Association of highly and weakly mycorrhizal seedlings can promote the extra- and intraradical development of a common mycorrhizal network.

Arbuscular mycorrhizal fungi (AMF) are key determinants of plant interactions in ecosystems. Through their effects on competition, they are regulators of the structure of communities. Conversely, the composition of plant assemblages may also influence the AMF colonization dynamics of plant species. Here, we tested under in vitro culture conditions the effects of Medicago truncatula, a highly mycorrhizal plant species, and Silene vulgaris, a weakly mycorrhizal plant species, grown single (monospecies treatments) or in combination (bispecies treatment) on the colonization dynamics of the AMF Rhizophagus irregularis MUCL 43194. The seedlings were placed in a pre-established hyphal network developing from a mature M. truncatula mycorrhizal donor plant. Extraradical mycelium (ERM) and root colonization parameters as well as root morphology were measured over a period of 12 days. An increased ERM length, total root colonization and proportion of arbuscules were noted in the bispecies treatment. Conversely, the bispecies treatment seemed to have no effect on root growth. This study also demonstrated the suitability of the in vitro culture system for studying the interactions between AMF and host plants grown as mono- and bispecies combinations.

Elemental composition in vesicles of an arbuscular mycorrhizal fungus, as revealed by PIXE analysis.

We investigated element accumulation in vesicles of the arbuscular mycorrhizal (AM) fungus Glomus intraradices, extracted from the roots of inoculated leek plants. The elemental composition (elements heavier than Mg) was quantified using particle-induced X-ray emission (PIXE), in combination with scanning transmission ion microscopy (STIM). In vesicles, P was the most abundant of the elements analysed, followed by Ca, S, Si and K. We analysed 12 vesicles from two root systems and found that the variation between vesicles was particularly high for P and Si. The P content related positively to Si, Zn and K, while its relation to Cl fitted to a negative power function. Vesicle transects showed that P and K were present in central parts, while Ca was present mainly near the vesicle surfaces. The results showed that P is an important part (0.5% of the dry weight) of the vesicle content and that the distribution of some elements, within mycelia, may be strongly correlated.

Fenpropimorph and fenhexamid impact phosphorus translocation by arbuscular mycorrhizal fungi.

Fenpropimorph and fenhexamid are sterol biosynthesis inhibitor (SBI) molecules widely used to control diseases in agriculture. Both molecules, at increasing concentrations, have been shown to impact on the non-target arbuscular mycorrhizal (AM) fungi. Root colonization, spore production and mycelium architecture, including the branched absorbing structures which are thought to be involved in phosphorus (P) uptake, were affected. In the present study, we investigated the capacity of Glomus sp. MUCL 43204 to take up, transfer and translocate labelled P to Medicago truncatula in the presence of these SBI molecules. We used a strict in vitro cultivation system associating an autotrophic plant of M. truncatula with the AM fungus. In addition, the effects of both SBI molecules on the proportion of hyphae with alkaline phosphatases (ALP), succinate dehydrogenase (SDH) activity and on the expression of the mycorrhiza-specific plant phosphate transporter MtPT4 gene were examined. We demonstrated that the two SBI molecules impacted the AM fungus. This was particularly evidenced for fenpropimorph. A decrease in P transport and ALP and SDH activities associated with the extraradical mycelium and MtPT4 expression level was noted. These three factors were closely related to the development of the AM fungus, suggesting a direct impact not only on the AM fungal growth but also on the physiology and metabolic activities of the AM fungus. These results further emphasized the interest on the autotrophic in vitro culture system as an alternative to pot experiments to investigate the mechanisms behind the impact of disease control molecules on the non-target AM fungal symbionts.

Genomics of pyrrolnitrin biosynthetic loci: evidence for conservation and whole-operon mobility within gram-negative bacteria.

Pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite produced by a narrow range of gram-negative bacteria. The PRN biosynthesis by rhizobacteria presumably has a key role in their life strategies and in the biocontrol of plant diseases. The biosynthetic operon that encodes the pathway that converts tryptophan to PRN is composed of four genes, prnA through D, whose diversity, genomic context and spread over bacterial genomes are poorly understood. Therefore, we launched an endeavour aimed at retrieving, by in vitro and in silico means, diverse bacteria carrying the prnABCD biosynthetic loci in their genomes. Analysis of polymorphisms of the prnD gene sequences revealed a high level of conservation between Burkholderia, Pseudomonas and Serratia spp. derived sequences. Whole-operon- and prnD-based phylogeny resulted in tree topologies that are incongruent with the taxonomic status of the evaluated strains as predicted by 16S rRNA gene phylogeny. The genomic composition of c. 20 kb DNA fragments containing the PRN operon varied in different strains. Highly conserved and distinct transposase-encoding genes surrounding the PRN biosynthetic operons of Burkholderia pseudomallei strains were found. A prnABCD-deprived genomic region in B. pseudomallei strain K96243 contained the same gene composition as, and shared high homology with, the flanking regions of the PRN operon in B. pseudomallei strains 668, 1106a and 1710b. Our results strongly suggest that the PRN biosynthetic operon is mobile. The extent, frequency and promiscuity of this mobility remain to be understood.

Two differentially regulated phosphate transporters from the symbiotic fungus Hebeloma cylindrosporum and phosphorus acquisition by ectomycorrhizal Pinus pinaster.

Ectomycorrhizal symbiosis markedly improves plant phosphate uptake, but the molecular mechanisms underlying this benefit are still poorly understood. We identified two ESTs in a cDNA library prepared from the ectomycorrhizal basidiomycete Hebeloma cylindrosporum with significant similarities to phosphate transporters from the endomycorrhizal fungus Glomus versiforme and from non-mycorrhizal fungi. The full-length cDNAs corresponding to these two ESTs complemented a yeast phosphate transport mutant (Deltapho84). Measurements of (33)P-phosphate influx into yeast expressing either cDNA demonstrated that the encoded proteins, named HcPT1 and HcPT2, were able to mediate Pi:H(+) symport with different affinities for Pi (K(m) values of 55 and 4 mum, respectively). Real-time RT-PCR showed that Pi starvation increased the levels of HcPT1 transcripts in H. cylindrosporum hyphae grown in pure culture. Transcript levels of HcPT2 were less dependent on Pi availability. The two transporters were expressed in H. cylindrosporum associated with its natural host plant, Pinus pinaster, grown under low or high P conditions. The presence of ectomycorrhizae increased net Pi uptake rates into intact Pinus pinaster roots at low or high soil P levels. The expression patterns of HcPT1 and HcPT2 indicate that the two fungal phosphate transporters may be involved in uptake of phosphate from the soil solution under the two soil P availability conditions used.

Fluorescent in situ RT-PCR to visualise the expression of a phosphate transporter gene from an ectomycorrhizal fungus.

Expression of a mycorrhizal fungal-specific phosphate (P) transporter gene (HcPT1) was studied in mycelium of the ectomycorrhizal fungus Hebeloma cylindrosporum, by in situ reverse transcriptase polymerase chain reaction using amplification of complementary DNA sequences. The expression of HcPT1 was visualised under two different P treatments. Mycelium was transferred to liquid medium with or without P and incubated for 5 days. Under P starvation, mycelium growth and vitality was reduced and the expression of HcPT1 up regulated. Enzyme-labelled fluorescent substrate was used to detect gene expression in situ with epi-fluorescence microscopy and to visualise it at the level of the individual hyphae both in starved and non-starved hyphae. Up-regulation of HcPT1 was observed as a more intense fluorescent signal and from the larger proportion of hyphae that showed expression.

The influence of external nitrogen on carbon allocation to Glomus intraradices in monoxenic arbuscular mycorrhiza.

The influence of external nitrogen (N) on carbon (C) allocation and processes related to phosphorus (P) metabolism were studied in monoxenic arbuscular mycorrhiza (AM) cultures of Daucus carota. Fungal hyphae of Glomus intraradices proliferated from colonized roots growing on solid medium into C-free liquid minimal medium with two different N and P levels. Furthermore, we exposed the colonized roots to high or low N availability and then studied the mycelial development. Roots were provided with (13)C-glucose in order to follow the C allocation. The mycelium was analysed for phosphatase activity and transcription levels of two nutrient regulated genes. High N availability to the monoxenic AM root reduced the C allocation to the AM fungus while N availability to the mycelium was important for the upregulation of the fungal inorganic phosphorus (Pi)-transporter GiPT. We found that N availability can regulate nutritional processes in arbuscular mycorrhiza. We conclude that negative impacts of N on AM abundance are caused by reduced C allocation from the plant. Upregulation of the fungal Pi-transporter GiPT indicated that increased N availability might induce P limitation in the mycelium.

13C incorporation into signature fatty acids as an assay for carbon allocation in arbuscular mycorrhiza.

The ubiquitous arbuscular mycorrhizal fungi consume significant amounts of plant assimilated C, but this C flow has been difficult to quantify. The neutral lipid fatty acid 16:1omega5 is a quantitative signature for most arbuscular mycorrhizal fungi in roots and soil. We measured carbon transfer from four plant species to the arbuscular mycorrhizal fungus Glomus intraradices by estimating (13)C enrichment of 16:1omega5 and compared it with (13)C enrichment of total root and mycelial C. Carbon allocation to mycelia was detected within 1 day in monoxenic arbuscular mycorrhizal root cultures labeled with [(13)C]glucose. The (13)C enrichment of neutral lipid fatty acid 16:1omega5 extracted from roots increased from 0.14% 1 day after labeling to 2.2% 7 days after labeling. The colonized roots usually were more enriched for (13)C in the arbuscular mycorrhizal fungal neutral lipid fatty acid 16:1omega5 than for the root specific neutral lipid fatty acid 18:2omega6,9. We labeled plant assimilates by using (13)CO(2) in whole-plant experiments. The extraradical mycelium often was more enriched for (13)C than was the intraradical mycelium, suggesting rapid translocation of carbon to and more active growth by the extraradical mycelium. Since there was a good correlation between (13)C enrichment in neutral lipid fatty acid 16:1omega5 and total (13)C in extraradical mycelia in different systems (r(2) = 0.94), we propose that the total amount of labeled C in intraradical and extraradical mycelium can be calculated from the (13)C enrichment of 16:1omega5. The method described enables evaluation of C flow from plants to arbuscular mycorrhizal fungi to be made without extraction, purification and identification of fungal mycelia.

Metabolic activity of Glomus intraradices in Arum- and Paris-type arbuscular mycorrhizal colonization.

Colonization of two plant species by Glomus intraradices was studied to investigate the two morphological types (Arum and Paris), their symbiotic interfaces and metabolic activities. Root pieces and sections were stained to observe the colonization and metabolic activity of all mycorrhizal structures. There were no growth responses observed in the plants caused by mycorrhizal symbiosis. The two morphological types had a similar percentage of root colonized, but the Arum-type had higher metabolic activity. Most of the mycorrhizal structures (88%) showed succinate dehydrogenase activity; about half showed acid phosphatase activity; and a small percentage showed alkaline phosphatase activity. Phosphatase activity was highest in arbuscules and low in intercellular hyphae in the Arum-type colonization. In the Paris-type, hyphal coils and arbusculate coils showed a similar intermediate percentage of phosphatase activity. We conclude that acid phosphatase is more important than alkaline phosphatase in both colonization types. We discuss the possibility that, whereas arbuscules in Arum-type are the main site for phosphorus release to the host plant, both the hyphal and arbusculate coils may be involved in the Paris-type.

Fungal lipid accumulation and development of mycelial structures by two arbuscular mycorrhizal fungi.

We monitored the development of intraradical and extraradical mycelia of the arbuscular mycorrhizal (AM) fungi Scutellospora calospora and Glomus intraradices when colonizing Plantago lanceolata. The occurrence of arbuscules (branched hyphal structures) and vesicles (lipid storage organs) was compared with the amounts of signature fatty acids. The fatty acid 16:1omega5 was used as a signature for both AM fungal phospholipids (membrane constituents) and neutral lipids (energy storage) in roots (intraradical mycelium) and in soil (extraradical mycelium). The formation of arbuscules and the accumulation of AM fungal phospholipids in intraradical mycelium followed each other closely in both fungal species. In contrast, the neutral lipids of G. intraradices increased continuously in the intraradical mycelium, while vesicle occurrence decreased after initial rapid root colonization by the fungus. S. calospora does not form vesicles and accumulated more neutral lipids in extraradical than in intraradical mycelium, while the opposite pattern was found for G. intraradices. G. intraradices allocated more of its lipids to storage than did S. calospora. Thus, within a species, the fatty acid 16:1omega5 is a good indicator for AM fungal development. The phospholipid fatty acid 16:1omega5 is especially suitable for indicating the frequency of arbuscules in the symbiosis. We propose that the ratio of neutral lipids to phospholipids is more important than is the presence of vesicles in determining the storage status of AM fungi.

Phosphorus effects on metabolic processes in monoxenic arbuscular mycorrhiza cultures.

The influence of external phosphorus (P) on carbon (C) allocation and metabolism as well as processes related to P metabolism was studied in monoxenic arbuscular mycorrhiza cultures of carrot (Daucus carota). Fungal hyphae of Glomus intraradices proliferated from the solid minimal medium containing the colonized roots into C-free liquid minimal medium with different P treatments. The fungus formed around three times higher biomass in P-free liquid medium than in medium with 2.5 mM inorganic P (high-P). Mycelium in the second experiment was harvested at an earlier growth stage to study metabolic processes when the mycelium was actively growing. P treatment influenced the root P content and [(13)C]glucose administered to the roots 7 d before harvest gave a negative correlation between root P content and (13)C enrichment in arbuscular mycorrhiza fungal storage lipids in the extraradical hyphae. Eighteen percent of the enriched (13)C in extraradical hyphae was recovered in the fatty acid 16:1omega5 from neutral lipids. Polyphosphate accumulated in hyphae even in P-free medium. No influence of P treatment on fungal acid phosphatase activity was observed, whereas the proportion of alkaline-phosphatase-active hyphae was highest in high-P medium. We demonstrated the presence of a motile tubular vacuolar system in G. intraradices. This system was rarely seen in hyphae subjected to the highest P treatment. We concluded that the direct responses of the extraradical hyphae to the P concentration in the medium are limited. The effects found in hyphae seemed instead to be related to increased availability of P to the host root.

Arbuscular mycorrhizal fungi respond to the substrate pH of their extraradical mycelium by altered growth and root colonization.

• To test the response of arbuscular mycorrhizal (AM) fungi to a difference in soil pH, the extraradical mycelium of Scutellospora calospora or Glomus intraradices, in association with Plantago lanceolata, was exposed to two different pH treatments, while the root substrate pH was left unchanged. • Seedlings of P. lanceolata, colonized by one or other of the fungal symbionts, and nonmycorrhizal controls, were grown in mesh bags placed in pots containing pH-buffered sand (pH around 5 or 6). The systems were harvested at approximately 2-wk intervals between 20 and 80 d. • Both fungi formed more extraradical mycelium at the higher pH. Glomus intraradices formed almost no detectable extraradical mycelium at lower pH. The extraradical mycelium of S. calospora had higher acid phosphatase activity than that of G. intraradices. Total AM root colonization decreased for both fungi at the higher pH, and high pH also reduced arbuscule and vesicle formation in G. intraradices. • In conclusion, soil pH influences AM root colonization as well as the growth and phosphatase activities of extraradical mycelium, although the two fungi responded differently.