Concepción Azcón-Aguilar

Arbuscular mycorrhizas and biological control of soil-borne plant pathogens – an overview of the mechanisms involved

Abstract Biological control of plant pathogens is currently accepted as a key practice in sustainable agriculture because it is based on the management of a natural resource, i.e. certain rhizosphere organisms, common components of ecosystems, known to develop antagonistic activities against harmful organisms (bacteria, fungi, nematodes etc.). Arbuscular mycorrhizal (AM) associations have been shown to reduce damage caused by soil-borne plant pathogens. Although few AM isolates have been tested in this regard, some appear to be more effective than others. Furthermore, the degree of protection varies with the pathogen involved and can be modified by soil and other environmental conditions. This prophylactic ability of AM fungi could be exploited in cooperation with other rhizospheric microbial angatonists to improve plant growth and health. Despite past achievements on the application of AM in plant protection, further research is needed for a better understanding of both the ecophysiological parameters contributing to effectiveness and of the mechanisms involved. Although the improvement of plant nutrition, compensation for pathogen damage, and competition for photosynthates or colonization/infection sites have been claimed to play a protective role in the AM symbiosis, information is scarce, fragmentary or even controversial, particularly concerning other mechanisms. Such mechanisms include (a) anatomical or morphological AM-induced changes in the root system, (b) microbial changes in rhizosphere populations of AM plants, and (c) local elicitation of plant defence mechanisms by AM fungi. Although compounds typically involved in plant defence reactions are elicited by AM only in low amounts, they could act locally or transiently by making the root more prone to react against pathogens. Current research based on molecular, immunological and histochemical techniques is providing new insights into these mechanisms.

Mycorrhizosphere interactions to improve plant fitness and soil quality

Arbuscular mycorrhizal fungi are key components of soil microbiota and obviously interact with other microorganisms in the rhizosphere, i.e. the zone of influence of plant roots on microbial populations and other soil constituents. Mycorrhiza formation changes several aspects of plant physiology and some nutritional and physical properties of the rhizospheric soil. These effects modify the colonization patterns of the root or mycorrhizas (mycorrhizosphere) by soil microorganisms. The rhizosphere of mycorrhizal plants, in practice a mycorrhizosphere, harbors a great array of microbial activities responsible for several key ecosystem processes. This paper summarizes the main conceptual principles and accepted statements on the microbial interactions between mycorrhizal fungi and other members of rhizosphere microbiota and discusses current developments and future trends concerning the following topics: (i) effect of soil microorganisms on mycorrhiza formation; (ii) mycorrhizosphere establishment; (iii) interactions involved in nutrient cycling and plant growth; (iv) interactions involved in the biological control of plant pathogens; and (v) interactions to improve soil quality. The main conclusion is that microbial interactions in the rhizosphere of mycorrhizal plants improve plant fitness and soil quality, critical issues for a sustainable agricultural development and ecosystem functioning.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

Nitrate depletion and pH changes induced by the extraradical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices grown in monoxenic culture

The effect of the extraradical mycelium of the arbuscular mycorrhizal (AM) fungus Glomus intraradices Smith & Schenck on nitrate uptake and on the pH of the medium was studied in a monoxenic culture with tomato (Lycopersicon esculentum Mill. var. Vendor) roots obtained from root organ culture. The symbiosis was established in compartmented Petri dishes containing agar media amended with the pH indicator bromocresol purple. A pattern of pH changes was revealed as the symbiosis progressed in the media of the Petri dish compartments containing the dual, arbuscular-mycorrhizal fungi/root, culture as well as in the media of the hyphae, root-free compartments, in which the extraradical hyphae developed extensively, coming from the compartment containing the symbiosis. The colour changes in the media were measured spectrophotometrically, whilst maintaining the monoxenic conditions. The extraradical hyphae of G. intraradices strongly increased the pH of nutrient-free medium when supplied with nitrate, whereas the pH decreased m the absence of this N source. The hyphae developing from germinated spores and growing in axenic, nitrate-amended media did not induce any increase in pH. Nitrogen analysis revealed that a depletion of nitrate in the media accompanied increased pH. These results point towards an active uptake of nitrate by the extraradical mycelium of G. intraradices, probably coupled to a H+ -symport mechanism. The pH changes induced by AM fungal hyphae and the possible influence of the establishment of a functional symbiosis on these pH changes are discussed.

Influence of arbuscular mycorrhizae on heavy metal (Zn and Pb) uptake and growth of Lygeum spartum and Anthyllis cytisoides

The influence of arbuscular mycorrhizae (AM) on plant growth and Zn and Pb uptake by Lygeum spartum and Anthyllis cytisoides was studied in soils with different levels of these heavy metals. A. cytisoides is highly dependent on AM for optimal growth, while L. spartum is a facultative mycotroph.Mycorrhizal and nonmycorrhizal plants were grown in soil supplemented with 0, 10, 100 and 1000 mg of Zn kg-1 soil or 0, 100 and 1000 mg of Pb kg-1 soil. Two different mycorrhizal fungi were separately studied: Glomus macrocarpum isolated from a non contaminated site and a strain of Glomus mosseae isolated from a soil contaminated with these metals. The infectivity of the fungi was not affected by the presence of Zn or Pb in the soil. In unamended soil, both fungi were equally effective in promoting plant growth, but when Zn or Pb were added to soils, G. mosseae was more efficient than G. macrocarpum in stimulating plant growth of A. cytisoides. A. cytisoides was unable to grow unless mycorrhizal. Metal addition to the soil induced a reduction in the biomass of L. spartum and of mycorrhizal A. cytisoides, and a decrease in shoot P concentration of mycorrhizal plants. The concentration of metals in the plants varied according to the amount added to the soil and to the inoculation treatment: at low doses, mycorrhizal plants showed equal or higher concentration of Zn or Pb than nonmycorrhizal ones; at higher doses, however, metal concentrations in the plants inoculated with G. mosseae were lower than those found in the corresponding controls, while the plants inoculated with G. macrocarpum showed similar (L. spartum) or even higher (A. cytisoides) levels than the controls.

Hormonal and transcriptional profiles highlight common and differential host responses to arbuscular mycorrhizal fungi and the regulation of the oxylipin pathway

Arbuscular mycorrhizal (AM) symbioses are mutualistic associations between soil fungi and most vascular plants. The symbiosis significantly affects the host physiology in terms of nutrition and stress resistance. Despite the lack of host range specificity of the interaction, functional diversity between AM fungal species exists. The interaction is finely regulated according to plant and fungal characters, and plant hormones are believed to orchestrate the modifications in the host plant. Using tomato as a model, an integrative analysis of the host response to different mycorrhizal fungi was performed combining multiple hormone determination and transcriptional profiling. Analysis of ethylene-, abscisic acid-, salicylic acid-, and jasmonate-related compounds evidenced common and divergent responses of tomato roots to Glomus mosseae and Glomus intraradices, two fungi differing in their colonization abilities and impact on the host. Both hormonal and transcriptional analyses revealed, among others, regulation of the oxylipin pathway during the AM symbiosis and point to a key regulatory role for jasmonates. In addition, the results suggest that specific responses to particular fungi underlie the differential impact of individual AM fungi on plant physiology, and particularly on its ability to cope with biotic stresses.

β-1,3-Glucanase activities in tomato roots inoculated with arbuscular mycorrhizal fungi and/or Phytophthora parasitica and their possible involvement in bioprotection

b-1,3-Glucanases in tomato roots were studied after arbuscular mycorrhizal (AM) symbiosis establishment and:or pathogenic infection by Phytophthora parasitica by polyacrylamide gel electrophoresis (PAGE). Two species of AM fungi, Glomus mosseae and Glomus intraradices were tested, and Phytophthora inoculation was performed on both non-mycorrhizal and mycorrhizal tomato pre-colonized for 4 weeks with either of the AM fungal species. The protective effect of both AM fungi on tomato plants against Phytophthora was assessed. In control roots two acidic b-1,3-glucanase isoforms were constitutively expressed, and their activity was higher in mycorrhizal roots. Two additional acidic isoforms were detected in extracts from G. mosseae-colonized tomato roots, but not in G. intraradices-colonized roots. Roots infected by P. parasitica displayed stronger activities but the pathogen did not induce the isoforms related to G. mosseae colonization. Only one basic glucanase isoform was detected whether the plants were non-inoculated or colonized by any of the fungi when inoculated singly. However, when plants were pre-inoculated with G. mosseae and post-infected with P. parasitica two additional basic isoforms were clearly revealed. Results are discussed in relation to the possible role of the additional acidic and basic b-1,3-glucanase isoforms in the establishment and development of the AM symbiosis, as well as their putative implication in plant bioprotection.

Analysis of the mycorrhizal potential in the rhizosphere of representative plant species from desertification-threatened Mediterranean shrublands

An evaluation of the mycorrhizal status of desertification-threatened ecosystems has been recommended as a first step in rehabilitation/restoration approaches based on revegetation strategies using arbuscular mycorrhizal (AM) technology. Representative desertified semiarid areas were selected from southeast Spain where the vegetation is dominated by grasses, with Stipa tenacissima usually present, and with some patches of the shrubs Pistacia lentiscus, Rhamnus lycioides, Olea europaea subsp. sylvestris and Retama sphaerocarpa. The objective of this study was to evaluate the mycorrhizal potential in these soils, the contribution of the different species established to the mycorrhizal potential of the soils and to assess the main mycorrhizal propagules involved. There were more AM fungal propagules in the rhizospheres of all the shrub species studied compared with adjacent fallow soils, suggesting that AM propagules can be considered as a functional component of the resource islands developing around plant roots. R. sphaerocarpa and O. europaea had a higher capacity to enhance the development of mycorrhizal propagules in their rhizospheres than R. lycioides and P. lentiscus. Correlation analyses showed that the number of spores of the most representative AM fungal species, i.e. Glomus constrictum, and the total length of extraradical AM mycelium are the propagule sources which were best correlated with the mycorrhizal potential in terms of the number of “infective” AM propagules in the rhizosphere of the target plant species. The contribution of AM symbiosis to the potentiality of S. tenacissima as nurse plant was site dependent. Diversity of AM fungi present in the test area is rather low, indicating the high degree of degradation of the ecosystem. At most, only four AM fungal spore morphoecotypes were consistently detected in the rhizosphere of the target plant species.

Phytohormones as integrators of environmental signals in the regulation of mycorrhizal symbioses

For survival, plants have to efficiently adjust their phenotype to environmental challenges, finely coordinating their responses to balance growth and defence. Such phenotypic plasticity can be modulated by their associated microbiota. The widespread mycorrhizal symbioses modify plant responses to external stimuli, generally improving the resilience of the symbiotic system to environmental stresses. Phytohormones, central regulators of plant development and immunity, are instrumental in orchestrating plant responses to the fluctuating environment, but also in the regulation of mycorrhizal symbioses. Exciting advances in the molecular regulation of phytohormone signalling are providing mechanistic insights into how plants coordinate their responses to environmental cues and mycorrhizal functioning. Here, we summarize how these mechanisms permit the fine-tuning of the symbiosis according to the ever-changing environment.

Assessing the tolerance to heavy metals of arbuscular mycorrhizal fungi isolated from sewage sludge-contaminated soils

Different fungal ecotypes were isolated from soils which had received long-term applications of metal-contaminated sewage sludge with the aim of studying the degree of tolerance and adaptation to heavy metals of arbuscular mycorrhizal (AM) fungi. The development and structural aspects of AM colonization produced by the different fungal isolates were studied using two host plants, Allium porrum and Sorghum bicolor, which were grown in either contaminated or non-contaminated soils. Four different AM fungi were successfully isolated from the experimental field plots: (i) Glomus claroideum, isolated from plots receiving only inorganic fertilizer; (ii) another apparently similar ecotype of Glomus claroideum, but isolated from plots with 300 m 3 ha ˇ1 year ˇ1 of contaminated sludge added, (iii) an unidentified Glomus sp., present only in the less contaminated plots (100 m 3 ha ˇ1 year ˇ1 of unamended sludge) and (iv) Glomus mosseae, isolated from plots receiving 100 or 300 m 3 ha ˇ1 year ˇ1 of amended or unamended sludge (intermediate rates of contamination). There were consistent differences in behaviour among the four AM fungi tested with regard to the colonization levels they produced in non-contaminated and contaminated soils. Both total and arbuscular colonization were affected by heavy metal contamination. The main conclusions of this study are that Glomus sp. and G. mosseae isolates are strongly inhibited by heavy metals, which acted mainly by interfering with the growth of the external mycelium, and also by limiting the production of arbuscules. Our results suggest that G. claroideum isolates, particularly the ecotype which was isolated from the plots receiving the highest dose of metal-contaminated sludge, shows a potential adaptation to increased metal concentration in soil. # 1999 Elsevier Science B.V.