Yves Piché

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.

Azoarcus Grass Endophytes Contribute Fixed Nitrogen to the Plant in an Unculturable State

The extent to which the N2-fixing bacterial endophyte Azoarcus sp. strain BH72 in the rhizosphere of Kallar grass can provide fixed nitrogen to the plant was assessed by evaluating inoculated plants grown in the greenhouse and uninoculated plants taken from the natural environment. The inoculum consisted of either wild-type bacteria or nifK- mutant strain BHNKD4. In N2-deficient conditions, plants inoculated with strain BH72 (N2-fixing test plants) grew better and accumulated more nitrogen with a lower delta15N signature after 8 months than did plants inoculated with the mutant strain (non-N2-fixing control plants). Polyadenylated or polymerase chain reaction-amplified BH72 nifH transcripts were retrieved from test but not from control plants. BH72 nifH transcripts were abundant. The inocula could not be reisolated. These results indicate that Azoarcus sp. BH72 can contribute combined N2 to the plant in an unculturable state. Abundant BH72 nifH transcripts were detected also in uninoculated plants taken from the natural environment, from which Azoarcus sp. BH72 also could not be isolated. Quantification of nitrogenase gene transcription indicated a high potential of strain BH72 for biological N2 fixation in association with roots. Phylogenetic analysis of nitrogenase sequences predicted that uncultured grass endophytes including Azoarcus spp. are ecologically dominant and play an important role in N2-fixation in natural grass ecosystems.

LIFE CYCLE OF GLOMUS INTRARADIX IN ROOT ORGAN CULTURE

The entire vegetative life cycle of the vesicular-arbuscular mycorrhizal fungus Glomus intraradix was followed in a simple monoxenic culture system using Ri T DNA-transformed carrot roots and nontransformed tomato roots as plant partners. Fungal development, from the growth of initial germ tubes to the formation of an external mycelium network and spore production, was observed nondestructively using light microscopy. Surface-sterilized spores isolated from pot culture constituted an excellent source of fungal inoculum. These spores germinated readily on the nutrient medium and only a low rate of contamination was recorded. Germ tubes approaching the surface of isolated roots changed their growth pattern and branched profusely in response to root factors. Throughout the growth process it was possible to observe directly the formation of terminal and intercalary secondary spores, numerous hyphal anastomoses and arbuscle-like structures, intracellular vesicles and spores. The extent of internal root colonization by G. intraradix was relatively limited, but it was sufficient to permit the in vitro formation of hundreds of new spores free of contaminants, which were viable and capable of colonizing host roots.

Flavonoid levels in roots ofMedicago sativa are modulated by the developmental stage of the symbiosis and the root colonizing arbuscular mycorrhizal fungus

Abundant data on the effect of flavonoids on spore germination, hyphal growth and root colonization by AMF are available. Moreover, the flavonoid pattern in mycorrhizal roots changes, thus flavonoids have been suggested as arbuscular mycorrhizal signalling compounds. In our work we studied the accumulation of flavonoids in roots of Medicago sativa i) after the exposure of uncolonized roots to sterile solutions containing Glomus intraradices tissue, ii) at three different stages of colonization by G. mosseae, iii) colonized by G. mosseae, G. intraradices or Gigaspora rosea. We could show that flavonoid accumulation in M. sativa roots i) is induced before root colonization, pointing towards the presence of a fungal-derived signal, ii) depends on the developmental stage of the symbiosis and iii) depends on the root-colonizing arbuscular mycorrhizal fungus. The data presented indicate not only a time-specificity of the flavonoid accumulation during the mycorrhizal association, but also an arbuscular mycorrhizal fungal-specificity. The possible functions of the flavonoid pattern changes are discussed.

Root colonization by arbuscular mycorrhizal fungi is affected by the salicylic acid content of the plant

Wild type, transgenic NahG tobacco plants with reduced levels of salicylic acid (SA) and transgenic CSA (constitutive SA biosynthesis) tobacco plants with enhanced SA levels were inoculated with the arbuscular mycorrhizal fungi (AMF) Glomus mosseae or Glomus intraradices. In a time course study the effect of SA content on root colonization by the fungal symbiont was determined. Throughout the experiment in NahG plants an enhanced root colonization level could be detected, whereas in CSA plants mycorrhization was reduced. At the end of the experiment with Glomus mosseae, root colonization was similar in wild type and in transgenic plants (NahG and CSA plants), indicating that enhanced SA levels in plants can have an effect on delay AMF root colonization, but do not affect the symbiotic potential of plants in terms of changes in maximal threshold of root colonization. Compared to non-mycorrhizal plants, in mycorrhizal wild type and NahG the SA concentration was reduced. The role of SA in the regulation of mycorrhization is discussed.

Architecture and developmental dynamics of the external mycelium of the arbuscular mycorrhizal fungus Glomus intraradices grown under monoxenic conditions

The structural development of arbuscular mycorrhiza extraradical mycelium is difficult to fol- low in soil-based systems. The use of dual arbuscular mycorrhizal fungi/in vitro root organ cultures (mon- oxenic AM cultures) allowed the nondestructive study of hyphal development following establishment of the symbiosis. The present study shows that the extraradical spreading of the arbuscular mycorrhizal fungus Glomus intraradices grown monoxenically with tomato roots can be divided into three stages: (i) pro- liferation of runner hyphae acting as conducting channels, which divide dichotomously and extend the fungal colony radially; (ii) development of arbus- cule-like structures, which are formed at regular in- tervals along the runner hyphae and which might play a preferential role in nutrient uptake; and (iii) formation of spores in zones already colonized by runner hyphae and arbuscule-like structures. The de- velopment of the mycorrhiza is accompanied by changes in the pH of the medium. In particular, pH decreases in zones of the medium in which a high number of arbuscular mycorrhizal fungal spores are formed. The intricate architecture shown by the ex- traradical mycelium highlights the potential for en- hanced nutrient uptake by mycorrhizal roots, and their role in the maintainance and amelioration of soil structure.

6 Establishment of Vesicular-arbuscular Mycorrhiza in Root Organ Culture: Review and Proposed Methodology

Publisher Summary Vesicular-arbuscular mycorrhizal fungi are obligate biotrophs that have so far resisted all attempts to be cultivated axenically (in pure culture). This lack of independent growth has not prevented vesicular-arbuscular mycorrhizal fungi from becoming distributed world-wide as a symbiotic partner of most vascular plants, under a wide variety of pedologic and climatic conditions. Cultivation of vesicular-arbuscular mycorrhizal fungi under axenic conditions continues to be a preoccupation and represents one of the most challenging goals of modern plant biology. The purpose of this chapter is to provide a detailed description of the procedures of the method and to suggest new avenues, to stimulate research on the biology of these symbiotic fungi. In this chapter, the use of transformed carrot roots inoculated with a single spore of G. margarita is proposed as a simple experimental system which allows reproducible observations of all stages of vesicular-arbuscular mycorrhizal development, including the extraradical phases. The control of colonization of selected root parts by G. margarita germ tubes is the key to making quantitative and consistent measurements of the initiation and development of a vesicular-arbuscular mycorrhizal symbiosis. The system has been useful for determining symbiotic factors provided by the root which govern fungal growth.

Signalling in arbuscular mycorrhiza: facts and hypotheses.

The arbuscular mycorrhizal symbiosis is an association between plant roots and fungi. Arbuscular mycorrhizal fungi (AMF) colonize roots improving plant nutrition mainly by transferring phosphate (P) from the soil to the plant, whereas plants provide the fungi with carbohydrates (Smith and Read, 1997). In contrast to the rhizobial symbiosis with a host range limited to the Leguminoseae, AMF form symbiotic associations with a wide range of plant species. Interestingly, there seem to be striking similarities between signalling in rhizobial and arbuscular mycorrhizal symbiosis (reviewed by Hirsch and Kapulnik, 1998). Apart from the effect of plant derived secondary plant compounds (SPC) on the bacterial and the fungal symbiont, SPC (e.g. flavonoids) are accumulated in the roots of the respective host plants during the establishment of both symbioses. Whereas there is some information on the role of SPC in the rhizobial symbiosis, the exact role of SPC during the establishment of the AM symbiosis still remains unclear.

FLAVONOIDS AND ARBUSCULAR-MYCORRHIZAL FUNGI

Arbuscular mycorrhizal fungi (AMF) are ancient Zygomycetes forming the most widespread plant-fungus symbiosis. The regulation of this association is still poorly understood in terms of the communication between the two partners. Compounds inside the root and released by the root, such as flavonoids, are hypothesized to play a role in this plant-fungus communication, as already demonstrated in other symbiotic associations (e.g. Rhizobium-leguminoseae). Here we give a general overview of the research concerning this question.

14C transfer between the spring ephemeral Erythronium americanum and sugar maple saplings via arbuscular mycorrhizal fungi in natural stands

Abstract. We investigated in the field the carbon (C) transfer between sugar maple (Acer saccharum) saplings and the spring ephemeral Erythronium americanum via the mycelium of arbuscular mycorrhizal (AM) fungi. Sugar maple saplings and E. americanum plants were planted together in pots placed in the ground of a maple forest in 1999. Ectomycorrhizal yellow birches (Betula alleghaniensis) were added as control plants. In spring 2000, during leaf expansion of sugar maple saplings, the leaves of E. americanum were labelled with 14CO2. Seven days after labelling, radioactivity was detected in leaves, stem and roots of sugar maples. Specific radioactivity in sugar maples was 13-fold higher than in yellow birches revealing the occurrence of a direct transfer of 14C between the AM plants. The quantity of 14C transferred to sugar maple saplings was negatively correlated with the percentage of 14C allocated to the storage organ of E. americanum. A second labelling was performed in autumn 2000 on sugar maple leaves during annual growth of E. americanum roots. Radioactivity was detected in 7 of 22 E.americanum root systems and absent in yellow birches. These results suggest that AM fungi connecting different understorey species can act as reciprocal C transfer bridges between plant species in relation with the phenology of the plants involved.