Plant-microbe interactions of selenium hyperaccumulators: Effects on plant growth and selenium metabolism

As a first step in elucidating the specific interactions of hyperaccumulator (HA) plants and microbes, I characterized the selenium (Se) localization and chemical speciation in roots of the HA Astragalus bisulcatus, commonly known as the two-grooved milk vetch (Fabaceae) and Stanleya pinnata, or Prince's plume (Brassicaceae), collected from a seleniferous area in Fort Collins, CO. The focus of this study was on the root since similar studies had already been done on all above-ground organs, and the root is of particular interest for rhizospheric plant-microbe interactions. Four fungi collected previously from HA roots were characterized with respect to their Se tolerance and ability to produce Se0, and then used to inoculate HA plants in a controlled greenhouse study. X-ray microprobe analysis showed that three of fungi could produce Se0. The roots of the greenhouse-grown HAs Astragalus racemosus and S. pinnata showed similar Se distribution patterns regardless of inoculation treatment, and contained almost exclusively C-Se-C. In fact, there was not even a minor fraction of Se0 detected in the greenhouse-grown plants, with the exception of areas associated with microbial activity. A substantial fraction of Se0 was found inside A. racemosus root nodules (that contain nitrogen-fixing bacteria), and some Se0 was observed on the surface of A. racemosus roots inoculated with Alternaria astragali. Thus, root Se speciation was strikingly different in HA plants collected from their natural habitat as compared to when grown from surface-sterilized seeds in the greenhouse.;In the greenhouse inoculation studies I also investigated the effect of the rhizosphere fungi on plant growth and Se accumulation. Two Astragalus species, HA A. racemosus and the non-HA Astragalus convallarius, were inoculated with A. astragali (A3) and Fusarium acuminatum (F30), which were originally isolated from Astragalus HA species. Inoculation did affect growth in both species; A3 enhanced growth of A. racemosus yet inhibited growth of A. convallarius. Selenium treatment negated these differences in growth, perhaps because the A3 fungus is inhibited by Se. F30 reduced shoot-to-root Se translocation in A. racemosus, perhaps because of fungal trapping of Se on the root surface. X-ray microprobe analysis revealed no apparent differences between the inoculation treatments, but showed that the two Astragalus species differed in Se localization as well as chemical Se speciation.;In a similar experimental approach, HA S. pinnata and related non-HA Stanleya elata were inoculated with Alternaria seleniiphila (A1) or Aspergillus leporis (AS117), two rhizoplane fungi isolated from S. pinnata. Growth of S. pinnata was not affected by inoculation or by Se. Stanleya elata growth was inhibited by the presence of AS117 and by Se, but the combination of both treatments did not reduce growth of this non-HA. Selenium translocation was reduced in inoculated S. pinnata, and inoculation reduced S translocation in both the HA and non-HA species. X-ray microprobe analysis did not reveal inoculation-related differences in root Se distribution and speciation in either species; both species accumulated mainly (90%) organic Se. Sulfur, interestingly, was present equally in organic and inorganic forms in S. pinnata roots, suggesting Se-specific assimilation in this species. In conclusion, these rhizosphere fungi can affect growth and Se and/or S accumulation in Stanleya, and the effects were dependent on host species.;In the earlier studies, it was hypothesized that the root Se0 was microbe-derived, yet none of the four rhizosphere fungi selected significantly affected plant Se speciation. Therefore, my attention shifted to another possible candidate. A fungus, shown to be an abundant seed endophyte in HA A. bisulcatus was identified as Alternaria tenuissima (A2) and was shown by X-ray microprobe analysis to be capable of producing Se0 when supplied with selenite as well as when growing on A. bisulcatus seed. Uninfected A. bisulcatus seeds contained 100% organic Se (MeSeCys), which was located in the seed embryo but not the seed coat. Seeds harboring A2 contained up to 22% Se0, as did the A2 mycelium growing on the seed. The production of Se0 by A2 likely serves as a fungal Se tolerance mechanism. Surprisingly, while A2 successfully colonized seeds containing 10,000 mg kg-1 MeSeCys, it was sensitive to 25 mg kg -1 Se from flower extract or when supplied in the form of pure MeSeCys. Thus, this fungus likely occupies low-Se areas of the plant, such as in the seed coat and the apoplast. (Abstract shortened by UMI.).