.\n tn vitro system using Ri T-DN.A transformed roots and the vesiculat-arbuscular mycorrhizal fungus Gigaspora margarita Becker & Hall has been developed to study the initial events of mycorrhiza formation. Sucrose, sodium and phosphorus were found to be critical components of the n-iedium used to establish the dual culture. L'sing a single spore as inoculum it vvas consistently possible to obtain colonization of a preselected point on the root and to time the colonization process (within . S days), .'\hundant viable and aseptic spores can be obtained. The systet-i-i IS especially appropriate for studying the triggering of the fungal biotrophy towards the root.

No abstract

We used the polymerase chain reaction (PCR) to amplify specific regions of the nuclear and mitochondrial genomes of fungi using DNA extracted from pure cultures as well as that directly from ectomycorrhizal rootlets. The internal transcribed spacer (ITS) of the nuclear ribosomal repeat unit and a portion of the mitochondrial large subunit ribosomal RNA gene were chosen as target sequences because both exist in high copy number and amplification primers for both discriminate between plant and fungal DNAs. These features provided a sensitivity and specificity sufficient for detection and analysis of a single mycorrhizal rootlet. We evaluated the variations in the amplified products with regard to the length, restriction endonuclease sites, and primary sequence for use in identification of genera, species, and strains of ectomycorrhizal fungi, with particular attention to selected Laccaria species. Accidental contamination of jack pine seedlings by Telephora terrestris was easily recognized. Amplification and direct DNA sequencing of a portion of the ITS were done for three strains of L. bicolor, one of L. laccata, one of L. proximo, and one of T. terrestris. The nucleotide sequence variation was 32% between L. bicolor and T. terrestris, and it ranged from 3 to 5% among the three Laccaria species examined and from 1 to 2% within L. bicolor. The degree of variation observed is sufficient to allow the use of specific oligonucleotides to characterize amplified ITS products. To demonstrate the feasibility of this approach we designed and tested a probe that enabled two isolates of L. bicolor to be distinguished by a single base-pair difference in a filter-based hybridization assay. In combination these methods now provide an important set of tools for the study of mycorrhizal ecology. Key words: internal transcribed spacer, LrDNA gene, mycorrhizal ecology, polymerase chain reaction, rDNA.

The growth of twenty plant species was compared under field conditions in a methyl bromide fumigated and non-fumigated soil. The non-fumigated soil had a wild endomycorrhizal flora and contained 100 ~tg/g of available phosphorus. No phosphorus was added to the soil but both fumigated and non-fumigated plots received a basal fertilization of 100 kg/ha N-NH4NO 3 and 100 kg/ha K-KCI. Based on plant growth responses, three groups of plants were distinguishable. Plants from group I were mycorrhizal and had better growth in non-fumigated than in the fumigated soil. This group was the most important, including sixteen plant species. Stunting of plants from group I following soil fumigation was mainly attribuable to the destruction of mycorrhizae. Plants from group II (oat and wheat) grew equally well in non-fumigated and fumigated soils. For these plants which were mycorrhizal in the non-fumigated plots, the P-content of the soil was sufficient for growth and therefore no stunting was observed in the absence ofmycorrhizae. Plants from group III (cabbage and garden beet) grew better in fumigated than in non-fumigated soil. Their better growth in fumigated soil was tentatively attributed to the destruction of soil-borne pathogens. They did not form mycorrhizae in non-fumigated soil.A new method of calculating mycorrhizal dependency is proposed, and the value calculated was named relative field mycorrhizal dependency (RFMD) index. It is also proposed that the acronym RFMD receive a superscript representing in ~tg/g the quantity of available P in the soil. Carrot with its characteristic root systems had the highest RFMD 1~176 index (99.2~o), but other plants with high phosphorus requirements for normal growth had a wide range of RFMD 1~176 index values.

The study of arbuscular mycorrhizal (AM) fungi and the AM symbiosis formed with host plant roots is complicated by the biotrophic and hypogeous nature of the mycobionts involved. To overcome this, several attempts have been made during the last three decades to obtain this symbiosis in vitro. The use of root-organ cultures has proved particularly successful. In this review, we describe the method by which root-organ cultures (transformed and nontransformed) have been obtained, together with the choice of host species, inoculation techniques, and culture media. We also outline the potential use of continuous cultures and cryopreservation of in vitro produced spores for long-term germ plasm storage. Furthermore, this review highlights the considerable impact that in vitro root-organ cultures have had on studies of AM fungal morphology, taxonomy, and phylogeny and how they have improved our understanding of the processes leading to root colonization and development of the extraradical mycelium. This is supported by a summary of some of the most important findings, regarding this symbiosis, that have been made at the physiological, biochemical, and molecular levels. We also summarize results from studies between AM fungi and certain pathogenic and nonpathogenic soil microorganisms. We describe some of the limitations of this in vitro system and propose diverse avenues of AM research that can now be undertaken, including the potential use of a similar system for ectomycorrhizal research.Key words: arbuscular mycorrhiza, root-organ cultures, Glomales, in vitro, root symbioses, source of inoculum, cryopreservation, intraradical and extraradical mycelium, mycorrhizosphere.

Arbuscular mycorrhizal (AM) fungi can reduce the incidence and importance of plant root diseases caused by pathogens. The mechanisms involved are not well characterized. We used an in vitro experimental system to test the hypothesis that the extraradical mycelium of AM fungi can interfere directly with microorganisms in the mycosphere and directly or indirectly reduce the population of plant pathogens. This system permitted the isolation of soluble substances released by the extraradical mycelium of Glomus intraradices. The AM fungus was grown on Daucus carota transformed roots in one compartment, while only the extraradical mycelium was allowed to grow in a second compartment. A freezing and centrifugation technique was developed for the extraction and concentration of substances present in the compartment containing only the AM fungal mycelium. Four soilinhabiting microorganisms were selected, and conidial germination (fungi) or growth (bacteria) of these was studied in the presence and absence (control) of the extract. In comparison with the control, the results indicated that both the growth of Pseudomonas chlororaphis and the conidial germination of Trichoderma harzianum were stimulated in the presence of the AM fungal extract. In contrast, conidial germination of Fusarium oxysporum f. sp. chrysanthemi was reduced while the growth of Clavibacter michiganensis subsp. michiganensis was not affected. The measured effects in general were directly correlated with extract concentration. Differences in pH were noted between the extract containing substances released by the AM fungus and the non-AM control, but no significant influence of the pH on growth or conidial germination was noted, confirming that substances released by the AM fungus in the growth medium is the main factor explaining differential growth of the microorganisms tested. The results suggest that direct interactions exist between AM fungi and soil microorganisms, which might lead to changes in microbial equilibrium detrimental to pathogens.

Plencette, C., Clermont-Dauphin, C., Meynard, J. M. and Fortin, J. A. 2005. Managing arbuscular mycorrhizal fungi in cropping systems. Can. J. Plant Sci. 85: 31-40. Market globalization, demographic pressure, and environmental degradation have led us to reconsider many of our current agricultural systems. The heavy use of chemical inputs, including fertilizers and pesticides, has resulted in pollution, decreased biodiversity in intensively-farmed regions, degradation of fragile agro-ecosystems, and prohibitive costs for many farmers. Low input sustainable cropping systems should replace conventional agriculture, but this requires a more comprehensive understanding of the biological interactions within agro-ecosystems. Mycorrhizal fungi appear to be the most important telluric organisms to consider. Mycorrhizae, which result from a symbiosis between these fungi and plant roots, are directly involved in plant mineral nutrition, the control of plant pathogens, and drought tolerance. Most horticultural and crop plants are symbiotic with arbuscular mycorrhizal fungi. Mycorrhizal literature is abundant, showing that stimulation of plant growth can be mainly attributed to improved phosphorous nutrition. Although the mycorrhizal potential of its symbiosis to improve crop production is widely recognized, it is not implemented in agricultural systems. There is an urgent need to improve and widely apply analytical methods to evaluate characteristics such as, relative field mycorrhizal dependency, soil mycorrhizal infectivity, and mycorrhizal receptivity of soil. Decreased use of fertilizers, pesticides, and tillage will favour arbuscular mycorrhizal fungi. However, shifting from one system to a more sustainable one is not easy since all components of the cropping system are closely linked. Different cases, from actual agricultural practices in different countries, are analyzed to highlight situations in which mycorrhizae might or might not play a role in developing more sustainable agriculture. Key words:Cropping systems, mycorrhizae, sustainability, technical itineraries, rotation Plencette, C., Clermont-Dauphin, C., Meynard, J. M. et Fortin, J. A. 2005. Gestion des champignons mycorchiziens a arbuscules dans les systemes de culture. Can. J. Plant Sci. 85: 31-40. La globalisation mondiale du marché, la pression démographique et la dégradation de l'environnement ont, dans plusieurs parties du monde, mené à une réévaluation des systèmes agricoles actuels. Les modifications de l'environnement montrent que l'utilisation des engrais et des pesticides a atteint ses limites : pollution et perte de biodiversité dans les régions d'agriculture intensive, dégradation des systèmes agricoles les plus fragiles, coûts prohibitifs pour les producteurs les plus pauvres. L'agriculture conventionnelle doit s'orienter vers des pratiques plus durables, mais les systèmes de culture durable à faibles intrants ne pourront être viables que par une meilleure connaissance et maîtrise des interactions biologiques dans ces agro-systèmes. Les champign...

A greenhouse experiment was carried out comparing the growth of various plant species in non-fumigated, fumigated, and fumigated-inoculated soils. The soil used contained numerous pieces of root of Broom-Corn Millet (Panicum miliaceum L.) that were found intensely colonized by indigenous endomycorrhizal fungi. The soil was fumigated with methyl bromide and the inoculum used was a mixture of VA mycorrhizal root fragment from plants grown in the field from which the soil was collected. Plants used were cabbage (Brassica oleracea L. var Copenhagen Market), carrot (Daucus earota L. var. Nantaise), leek (Allium porrum L. var. American Flag), marigold (Tagetes patulus L. vat. Golden Boy), tomato (Lycopersicum esculentum Mill. var. Michigan Ohio), sweet corn (Zea mays L. var. Span Cross) and wheat (Triticum aestivum L. var. Glenlea). No phosphorus was added to the soil which contained 93 ~tg/g of available P (bray II). All plants tested formed mycorrhizae except cabbage. Generally, values of the root endomycorrhizal colonization (REC) index were higher in fumigated-inoculated soil than in non-fumigated soil. Cabbage grew equally well in fumigated and fumigated-inoculated soil, but better than in non-fumigated soil. Cabbage did not form VA mycorrhizae and its better growth in fumigated soil was tentatively attributed to the destruction of soil-borne pathogens and the absence of competition. Wheat grew equally well in the three treatments, because 93 I~g/g of available P is sufficient for wheat growth and thus the mycorrhizae were not efficient. The five other plant species used were severely stunted in fumigated soil and the inoculation permitted the reestablishment of normal growth as in non-fumigated soil. Growth stimulation is attributed to the efficiency of VA mycorrhizae since these plants were mycorrhizal in non-fumigated soil and in fumigated-inoculated soil. Stunting of these plants in fumigated soil was due to the destruction of VA mycorrhizae since results show that this stunting cannot be attributed to methylbromide residues in the soil. Moreover soil pH and nutrient content were not markedly changed after fumigation. * To be submitted by C. Plenchette in partial fulfillment for the Ph.D. degree at Laval University. Soil Research Service contribution # 315. M.A.P.A.Q.