar maple saplings was negatively correlated with the percentage of 14C allocated to the storage organ of E. americanum. A second labelling was performed in autumn 2000 on sugar maple leaves during annual growth of E. americanum roots. Radioactivity was detected in 7 of 22 E. americanum root systems and absent in yellow birches. These results suggest that AM fungi connecting different understorey species can act as reciprocal C transfer bridges between plant species in relation with the phenology of the plants involved.

Lophodermium macci sp. nov., a new species on senesced foliage of five-needle pines.

The new species Lophodermium macci is described. It is similar in its morphology, habitat, geographic range and ecology to L. pini-excelsae, L. staleyi and L. nitens and often is misidentified as L. pinastri on Pinus strobus in herbaria. A modified technique was used to extract DNA from minute ascomata on herbarium specimens, and new primers were made to amplify the damaged DNA from these specimens. It provides added evidence to separate L. macci from L. pini-excelsae, its closest morphological taxon.

Differential morphogenesis of the extraradical mycelium of an arbuscular mycorrhizal fungus grown monoxenically on spatially heterogeneous culture media.

A new in vitro experimental system was developed to study the morphogenesis of discrete regions of a single extraradical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices, growing simultaneously in six different agar-based media. The media were (i) unamended water agar (WA), (ii) WA+PO(4)(3-) (PO(4)(3-)), (iii) WA+NO(3)(-) (NO(3)(-)), (iv) WA+NH(4)(+) (NH(4)(+)), (v) WA+NH(4)(+)+MES (NH(4)(+)+MES) and (vi) minimal medium (M, complete nutrients). Each medium was amended with the pH indicator bromocresol purple. The extraradical mycelium of the fungus showed between-treatment differences in morphogenesis, architecture, formation of branched absorbing structures (BAS) and sporulation. Extraradical hyphae that developed in WA or PO(4)(3-) compartments exhibited an economic development pattern, in which runner hyphae radially extended the external colony. Extraradical hyphal growth in the NO(3)(-) compartments was characterized by increased formation of runner hyphae, BAS and spores and an alkalinization of the medium. In the two NH(4)(+)-amended media (NH(4)(+), NH(4)(+)+MES), sporulation was suppressed and considerable morphological changes were noted. These results show the plasticity of G. intraradices that lets it efficiently exploit an heterogeneous substrate.

A fungal endophyte of black spruce (Picea mariana) needles is also an aquatic hyphomycete.

An aquatic hyphomycete, Dwayaangam sp., was isolated from superficially sterilized black spruce (Picea mariana) needles submerged in aerated water in a small glass chamber (microcosm). The internal transcribed spacer (ITS) sequence of this fungus and of a commonly encountered foliar endophyte isolated from P. mariana showed a high degree of similarity. When sporulation was induced in the microcosm, both the aquatic hyphomycete and the endophyte isolate produced similar aquatic conidia after 30 days, which is longer than previously documented in similar studies. Without the use of molecular tools, the link between the aquatic and endophytic phases of the fungus would have gone unnoticed. This is the first time that a fungal endophyte of conifer needles has been shown to have an aquatic phase. Its presence both as a foliar endophyte and a sporulating aquatic fungus suggests an alternating life cycle between the two environments.

Stereoselective reduction of ketones by Daucus carota hairy root cultures.

Reduction of acetophenone by Daucus carota hairy root cultures afforded (S)-phenylethanol in high yield (96%) and excellent enantiomeric excess (ee>or=98%). Aromatic ketones, keto esters, and a simple aliphatic ketone were reduced with good stereoselectivity (ee=62-98%) and moderate to high chemical yields (25-90%).

Suppression of arbuscular mycorrhizal colonization and nodulation in split-root systems of alfalfa after pre-inoculation and treatment with Nod factors.

Roots of legumes establish symbiosis with arbuscular mycorrhizal fungi (AMF) and nodule-inducing rhizobia. The existing nodules systemically suppress subsequent nodule formation in other parts of the root, a phenomenon termed autoregulation. Similarly, mycorrhizal roots reduce further AMF colonization on other parts of the root system. In this work, split- root systems of alfalfa (Medicago sativa) were used to study the autoregulation of symbiosis with Sinorhizobium meliloti and the mycorrhizal fungus Glomus mosseae. It is shown that nodulation systemically influences AMF root colonization and vice versa. Nodules on one half of the split-root system suppressed subsequent AMF colonization on the other half. Conversely, root systems pre-colonized on one side by AMF exhibited reduced nodule formation on the other side. An inhibition effect was also observed with Nod factors (lipo-chito-oligosaccharides). NodSm-IV(C16:2, S) purified from S. meliloti systemically suppressed both nodule formation and AMF colonization. The application of Nod factors, however, did not influence the allocation of (14)C within the split-root system, excluding competition for carbohydrates as the regulatory mechanism. These results indicate a systemic regulatory mechanism in the rhizobial and the arbuscular mycorrhizal association, which is similar in both symbioses.

Breaking dormancy is spores of the arbuscular mycorrhizal fungus Glomus intraradices: a critical cold-storage period.

To elucidate the effect of cold storage on spore dormancy in the arbuscular mycorrhizal (AM) fungus Glomus intraradices, spores were cold stratified at 4 degrees C, for either 0, 3, 7, 14, 90 or 120 days, prior to germination tests at 25 degrees C. The results showed that cold stratification longer than 14 days significantly increased spore germination. Moreover, the longer cold storage periods clearly reduced spore mortality from 90% to 50% and considerably altered the hyphal growth pattern. Long polarized hyphae were only observed after cold stratification periods longer than 14 days, involving consequences for root infectivity. The results clearly show that environmental factors, e.g., coldness, can affect the physiology of AM fungal spores.