Diversity of frankiae in soils from five continents.

Clone libraries of nifH gene fragments specific for the nitrogen-fixing actinomycete Frankia were generated from six soils obtained from five continents using a nested PCR. Comparative sequence analyses of all libraries (n=247 clones) using 96 to 97% similarity thresholds revealed the presence of three and four clusters of frankiae representing the Elaeagnus and the Alnus host infection groups, respectively. Diversity of frankiae was represented by fewer clusters (i.e., up to four in total) within individual libraries, with one cluster generally harboring the vast majority of sequences. Meta-analysis including sequences previously published for cultures (n=48) and for uncultured frankiae in root nodules of Morella pensylvanica formed in bioassays with the respective soils (n=121) revealed a higher overall diversity with four and six clusters of frankiae representing the Elaeagnus and the Alnus host infection groups, respectively, and displayed large differences in cluster assignments between sequences retrieved from clone libraries and those obtained from nodules, with assignments to the same cluster only rarely encountered for individual soils. These results demonstrate large differences between detectable Frankia populations in soil and those in root nodules indicating the inadequacy of bioassays for the analysis of frankiae in soil and the role of plants in the selection of frankiae from soil for root nodule formation.

Variation in Frankia populations of the Elaeagnus host infection group in nodules of six host plant species after inoculation with soil.

The potential role of host plant species in the selection of symbiotic, nitrogen-fixing Frankia strains belonging to the Elaeagnus host infection group was assessed in bioassays with two Morella, three Elaeagnus, and one Shepherdia species as capture plants, inoculated with soil slurries made with soil collected from a mixed pine/grassland area in central Wisconsin, USA. Comparative sequence analysis of nifH gene fragments amplified from homogenates of at least 20 individual lobes of root nodules harvested from capture plants of each species confirmed the more promiscuous character of Morella cerifera and Morella pensylvanica that formed nodules with frankiae of the Alnus and the Elaeagnus host infection groups, while frankiae in nodules formed on Elaeagnus umbellata, Elaeagnus angustifolia, Elaeagnus commutata, and Shepherdia argentea generally belonged to the Elaeagnus host infection group. Diversity of frankiae of the Elaeagnus host infection groups was larger in nodules on both Morella species than in nodules formed on the other plant species. None of the plants, however, captured the entire diversity of nodule-forming frankiae. The distribution of clusters of Frankia populations and their abundance in nodules was unique for each of the plant species, with only one cluster being ubiquitous and most abundant while the remaining clusters were only present in nodules of one (six clusters) or two (two clusters) host plant species. These results demonstrate large effects of the host plant species in the selection of Frankia strains from soil for potential nodule formation and thus the significant effect of the choice of capture plant species in bioassays on diversity estimates in soil.

Diversity of frankiae in root nodules of Morella pensylvanica grown in soils from five continents.

Bioassays with Morella pensylvanica as capture plant and comparative sequence analyses of nifH gene fragments of Frankia populations in nodules formed were used to investigate the diversity of Frankia in soils over a broad geographic range, i.e., from sites in five continents (Africa, Europe, Asia, North America, and South America). Phylogenetic analyses of 522-bp nifH gene fragments of 100 uncultured frankiae from root nodules of M. pensylvanica and of 58 Frankia strains resulted in a clear differentiation between frankiae of the Elaeagnus and the Alnus host infection groups, with sequences from each group found in all soils and the assignment of all sequences to four and five clusters within these groups, respectively. All clusters were formed or dominated by frankiae obtained from one or two soils with single sequences occasionally present from frankiae of other soils. Variation within a cluster was generally low for sequences representing frankiae in nodules induced by the same soil, but large between sequences of frankiae originating from different soils. Three clusters, one within the Elaeagnus and two within the Alnus host infection groups, were represented entirely by uncultured frankiae with no sequences from cultured relatives available. These results demonstrate large differences in nodule-forming frankiae in five soils from a broad geographic range, but low diversity of nodule-forming Frankia populations within any of these soils.

Phytotoxic polyacetylenes from roots of Russian knapweed (Acroptilon repens (L.) DC.).

There are several factors thought to assist invasive weeds in colonization of ecosystems. One of these factors is allelopathy, the negative effect of chemicals produced by one plant on neighboring plants, frequently mediated through root exudates and other plant leachates. Acroptilon repens (Asteraceae) is one of the most invasive and ecologically threatening weed species in western North America. A bioassay-guided fractionation of the root extracts of this plant led to the isolation of five polyacetylenic compounds, of which one [5'-methoxy-1'-(5-prop-1-yn-1-yl-2-thienyl)-hexa-2',4'-diyin-6'-yl acetate] was hitherto unknown. The structures of these compounds were elucidated on the basis of spectroscopic analysis (IR, ESIMS, (1)H, (13)C NMR and 2D NMR). All of the compounds obtained, except 1-chloro-4-(5-penta-1,3-diyn-1-yl-2-thienyl)but-3-yn-2-ol, showed phytotoxic activity against Arabidopsis thaliana seedlings. The presence of 4'-chloro-1'-(5-penta-1,3-diyn-1-yl-2-thienyl)-but-2'-yn-3'-ol was detected in the root exudates of aeroponically grown A. repens plants. None of the polyacetylenes isolated in this study were found in Colorado soils collected between September 2006 and July 2007 in an A. repens colonized site. However, polyacetylene 5 in A. repens infested soil from Washington was found in June, 2007. Contrary to our previous report, the compound 7,8-benzoflavone (6) was not detected in root exudates, nor was it encountered in extracts of roots, aerial parts or infested soil. Since we could not repeat this work, the original report has been retracted [Stermitz, F.R., Bais, H.P., Foderaro, T.A., Vivanco, J.M., 2003. 7,8-Benzoflavone: a phytotoxin from root exudates of invasive Russian knapweed [A retraction]. Phytochemistry 64, 493-497.].