Silvia Perotto

The contribution of arbuscular mycorrhizal fungi in sustainable maintenance of plant health and soil fertility

Abstract. Beneficial plant–microbe interactions in the rhizosphere are primary determinants of plant health and soil fertility. Arbuscular mycorrhizas are the most important microbial symbioses for the majority of plants and, under conditions of P-limitation, influence plant community development, nutrient uptake, water relations and above-ground productivity. They also act as bioprotectants against pathogens and toxic stresses. This review discusses the mechanism by which these benefits are conferred through abiotic and biotic interactions in the rhizosphere. Attention is paid to the conservation of biodiversity in arbuscular mycorrhizal fungi (AMF). Examples are provided in which the ecology of AMF has been taken into account and has had an impact in landscape regeneration, horticulture, alleviation of desertification and in the bioremediation of contaminated soils. It is vital that soil scientists and agriculturalists pay due attention to the management of AMF in any schemes to increase, restore or maintain soil fertility.

Detection and Identification of Bacterial Endosymbionts in Arbuscular Mycorrhizal Fungi Belonging to the Family Gigasporaceae

ABSTRACT Intracellular bacteria have been found previously in one isolate of the arbuscular mycorrhizal (AM) fungus Gigaspora margaritaBEG 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 ofGigaspora 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 ofScutellospora persica, Scutellospora castanea, and G. margarita revealed a single, strongly supported branch nested in the genus Burkholderia.

Cellulose and pectin localization in roots of mycorrhizalAllium porrum: labelling continuity between host cell wall and interfacial material

Two different types of contacts (or interfaces) exist between the plant host and the fungus during the vesicular-arbuscular mycorrhizal symbiosis, depending on whether the fungus is intercellular or intracellular. In the first case, the walls of the partners are in contact, while in the second case the fungal wall is separated from the host cytoplasm by the invaginated host plasmamembrane and by an interfacial material. In order to verify the origin of the interfacial material, affinity techniques which allow identification in situ of cell-wall components, were used. Cellobiohydrolase (CBH I) that binds to cellulose and a monoclonal antibody (JIM 5) that reacts with pectic components were tested on roots ofAllium porrum L. (leek) colonized byGlomus versiforme (Karst.) Berch. Both probes gave a labelling specific for the host cell wall, but each probe labelled over specific and distinct areas. The CBH I-colloidal gold complex heavily labelled the thick epidermal cell walls, whereas JIM 5 only labelled this area weakly. Labelling of the hypodermis was mostly on intercellular material after treatment with JIM 5 and only on the wall when CBH I was used. Suberin bands found on the radial walls were never labelled. Cortical cells were mostly labelled on the middle lamella with JIM 5 and on the wall with CBH I. Gold granules from the two probes were found in interfacial material both near the point where the fungus enters the cell and around the thin hyphae penetrating deep into the cell. The ultrastructural observations demonstrate that cellulose and pectic components have different but complementary distributions in the walls of root cells involved in the mycorrhizal symbiosis. These components show a similar distribution in the interfacial material laid down around the vesicular-arbuscular mycorrhizal fungus indicating that the interfacial material is of host origin.

Inefficient photosynthesis in the Mediterranean orchid Limodorum abortivum is mirrored by specific association to ectomycorrhizal Russulaceae

Among European Neottieae, Limodorum abortivum is a common Mediterranean orchid. It forms small populations with a patchy distribution in woodlands, and is characterized by much reduced leaves, suggesting a partial mycoheterotrophy. We have investigated both the photosynthetic abilities of L. abortivum adult plants and the diversity of mycorrhizal fungi in Limodorum plants growing in different environments and plant communities (coniferous and broadleaf forests) over a wide geographical and altitudinal range. Despite the presence of photosynthetic pigments, CO2 fixation was found to be insufficient to compensate for respiration in adult plants. Fungal diversity was assessed by morphological and molecular methods in L. abortivum as well as in the related rare species Limodorum trabutianum and Limodorum brulloi. Phylogenetic analyses of the fungal internal transcribed spacer (ITS) sequences, obtained from root samples of about 80 plants, revealed a tendency to associate predominantly with fungal symbionts of the genus Russula. Based on sequence similarities with known species, most root endophytes could be ascribed to the species complex encompassing Russula delica, Russula chloroides, and Russula brevipes. Few sequences clustered in separate groups nested within Russula, a genus of ectomycorrhizal fungi. The morphotypes of ectomycorrhizal root tips of surrounding trees yielded sequences similar or identical to those obtained from L. abortivum. These results demonstrate that Limodorum species with inefficient photosynthesis specifically associate with ectomycorrhizal fungi, and appear to have adopted a nutrition strategy similar to that known from achlorophyllous orchids.

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.

Molecular diversity of fungi from ericoid mycorrhizal roots

In order to investigate the diversity of fungal endophytes in ericoid mycorrhizal roots, about 150 mycelia were isolated from surface‐sterilized roots of 10 plants of Calluna vulgaris. Each mycelium was reinoculated to C. vulgaris seedlings under axenic conditions, and the phenotype of the plant‐fungus association assessed by light and electron microscopy. Many isolates that were able in vitro to produce typical ericoid mycorrhizae did not form reproductive structures under our culture conditions, whereas others could be identified as belonging to the species Oidiodendron maius. Morphological and molecular analysis of the fungal isolates showed that the root system of a single plant of C. vulgaris is a complex mosaic of several populations of mycorrhizal and non mycorrhizal fungi. PCR‐RFLP techniques, used to investigate the mycorrhizal endophytes, revealed up to four groups of fungi with different PCR‐RFLP patterns of the ITS ribosomal region from a single plant. Some of the mycorrhizal fungi sharing the same PCR‐RFLP pattern showed high degree of genetic polymorphism when analysed with the more sensitive RAPD technique; this technique may prove a useful tool to trace the spread of individual mycorrhizal mycelia, as it has allowed us to identify isolates with identical RAPD fingerprints on different plants.

Ericoid mycorrhizal fungi are common root associates of a Mediterranean ectomycorrhizal plant (Quercus ilex).

Mycorrhiza samples of neighbouring Quercus ilex and Erica arborea plants collected in a postcutting habitat were processed to see whether plants differing in mycorrhizal status harbour the same root endophytes. Three experiments were performed in parallel: (i) isolation, identification and molecular characterization of fungi from surface‐sterilized roots of both plant species; (ii) re‐inoculation of fungal isolates on axenic E. arborea and Q. ilex seedlings; (iii) direct inoculation of field‐collected Q. ilex ectomycorrhizas onto E. arborea seedlings. About 70 and 150 fungal isolates were obtained from roots of Q. ilex and E. arborea, respectively. Among them, Oidiodendron species and five cultural morphotypes of sterile isolates formed typical ericoid mycorrhizas on E. arborea in vitro. Fungi with such mycorrhizal ability were derived from both host plants. Isolates belonging to one of these morphotypes (sd9) also exhibited an unusual pattern of colonization, with an additional extracellular hyphal net. Ericoid mycorrhizas were also readily obtained by direct inoculation of E. arborea seedlings with Q. ilex ectomycorrhizal tips. Polymerase chain–restriction fragment length polymorphism and random amplified polymorphic DNA analyses of the shared sterile morphotypes demonstrate, in the case of sd9, the occurrence of the same genet on the two host plants. These results indicate that ericoid mycorrhizal fungi associate with ectomycorrhizal roots, and the ecological significance of this finding is discussed.

Cellular interactions between arbuscular mycorrhizal fungi and rhizosphere bacteria

SummaryWe have investigated whether direct physical interactions occur between arbuscular mycorrhizal (AM) fungi and plant growth promoting rhizobacteria (PGPRs), some of which are used as biocontrol agents. Attachment of rhizobia and pseudomonads to the spores and fungal mycelium ofGigaspora margarita has been assessed in vitro and visualized by a combination of electron and confocal microscopy. The results showed that both rhizobia and pseudomonads adhere to spores and hyphae of AM fungi germinated under sterile conditions, although the degree of attachment depended upon the strain.Pseudomonas fluorescens strain WCS 365 andRhizobium leguminosarum strains B556 and 3841 were the most effective colonizers. Extracellular material of bacterial origin containing cellulose produced around the attached bacteria may mediate fungal/bacterial interactions. These results suggest that antagonistic and synergistic interactions between AM fungi and rhizosphere bacteria may be mediated by soluble factors or physical contact. They also support the view that AM fungi are a vehicle for the colonization of plant roots by soil rhizobacteria.

Correlation between chitin distribution and cell wall morphology in the mycorrhizal fungus Glomus versiforme.

The distribution of N-acetylglucosamine residues in the cell wall of Glomus versiforme was studied by using fluorescein- and gold-labelled wheat germ agglutinin (WGA). This lectin is considered to possess specific binding sites for GlcNAc and a strong affinity for its oligomers and polymers, especially chitin. The affinity techniques provide a spatial description of the occurrence of the sugar residues. Chitin always occurred in the cell walls of the fungus studied throughout the whole duration of its symbiotic development. Gold granules conjugated with wheat germ agglutinin were found both on the thick walls of extraradical and intraradical hyphae as well as on the thin walls of arbuscular branches, irrespective of wall texture which was fibrillar or amorphous. However, when the fluorescein-labelled lectin was used, only the thin arbuscular branches were labelled. These observations lead to the conclusion that the probe accessibility to the substrate changes on the in toto hyphae and on the thin sections and that the thick-walled hyphae have their chitin partially hidden by non-chitinous cell wall components. These components were soluble to heat treatment alkali and oxidizing agent.

Solubilization of insoluble inorganic zinc compounds by ericoid mycorrhizal fungi derived from heavy metal polluted sites

Abstract Ericoid mycorrhizal fungi increase the ability of their host plants to colonize soils polluted with toxic metals, although the underlying mechanisms are not clearly understood. Two mycorrhizal strains of Oidiodendron maius isolated from contaminated soil were previously shown to tolerate high concentrations of toxic metals. We investigated further the biological mechanisms that may explain metal tolerance, focussing on the interactions between insoluble metal species and extracellular fungal metabolites. In particular, we demonstrate that fungal strains derived from polluted and unpolluted soils mobilize insoluble inorganic zinc compounds to different extents. Strains from polluted soils showed in fact little ability to solubilize Zn from both ZnO and Zn 3 (PO 4 ) 2 , whereas strains from unpolluted soils showed a higher solubilization potential. This different behaviour was confirmed when the solubilization abilities of a wider range of fungal strains (25 isolates) was examined. Induction of organic acids (malate and citrate) by the metal compounds was at least in part responsible for metal solubilization. Our results suggest that ericoid mycorrhizal strains from polluted and unpolluted soils may interact differently with metal compounds. We speculate that this may reflect specific strategies to maintain homeostasis of essential metals under different soil conditions.