Arbuscular mycorrhizal fungal community differences among European long-term observatories.

Arbuscular mycorrhizal fungal (AMF) communities have been demonstrated to respond to a variety of biotic and abiotic factors, including various aspects of land management. Numerous studies have specifically addressed the impact of land use on AMF communities, but usually have been confined to one or a few sites. In this study, soil AMF assemblages were described in four different long-term observatories (LTOs) across Europe, each of which included a site-specific high-intensity and a low-intensity land use. AMF communities were characterized on the basis of 454 sequencing of the internal transcribed spacer 2 (ITS2) rDNA region. The primary goals of this study were (i) to determine the main factors that shape AMF communities in differentially managed sites in Europe and (ii) to identify individual AMF taxa or combinations of taxa suitable for use as biomarkers of land use intensification. AMF communities were distinct among LTOs, and we detected significant effects of management type and soil properties within the sites, but not across all sites. Similarly, indicator species were identified for specific LTOs and land use types but not universally for high- or low-intensity land uses. Different subsets of soil properties, including several chemical and physical variables, were found to be able to explain an important fraction of AMF community variation alone or together with other examined factors in most sites. The important factors were different from those for other microorganisms studied in the same sites, highlighting particularities of AMF biology.

The impact of tillage practices on arbuscular mycorrhizal fungal diversity in subtropical crops.

Arbuscular mycorrhizal fungi (AMF) are a main component of soil microbiota in most agrosystems. As obligately mutualistic symbionts, they colonize the roots of the majority of plants, including crop plants. We used molecular techniques to investigate how different tillage systems (moldboard, shred-bedding, subsoil-bedding, and no tillage) can influence the AM fungal community colonizing maize, bean, and sorghum roots in an experimental site located in northern Tamaulipas, Mexico. Roots from 36 plants were analyzed using AM fungal-specific primers to partially amplify the small subunit (SSU) of the ribosomal DNA genes. More than 880 clones were screened for restriction fragment length polymorphism (RFLP) variation, and 173 of these were sequenced. Ten AM fungal types were identified and clustered into three AM fungal families: Gigasporaceae, Glomaceae, and Paraglomaceae. Glomus was the dominating taxon in all the samples. Four of the 10 identified types were distinct from any previously published sequences and could correspond to either known unsequenced species or unknown species. The fungal diversity was low in the four agriculture management systems, but the multidimensional scaling (MDS) analysis and log-linear-saturated model indicated that the composition of the AMF community was significantly affected by the tillage system. In conclusion, since some fungal types were treatment specific, agricultural practices could directly or indirectly influence AM biodiversity.

Mucoid mutants of the biocontrol strain pseudomonas fluorescens CHA0 show increased ability in biofilm formation on mycorrhizal and nonmycorrhizal carrot roots.

Extracellular polysaccharides play an important role in the formation of bacterial biofilms. We tested the biofilm-forming ability of two mutant strains with increased production of acidic extracellular polysaccharides compared with the wild-type biocontrol strain Pseudomonas fluorescens CHA0. The anchoring of bacteria to axenic nonmycorrhizal and mycorrhizal roots as well as on extraradical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices was investigated. The nonmucoid wild-type strain P. fluorescens CHA0 adhered very little on all surfaces, whereas both mucoid strains formed a dense and patchy bacterial layer on the roots and fungal structures. Increased adhesive properties of plant-growth-promoting bacteria may lead to more stable interactions in mixed inocula and the rhizosphere.

Impact on arbuscular mycorrhiza formation of pseudomonas strains used as inoculants for biocontrol of soil-borne fungal plant pathogens

The arbuscular mycorrhizal symbiosis, a key component of agroecosystems, was assayed as a rhizosphere biosensor for evaluation of the impact of certain antifungal Pseudomonas inoculants used to control soil-borne plant pathogens. The following three Pseudomonas strains were tested: wild-type strain F113, which produces the antifungal compound 2,4-diacetylphloroglucinol (DAPG); strain F113G22, a DAPG-negative mutant of F113; and strain F113(pCU203), a DAPG overproducer. Wild-type strain F113 and mutant strain F113G22 stimulated both mycelial development from Glomus mosseae spores germinating in soil and tomato root colonization. Strain F113(pCU203) did not adversely affect G. mosseae performance. Mycelial development, but not spore germination, is sensitive to 10 &mgr;M DAPG, a concentration that might be present in the rhizosphere. The results of scanning electron and confocal microscopy demonstrated that strain F113 and its derivatives adhered to G. mosseae spores independent of the ability to produce DAPG.

A combined morphological and molecular approach to characterize isolates of arbuscular mycorrhizal fungi in Gigaspora (Glomales).

• Morphological features of resting spores and information from nucleotide sequences of ribosomal RNA were used to characterize seven mycorrhizal fungal isolates in Gigaspora from different geographical areas. • Detailed observations were made under the light microscope on single spores mounted in Melzer's reagent and polyvinyl alcohol-lactic acid-glycerol medium to resolve size, colour and cell wall structures. Neighbour-joining analyses were carried out on a portion of the 18S gene and on the internal transcribed spacer (ITS) region amplified by PCR from multisporal DNA preparations. • Combined data allowed us to design oligonucleotides that unambiguously distinguished Gi. rosea from Gi. margarita and Gi. gigantea and also identified two isolates as Gi. rosea that had been previously diagnosed as Gi. margarita. ITS sequences revealed substantial genetic variability within clones of a single isolate of Gi. rosea as well as among geographically disjunct Gi. rosea isolates. • The results show how complementary morphological and molecular data can clarify relationships among species of low morphological divergence. Sequence information allowed the extent of genetic divergence within these species to be investigated and provided useful PCR primers for detection and identification.

Extracellular polysaccharides are involved in the attachment of Azospirillum brasilense and Rhizobium leguminosarum to arbuscular mycorrhizal structures.

Arbuscular mycorrhizal (AM) fungi, one of the most important component of the soil microbial community, establish physical interactions with naturally occurring and genetically modified bacterial biofertilizers and biopesticides, commonly referred to as plant growth-promoting rhizobacteria (PGPR). We have used a genetic approach to investigate the bacterial components possibly involved in the attachment of two PGPR (Azospirillum and Rhizobium) to AM roots and AM fungal structures. Mutants affected in extracellular polysaccharides (EPS) have been tested in in vitro adhesion assays and shown to be strongly impaired in the attachment to both types of surfaces as well as to quartz fibers. Anchoring of rhizobacteria to AM fungal structures may have special ecological and biotechnological significance because it may facilitate colonisation of new rhizospheres by the bacteria, and may be an essential trait for the development of mixed inocula.

Lipopolysaccharide epitope expression of Rhizobium bacteroids as revealed by in situ immunolabelling of pea root nodule sections.

To investigate the in situ expression of lipopolysaccharide (LPS) epitopes on nodule bacteria of Rhizobium leguminosarum, monoclonal antibodies recognizing LPS macromolecules were used for immunocytochemical staining of pea nodule tissue. Many LPS epitopes were constitutively expressed, and the corresponding antibodies reacted in nodule sections with bacteria at all stages of tissue infection and cell invasion. Some antibodies, however, recognized epitopes that were only expressed in particular regions of the nodule. Two general patterns of regulated LPS epitope expression could be distinguished on longitudinal sections of nodules. A radial pattern probably reflected the local physiological conditions experienced by endosymbiotic bacteria as a result of oxygen diffusion into the nodule tissue. The other pattern of expression, which followed a linear axis of symmetry along a longitudinal section of the pea nodule, was apparently associated with the differentiation of nodule bacteria and the development of the nitrogen-fixing capacity in bacteroids. Basically similar patterns of LPS epitope expression were observed for pea nodules harboring either of two immunologically distinct strains of R. leguminosarum bv. viciae, although these epitopes were recognized by different sets of strain-specific monoclonal antibodies. Furthermore, LPS epitope expression of rhizobia in pea nodules was compared with that of equivalent strains in nodules of French bean (Phaseolus vulgaris). From these observations, it is suggested that structural modifications of Rhizobium LPS may play an important role in the adaptation of endosymbiotic rhizobia to the surrounding microenvironment.

Vertical transmission of endobacteria in the arbuscular mycorrhizal fungus Gigaspora margarita through generation of vegetative spores.

Arbuscular mycorrhizal (AM) fungi living in symbiotic association with the roots of vascular plants have also been shown to host endocellular rod-shaped bacteria. Based on their ribosomal sequences, these endobacteria have recently been identified as a new taxon, Candidatus Glomeribacter gigasporarum. In order to investigate the cytoplasmic stability of the endobacteria in their fungal host and their transmission during AM fungal reproduction (asexual), a system based on transformed carrot roots and single-spore inocula of Gigaspora margarita was used. Under these in vitro sterile conditions, with no risk of horizontal contamination, the propagation of endobacteria could be monitored, and it was shown, by using primers designed for both 16S and 23S ribosomal DNAs, to occur through several vegetative spore generations (SG0 to SG4). A method of confocal microscopy for quantifying the density of endobacteria in spore cytoplasm was designed and applied; endobacteria were consistently found in all of the spore generations, although their number rapidly decreased from SG0 to SG4. The study demonstrates that a vertical transmission of endobacteria takes place through the fungal vegetative generations (sporulation) of an AM fungus, indicating that active bacterial proliferation occurs in the coenocytic mycelium of the fungus, and suggests that these bacteria are obligate endocellular components of their AM fungal host.

Detection and identification of bacterial endosymbionts in arbuscular mycorrhizal fungi belonging to the family Gigasporaceae.

Intracellular bacteria have been found previously in one isolate of the arbuscular mycorrhizal (AM) fungus Gigaspora margarita BEG 34. In this study, we extended our investigation to 11 fungal isolates obtained from different geographic areas and belonging to six different species of the family Gigasporaceae. With the exception of Gigaspora rosea, isolates of all of the AM species harbored bacteria, and their DNA could be PCR amplified with universal bacterial primers. Primers specific for the endosymbiotic bacteria of BEG 34 could also amplify spore DNA from four species. These specific primers were successfully used as probes for in situ hybridization of endobacteria in G. margarita spores. Neighbor-joining analysis of the 16S ribosomal DNA sequences obtained from isolates of Scutellospora persica, Scutellospora castanea, and G. margarita revealed a single, strongly supported branch nested in the genus Burkholderia.

An obligately endosymbiotic mycorrhizal fungus itself harbors obligately intracellular bacteria.

Arbuscular-mycorrhizal fungi are obligate endosymbionts that colonize the roots of almost 80% of land plants. This paper describes the employment of a combined morphological and molecular approach to demonstrate that the cytoplasm of the arbuscular-mycorrhizal fungus Gigaspora margarita harbors a further bacterial endosymbiont. Intracytoplasmic bacterium-like organisms (BLOs) were detected ultrastructurally in its spores and germinating and symbiotic mycelia. Morphological observations with a fluorescent stain revealed about 250,000 live bacteria inside each spore. The sequence for the small-subunit rRNA gene obtained for the BLOs from the spores was compared with those for representatives of the eubacterial lineages. Molecular phylogenetic analysis unambiguously showed that the endosymbiont of G. margarita was an rRNA group II pseudomanad (genus Burkholderia). PCR assays with specifically designed oligonucleotides were used to check that the sequence came from the BLOs. Successful amplification was obtained when templates from both the spores and the symbiotic mycelia were used. A band of the expected length was also obtained from spores of a Scutellospora sp. No bands were given by the negative controls. These findings indicate that mycorrhizal systems can include plant, fungal, and bacterial cells.