S. Gianinazzi

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.

First report of non-mycorrhizal plant mutants (Myc^-) obtained in pea (Pisum sativum L.) and fababean (Vicia faba L.)

Abstract Genetic resistance to vesicular-arbuscular (VA) mycorrhiza formation has been obtained in spontaneous or chemically induced mutants of two mycorrhiza-forming species (Pisum sativum L. and Vicia faba L.). The eight mutants, termed myc−, are characterized by aborted infections limited to one or two host cells. Expression of the myc− character is associated with that of the nod− character in both legumes, and is likewise under recessive genetic control. Preliminary analysis of the genetic behaviour of the myc− mutants in diallel crosses has shown that at least three genes are involved in VA mycorrhiza infection.

Differential activation of H+-ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco

Abstract. In arbuscular mycorrhizas, H+-ATPase is active in the plant membrane around arbuscules but absent from plant mutants defective in arbuscule development (Gianinazzi-Pearson etu2009al. 1995, Can J Bot 73: S526–S532). The proton-pumping H+-ATPase is encoded by a family of genes in plants. Immunocytochemical studies and promoter-gusA fusion assays were performed in transgenic tobacco (Nicotiana tabacum L.) to determine whether the periarbuscular enzyme activity results from de-novo activation of plant genes by an arbuscular mycorrhizal fungus. The H+-ATPase protein was localized in the plant membrane around arbuscule hyphae. The enzyme was absent from non-colonized cortical cells. Regulation of seven H+-ATPase genes (pma) was compared in non-mycorrhizal and mycorrhizal roots by histochemical detection of β-glucuronidase (GUS) activity. Two genes (pma2, pma4) were induced in arbuscule-containing cells of mycorrhizal roots but not in non-mycorrhizal cortical tissues or senescent mycorrhiza. It is concluded that de-novo H+-ATPase activity in the periarbuscular membrane results from selective induction of two H+-ATPase genes, which can have diverse roles in plant-fungal interactions at the symbiotic interface.

Expression profiling of up-regulated plant and fungal genes in early and late stages of Medicago truncatula-Glomus mosseae interactions.

Suppression subtractive hybridization (SSH), expression profiling and EST sequencing identified 12 plant genes and six fungal genes that are expressed in the arbuscular mycorrhizal symbiosis between Medicago truncatula and Glomus mosseae. All the plant genes and three of the fungal genes were up-regulated in symbiotic tissues. Expression of 15 of the genes is described for the first time in mycorrhizal roots and two are novel sequences. Six M. truncatula genes were also activated during appressorium formation at the root surface, suggesting a role in this early stage of mycorrhiza establishment, whilst the other six plant genes were only induced in the late stages of mycorrhization and could be involved in the development or functioning of the symbiosis. Phosphate fertilization had no significant influence on expression of any of the plant genes. Expression profiling of G. mosseae genes indicated that two of them may be associated with appressorium development on roots and one with arbuscule formation or function. The other three fungal genes were expressed throughout the life-cycle of G. mosseae.

Genetic and Cellular Analysis of Resistance to Vesicular Arbuscular (VA) Mycorrhizal Fungi in Pea Mutants

Screening of nodulation mutants of Pisum sativum has yielded mutants showing resistance to VA fungi (termed myc-). Most of these have aborted infections (myc-(1) phenotype) which are characterised by host cell reactions recalling those in certain pathogen infections. Mutants affected in later steps of mycorrhiza development (myc-(2) phenotype), with blocking of arbuscule formation, were less frequent. The myc-(1) character is recessive, segregates monogenically and occurs on at least five different, independently mutated loci, indicating that VA endomycorrhiza formation is under multiple gene control. Expression of the myc-(1) character is indissociable from that of nod’ in mutants and appears to be the result of pleiotropic effects of single genes. In late mutants where arbuscule formation is blocked, nodules develop but are inefficient (nod+,fix-). Coincidences between myc and nod characters may reflect common mechanisms in plant control over some step(s) in endomycorrhiza and nodule symbioses. Since chemical mutagenesis generally causes loss of gene function, inactivation of symbiosis-specific susceptibility genes in the mutants could affect production of signal molecules and somehow lead to stronger expression of plant resistance mechanisms to the symbionts.

b-protein as a constitutive component in highly (TMV) resistant interspecific hybrids of Nicotiana glutinosa × Nicotiana debneyi

Abstract Leaf-protein analysis of two reciprocal hybrids of Nicotiana glutinosa × N. debneyi, grown either in a greenhouse or in vitro, revealed that they contain a protein which is not normally present in the uninfected parent species. This protein has the same charge and the same mol. wt. of 13 800 as the b1″ protein induced in N. glutinosa and in N. debneyi during the hypersensitive reaction to tobacco mosaic virus (TMV) and tobacco necrosis virus (TNV), respectively (Ah et al., Phytophatology, 72 (1982) 80); it is not present in root extracts. Both hybrids are highly resistant to TMV, as compared to N. glutinosa, even though they are heterozygote for the resistance gene (N) of this species. Following inoculation, they develop smaller local lesions, from which only an extremely low level of infectivity can be obtained, and at higher temperatures (31°C) no generalization of the TMV infection occurs after 2 days, in contrast to N. glutinosa. The presence of b-proteins as constitutive components in these interspecific hybrids and the different possible pathways leading to their appearance are discussed, together with their possible relationship to the high level of resistance to TMV in these plants.

Cytochemical modifications in the host-fungus interface during intracellular interactions in vesicular- arbuscular mycorrhizae

Abstract The cytochemical modifications occuring in the host-fungus interface during intracellular interactions in two different vesicular-arbuscular mycorrhizae (VAM) ( Allium cepa/Glomus mosseae and Ornithogalum umbellatum/ Glomus fasciculatus ) are very similar; marked differences were only found in the wall cytochemistry of the two fungi involved. In both mycorrhizae, host primary wall material which is deposited around hyphae penetrating cells and which seems to contain a glycoprotein complex, strongly diminishes with development of the finer arbuscule branches and appears to change in nature. Host plasmalemma is not structurally or cytochemically modified with its invagination during arbuscule development. The analogies and differences in these observations to those reported for pathogenic infections are discussed in relation to the hypothesis that the arbuscule is the preferential site of bi-directional nutrient transfer between the fungus and plant in VAM.

Proteomics as a tool to monitor plant-microbe endosymbioses in the rhizosphere

In recent years, outstanding molecular approaches have been used to investigate genes and functions involved in plant-microbe endosymbioses. In this review, we outline the use of proteomic analysis, based on two-dimensional electrophoresis and mass spectrometry, to characterize symbiosis-related proteins. During the last decade, proteomics succeeded in identifying about 400 proteins associated with the development and functioning of both mycorrhizal and rhizobial symbioses. Further progress in prefractionation procedures is expected to allow the detection of symbiotic proteins showing low abundance or being present in certain cell compartments.

Ultrastructural Cytochemistry of the Host-Fungus Interfaces in the Endornycorrhizal Association Glomus mosseae / Allium cepa

Summary The structure of the host-fungus interface in the VA mycorrhizal association Glomus mosseae / Allium cepa has been investigated cytochemically (Thiery test; differential DMSO and EDTA extraction; phosphotungstic acid staining) and using different fixation procedures (glutaraldehyde and OsO 4 post-fixation; simultaneous glutaraldehyde — OSO 4 fixation). The continous, highly invaginated host plasmalemma surrounding the intracellular fungal structures retains its affinity for phosphotungstic acid and continues to produce polysaccharide fibrils, but in the presence of the endophyte it progressively loses the capacity to organise these into well-structured wall material. At the point of cell penetration and in the primary arbuscule branches the fungal wall, which is stained by the Thiery reaction and phosphotungstic acid but unaffected by DMSO or EDTA, is surrounded by a collar of host wall material which has the same cytochemical properties as the primary walls of the plant cell. The fine arbuscule branches are surrounded by a layer of condensed matrical fibrils. When the arbuscule branches senesce and collapse, they become encased by the polysaccharide fibrillar material derived from the host. The appearance of the interfacial matrix separating the host plasmalemma from the fungal wall is always the same, no matter which fixation procedure is used. It contains membranous vesicles and scattered polysaccharide fibrils, both of which are elaborated by the host plasmalemma. The importance of the host and endophyte plasmalemma formations is emphasised. The vesicles arising from the host plasmalemma are often in contact with the walls of the endophytic hyphae. In the fungus, these bulbous or tubular configurations of the plasmalemma can become very numerous and can occupy the whole hyphal width. Although these structures could be involved in host-fungus exchanges, their eventual participation in wall synthesis is also orooosed.

Conceptual approaches for the rational use of VA endomycorrhizae in agriculture: Possibilities and limitations

Abstract A strategy is proposed of plant inoculation with vesicular-arbuscular mycorrhizal (VAM) fungi based on appropriate biological tests (soil endomycorrhizal potential and fungal effectiveness/soil receptivity test) that provide information on whether it is necessary to inoculate and which fungi to use. It is suggested that the inoculum production methods should be chosen in function of the techniques used for plant production and of the economic criterion. Examples of successful inoculation of micropropagated apple root-stocks and ornamental plants sown in commercial nurseries are presented.