A method to identify arbuscular endomycorrhizal fungi based on the amplification of portions of the nuclear gene coding for the small subunit rRNA is presented. By coupling the sensitivity of the polymerase chain reaction and the specificity afforded by taxon-specific primers, a variety of samples can be analyzed, including small amounts of colonized roots. Family-specific primers as well as generic primers are described and can be used to amplify small subunit rRNA fragments from endomycorrhizal fungi by polymerase chain reaction. The amplified products are then subjected to single-strand conformation polymorphism analysis to detect sequence differences. Among the advantages of this approach is the possibility of directly identifying the fungi inside field-collected roots, without having to rely on the fortuitous presence of spores. This technique should have obvious applications in the study of arbuscular endomycorrhizal fungi populations and allow closer examination of their host specificity.

In situ gene amplification (in situ PCR) is a recent, powerful molecular technique which allows the localization of low abundance nucleic acids targets directly within tissue sections. The work presented here is, to our knowledge, the first report of successful direct detection of in situ PCR amplification with fluorescently-labelled primers, and the first successful in situ PCR performed on arbuscular mycorrhizal (AM) fungi. Ribosomal SSU genes within AM fungal spore sections were amplified by using fluorescent, glomalean-specific primers, then directly detected by means of epifluorescence microscopy. Different controls confirmed the successfulness of the in situ amplification. These results open new avenues in the study of arbuscular mycorrhizas, where genetic processes seem to be transient and very localized.

Cadmium (Cd) accumulation and distribution was studied in sunflower (Helianthus annuus L., public line HA-89) plant. From an uncontaminated sandy loam brown forest soil with 162 mu g kg(-1) HNO3/H2O2 extractable Cd the HA-89 sunflower public line accumulated 114 mu g kg(-1) Cd in its kernels under open field conditions. This value is rather low as compared to data found by others. Sandy loam brown forest soil was treated with 0, 1 or 10 mg kg(-1) of Cd to study the interaction of this heavy metal with young sunflower plants in a greenhouse pot experiment. The fresh weight and dry matter accumulation of sunflower plant organs (roots, shoots, leaves or heads) was unaffected by cadmium treatment of soil. The nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), copper (Cu), iron (Fe), manganese (Mn), or zinc (Zn) uptake of sunflower plant organs was not influenced by lower or higher Cd-doses, except sunflower heads where 10 mg kg(-1) of Cd treatment of soil significantly reduced the uptake of Ca, Fe, and Mn. Although Cd reduced the Zn uptake of roots, its rate was statistically not significant. Cadmium was accumulated prevalently in roots (1.21 mg kg(-1), 4.97 mg kg(-1), or 13.69 mg kg(-1) depending on Cd-dose), and its concentration increased also in shoots or leaves. In spite of the short interaction time, elevated concentrations of cadmium (0.78 mg kg(-1), 1.34 mg kg(-1), or 3.02 mg kg(-1) depending on Cd-dose) were detected in just emerged generative organs (heads) of young sunflower plants.

This work presents a cooperative effort to integrate new molecular (isozyme and SSU analyses) characters into the morphological taxonomy of the genus Gigaspora (Glomales). Previous analyses of published Gigaspora SSU sequences indicated the presence of a few polymorphic nucleotides in the region delimited by primers NS71‐SSU 1492′. In our study, the SSU of 24 isolates of arbuscular mycorrhizal (AM) fungi from the Gigasporaceae were amplified and the NS71‐SSU 1492′ region was directly sequenced. The corresponding sequences of four more isolates of AM fungi from Gigasporaceae, already published, were also included in our analyses. Three Gigaspora groups were identified on the basis of a 6 nucleotide‐long ‘molecular signature’: Gigaspora rosea group (G. rosea+G. albida), Gigaspora margarita group (G. margarita+G. decipiens) and Gigaspora gigantea, which constituted a group by itself. The isozyme profiles (malate dehydrogenase, MDH) of 12 of these 28 isolates, and seven other isolates not sequenced, were compared. The results obtained further supported the grouping of isolates provided by the SSU analysis. Both SSU and MDH analysis indicated that two out of the 35 isolates had been misidentified, which was confirmed when their morphology was reassessed. The use of the Gigaspora intrageneric molecular signature as a quick, unambiguous and objective method to recognize Gigaspora isolates under any (field or laboratory) experimental conditions is suggested.

The sensitivity of the polymerase chain reaction (PCR) (,), coupled to the specificity afforded by taxon-specific primers allows the analysis of a variety of samples, like minute amounts of cultured organisms (, ), or even endosymbionts like the endomycorrhizal fungi present in the roots of field collected plants (, , , ).

Summary The Glomales comprise more than 130 described species, presently classified in three families and five genera. Previous analysis of the sequence of the nuclear genes coding for the ribosornal small subunit rRNA (SSU) obtained from 12 isolates representing as many species generally corroborated the taxonomy based on morphological characters. As part of an ongoing effort to understand better the relationship between glomalean species, four new glomalean SSU sequences obtained in our laboratory are described, along with partial sequences from other laboratories. Using this enlarged dataset, a current phylogenetic tree of the Glomales was reconstructed. The dataset was also analysed to locate informative regions of the glomalean SSU that could be targeted by molecular ecologists studying the taxonomy, identification and ecology of arbuscular mycorrhizal fungi.

The symbiosis between vesicular-arbuscular mycorrhizal (VAM) fungi and host plants develops after successful interactions between both partners. These interactions probably involve signal molecules produced by the host plant, by the fungi, or by both. So far the biotrophic status of VAM fungi has hampered the understanding of the processes regulating their physiology. However, among different methods for co-cultivating VAM fungi, root organ cultures (ROC) appear to be a useful technique for studying VAM development. This system has been useful in defining the nutritional requirements of VAM fungi in the precolonization stage and in obtaining axenic fungal material in various developmental stages.

AMONG the Eukaryota, the true fungi comprise four divisions (Chytridiomycota, Zymogomycota, Ascomycota and Basidiomycota) that constitute a natural group which is thought to have diverged about 1 billion (10(9)) years ago, believed also to be the time of divergence between metaphyta and metazoa lineages1. The endosymbionts responsible for the most prevalent plant root symbiosis, the vesicular-arbuscular mycorrhizae (VAM) or, more appropriately, arbuscular mycorrhizae, comprise 130 species of fungi classified in the Zygomycotina, order Glomales2-4. The arbuscular endomycorrhizae are considered to be ecologically important for most vascular plants5-8 in view of their beneficial effects on plant growth and survival. They are one of the few plant-fungus associations with a fossil record and may even have facilitated the origin of land flora. But the biochemical and genetic characterization of these microsymbionts has been hampered by the inability to grow them in pure culture. To investigate the origin and clarify the phylogenetic relationships of these organisms, we have sequenced ribosomal DNA genes from twelve species. Our phylogenetic analyses confirm the existence of three families within arbuscular fungi on the basis of morphological characters. We obtain approximate dates for the divergence of major branches on the phylogenetic tree. These include an estimate for the origin of VAM-like fungi of 353-462 Myr ago, which is consistent with the hypothesis that VAM were instrumental in the colonization of land by ancient plants.

The first DNA sequences obtained from arbuscular endomycorrhizal fungi are reported. They were obtained by directly sequencing overlapping amplified fragments of the nuclear genes coding for the small subunit rRNA. These sequences were used to develop a polymerase chain reaction primer (VANS1) that enables the specific amplification of a portion of the vesicular-arbuscular endomycorrhizal fungus small subunit rRNA directly from a mixture of plant and fungal tissues. The specificity of this primer for arbuscular endomycorrhizal fungi was demonstrated by testing it on a number of organisms and by sequencing the fragment amplified from colonized leek (Allium porum) roots. This approach, coupled with other molecular techniques, will facilitate rapid detection, identification, and possibly quantitation of arbuscular endomycorrhizal fungi.

Nucleotide sequences of the small subunit ribosomal RNA (18S) gene were used to investigate evolutionary relationships within the Fungi. The inferred tree topologies are in general agreement with traditional classifications in the following ways: (1) the Chytridiomycota and Zygomycota appear to be basal groups within the Fungi. (2) The Ascomycota and Basidiomycota are a derived monophyletic group. (3) Relationships within the Ascomycota are concordant with traditional orders and divide the hemi- and euascomycetes into distinct lineages. (4) The Basidiomycota is divided between the holobasidiomycetes and phragmobasidiomycetes. Conflicts with traditional classification were limited to weakly supported branches of the tree. Strongly supported relationships were robust to minor changes in alignment, method of analysis, and various weighting schemes. Weighting, either of transversions or by site, did not convincingly improve the status of poorly supported portions of the tree. The rate of variation at particular sites does not appear to be independent of lineage, suggesting that covariation of sites may be an important phenomenon in these genes.

The VANS1/NS21 primer pair is useful for specifically amplifying a 550-bp ribosomal (r) DNA fragment from arbuscular endomycorrhizal fungi, directly from colonized root extracts. A procedure to quantitate these obligatory biotrophs rapidly, based on competitive PCR, was developed by constructing a suitable internal standard to be used with these primers. A 130-bp deletion in the Glomus mossae VANS1/NS21 amplified rDNA fragment was produced by amplifying separately external portions of that fragment, followed by ligation and amplification using the original external primers. When this deleted fragment was added to G. mossae rDNA, amplification using VANS1/NS21 primers yielded the two expected products of 430 bp and 550 bp, respectively, resolved by agarose electrophoresis. This fragment was cloned into the pCL1920 plasmid, a low-copy-number vector (five copies per cell), and mixed with the roots to be analyzed. This provides for a rapid quantitative assay because both steps--extraction of DNA from colonized roots and PCR amplification--are taken into account by the same internal standard. Using this procedure, a sample of colonized leek roots (Allium porum x Glomus vesiculiferum) was shown to contain 5 x 10(4) copies of arbuscular endomycorrhizal fungi rDNA genes per milligram of fresh weight.

A simple protocol for the extraction of total DNA from minute amounts of tissues and subsequent amplification of specific sequences by polymerase chain reaction is presented. The method is applicable to a wide variety of vegetative tissues such as leaves, single needles and rootlets, cell suspensions, and also single sexual embryos and megagametophytes derived from a variety of gymnosperms and perennial angiosperms. Amplification of DNA is shown using pairs of primers specific to genes that encode the small ribosomal subunit.

The fatty acid methyl esters of 30 selected Frankia strains were analysed by capillary GLC. The temporal stability of the resulting profiles was demonstrated on a subset of strains. Multivariate statistics were used to analyse the data. The Frankia strains belonging to the species F. elaeagni and those of F. alni subsp. pommerii could be separated in different clusters. Furthermore, strains classified as F. alni subsp. vandijkii were shown to be outside the F. alni subsp. pommerii cluster. This was confirmed by reanalysing isozyme data available for the same strains. The clustering of strains achieved using fatty acid profiles thus corroborates the recently proposed taxonomy of Frankia.

Green algae (Chorella pyrenoidosa) were grown in iron‐deficient and normal iron supplied nutrient solution and treated with 1 uM gallium. Treatment with gallium enhanced the growth rate and chlorophyll content of algae cultures. Significant changes were recorded in individual photosynthetic pigment composition of treated cultures. In both iron‐deficient and normal iron supplied cultures, gallium treatment enhanced peroxidase enzyme activity. Partially purified peroxidase showed different kinetic properties in the samples treated with gallium. An iron‐gallium interaction is proposed that affects chlorophyll metabolism and peroxidase activity in green algae.

Actinorhizae were synthetized on in vitro propagated Elaeagnus angustifolia L. clonal plantlets, by inoculation with combinations of pure cultures of three effective Frankia strains and an ineffective one. Using the OsO4 isolation method, 774 nodule fragments from 79 plantlets were treated and 152 Frankia reisolates were obtained. The soluble protein patterns of 121 reisolates were analyzed by one-dimensional SDS-PAGE, which permitted their positive identification. The protein patterns of all reisolates obtained from plants inoculated with a single strain were indistinguishable from those of the original strain. In six different dual strain inoculations, only one of the two Frankia strains could be reisolated. Thus, some Frankia strains appeared to be more competitive than others toward the Elaeagnus root system.