David D. Douds

Nitrogen transfer in the arbuscular mycorrhizal symbiosis

Most land plants are symbiotic with arbuscular mycorrhizal fungi (AMF), which take up mineral nutrients from the soil and exchange them with plants for photosynthetically fixed carbon. This exchange is a significant factor in global nutrient cycles as well as in the ecology, evolution and physiology of plants. Despite its importance as a nutrient, very little is known about how AMF take up nitrogen and transfer it to their host plants. Here we report the results of stable isotope labelling experiments showing that inorganic nitrogen taken up by the fungus outside the roots is incorporated into amino acids, translocated from the extraradical to the intraradical mycelium as arginine, but transferred to the plant without carbon. Consistent with this mechanism, the genes of primary nitrogen assimilation are preferentially expressed in the extraradical tissues, whereas genes associated with arginine breakdown are more highly expressed in the intraradical mycelium. Strong changes in the expression of these genes in response to nitrogen availability and form also support the operation of this novel metabolic pathway in the arbuscular mycorrhizal symbiosis.

Partitioning of Intermediary Carbon Metabolism in Vesicular-Arbuscular Mycorrhizal Leek.

Vesicular-arbuscular mycorrhizal fungi are symbionts for a large variety of crop plants; however, the form in which they take up carbon from the host is not established. To trace the course of carbon metabolism, we have used nuclear magnetic resonance spectroscopy with [13C]glucose labeling in vivo and in extracts to examine leek (Allium porrum) roots colonized by Glomus etunicatum (and uncolonized controls) as well as germinating spores. These studies implicate glucose as a likely substrate for vesicular-arbuscular mycorrhizal fungi in the symbiotic state. Root feeding of 0.6 mM 1-[13C]glucose labeled only the fungal metabolites trehalose and glycogen. The time course of this labeling was dependent on the status of the host. Incubation with 50 mM 1-[13C]glucose caused labeling of sucrose (in addition to fungal metabolites) with twice as much labeling in uncolonized plants. There was no detectable scrambling of the label from C1 glucose to the C6 position of glucose moieties in trehalose or glycogen. Labeling of mannitol C1,6 in the colonized root tissue was much less than in axenically germinating spores. Thus, carbohydrate metabolism of host and fungus are significantly altered in the symbiotic state.

Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid.

Arbuscular mycorrhizal (AM) fungi take up photosynthetically fixed carbon from plant roots and translocate it to their external mycelium. Previous experiments have shown that fungal lipid synthesized from carbohydrate in the root is one form of exported carbon. In this study, an analysis of the labeling in storage and structural carbohydrates after 13C1 glucose was provided to AM roots shows that this is not the only pathway for the flow of carbon from the intraradical to the extraradical mycelium (ERM). Labeling patterns in glycogen, chitin, and trehalose during the development of the symbiosis are consistent with a significant flux of exported glycogen. The identification, among expressed genes, of putative sequences for glycogen synthase, glycogen branching enzyme, chitin synthase, and for the first enzyme in chitin synthesis (glutamine fructose-6-phosphate aminotransferase) is reported. The results of quantifying glycogen synthase gene expression within mycorrhizal roots, germinating spores, and ERM are consistent with labeling observations using 13C-labeled acetate and glycerol, both of which indicate that glycogen is synthesized by the fungus in germinating spores and during symbiosis. Implications of the labeling analyses and gene sequences for the regulation of carbohydrate metabolism are discussed, and a 4-fold role for glycogen in the AM symbiosis is proposed: sequestration of hexose taken from the host, long-term storage in spores, translocation from intraradical mycelium to ERM, and buffering of intracellular hexose levels throughout the life cycle.

Partial separation of root exudate components and their effects upon the growth of germinated spores of AM fungi

Aseptic root exudates were collected from the liquid culture of roots of two host (Daucus carota and Lycopersicum esculentum) and one non-host plant (Beta vulgaris) of arbuscular mycorrhizal (AM) fungi. Exudate was also collected from maize (Zea mays FRB6) seedlings which were grown hydroponically under aseptic conditions. Exudate fractions of host roots stimulated hyphal branching behind any actively growing hyphal tip of three AM fungi tested (Gigaspora gigantea, G. rosea, and Glomus intraradices). Fractionation patterns obtained from C18 Sepak cartridges loaded with carrot root exudates isolated from roots grown under various phosphorus regimes, TLC analyses, and solubility properties of fractionated components, indicated a range of hydrophilic to hydrophobic hyphal branching stimulators. The 50/70% methanol fraction from a C18 cartridge induced hyphal branching patterns of G. gigantea that were dose dependent and were identical to those observed when germinated G. gigantea spores were grown with host roots in dual culture. Exudate fractions from B. vulgaris inhibited hyphal tip growth, but inhibited hyphal tips formed recovery branches which would allow continued fungal growth. These recovery hyphae were also formed when germinated G. gigantea spores were grown in dual culture with sugar beet roots. The recovery branches induced by non-host roots and the prolific branching induced by host roots have ecological implications.

The glyoxylate cycle in an arbuscular mycorrhizal fungus. Carbon flux and gene expression.

The arbuscular mycorrhizal (AM) symbiosis is responsible for huge fluxes of photosynthetically fixed carbon from plants to the soil. Lipid, which is the dominant form of stored carbon in the fungal partner and which fuels spore germination, is made by the fungus within the root and is exported to the extraradical mycelium. We tested the hypothesis that the glyoxylate cycle is central to the flow of carbon in the AM symbiosis. The results of (13)C labeling of germinating spores and extraradical mycelium with (13)C(2)-acetate and (13)C(2)-glycerol and analysis by nuclear magnetic resonance spectroscopy indicate that there are very substantial fluxes through the glyoxylate cycle in the fungal partner. Full-length sequences obtained by polymerase chain reaction from a cDNA library from germinating spores of the AM fungus Glomus intraradices showed strong homology to gene sequences for isocitrate lyase and malate synthase from plants and other fungal species. Quantitative real-time polymerase chain reaction measurements show that these genes are expressed at significant levels during the symbiosis. Glyoxysome-like bodies were observed by electron microscopy in fungal structures where the glyoxylate cycle is expected to be active, which is consistent with the presence in both enzyme sequences of motifs associated with glyoxysomal targeting. We also identified among several hundred expressed sequence tags several enzymes of primary metabolism whose expression during spore germination is consistent with previous labeling studies and with fluxes into and out of the glyoxylate cycle.

Purification of commercial gellan to monovalent cation salts results in acute modification of solution and gel-forming properties

Lithium, sodium, potassium, and ammonium salts of the industrial polysaccharide gellan were prepared. The salts were freely soluble in water at room temperature (25 degrees C). The opinion had been generally held that heating to 100 degrees C was necessary for gellan to achieve complete solubility in the presence of mono- or multivalent cations. Then, upon cooling, the solutions would form gels. These conclusions were based on the properties imposed upon gellan samples by the presence of contaminating divalent cations. Commercial gellan samples contain calcium and magnesium at levels exceeding 0.9%, sufficient for counterion formation with over one-third of gellans carboxyl groups. Purification was rapid and included sequential treatments with a cation-exchange (H+) resin, LiOH, NaOH, KOH, or NH4OH, and an anion-exchange (Cl-) resin. About 95% of the divalent cations and nearly 90% of the phosphate that contaminated commercial gellan were removed. The purified monovalent salts of gellan set in the presence of divalent cations and provide well-defined agents for gelling media used for propagation of microbes and plants. In a manner analogous to sodium alginate, solutions of lithium, sodium, potassium, or ammonium gellanate form beads when dropped into solutions of divalent cations. This property was exploited for entrapment of enzymes and cells in beads.

Inoculation with Arbuscular Mycorrhizal Fungi Increases the Yield of Potatoes in a High P Soil

ABSTRACT Arbuscular mycorrhizal (AM) fungi are potentially important tools in agricultural systems that reduce or eliminate chemical inputs common in modem agriculture. We tested the response of potato (Solanum tuberosum L. cv. Superior) to inoculation with AM fungi in a field with very high available P (375 μg g−l soil) in two growing seasons. Inoculation treatments included a commercially available inoculum containing Glomus intraradices, mixed species inocula produced on-farm in mixtures of compost and vermiculite, and a control treatment consisting of a freshly prepared compost and vermiculite mixture. In addition, two farming systems were imposed: conventional chemical fertilizers or dairy manure composted with leaves were applied to meet recommended nutrient requirements. Yields of tubers on a fresh weight basis in the first year were significantly increased by AM fungus inoculum, 33% under conventional fertilizer application and 45% with compost addition vs. controls in each system. The response to inoculation the second year was less; however yields of inoculated plants were 10 to 20% greater than controls. There was a significant positive treatment effect of inoculation upon production of larger sized potatoes in the second year. Neither year saw a marked difference in yield response among AM fungus inocula. These results demonstrate the potential yield benefits of inoculation of potatoes with AM fungi produced on the farm.

On-farm production of inoculum of indigenous arbuscular mycorrhizal fungi and assessment of diluents of compost for inoculum production.

On-farm production of arbuscular mycorrhizal [AM] fungus inoculum can be employed to make the benefits of the symbiosis more available to vegetable farmers. Experiments were conducted to modify an existing method for the production of inoculum in temperate climates to make it more readily adoptable by farmers. Perlite, vermiculite, and peat based potting media were tested as diluents of yard clippings compost for the media in which the inoculum was produced using bahiagrass (Paspalum notatum Flugge) as host plant. All produced satisfactory concentrations of AM fungus propagules, though vermiculite proved to be better than potting media (89 vs. 25 propagules cm(-3), respectively). Two methods were tested for the growth of AM fungi indigenous to the farm: (1) adding field soil into the vermiculite and compost mixture and (2) pre-colonizing the bahiagrass seedlings in media inoculated with field soil prior to transplant into that mixture. Adding 100 cm(3) of field soil to the compost and vermiculite produced 465 compared to 137 propagules cm(-3) for the pre-colonization method. The greater flexibility these modifications give will make it easier for farmers to produce inoculum of AM fungi on-the-farm.

Choosing a Mixture Ratio for the On-Farm Production of AM Fungus Inoculum In Mixtures of Compost and Vermiculite

Arbuscular mycorrhizal [AM] fungi are potentially important tools in sustainable agriculture due to their roles in crop nutrient uptake, disease resistance, and water relations and in stabilizing soil aggregates. Inocula of these fungi can be effectively produced on-farm in mixtures of compost and vermiculite with a suitable plant host, such as bahiagrass (Paspalum notatum Flugge). Success of this method, however, depends upon utilizing the optimal compost and vermiculite mixture ratio. Experiments were conducted over two years utilizing a complete factorial design with three composts, four mixture ratios, and three AM fungi with the objective of producing regression equations to predict optimal mixture ratios using routine measures of compost nutrient analyses as independent variables. Growth of colonized P. notatum in yard clippings and dairy manure + leaf composts; which were high in N, low in P, with moderate K levels; produced more spores of AM fungi at mixture ratios of 1:2 to 1:4 [v/v compost: vermiculite] relative to higher dilutions. Dilution ratios of 1:19 and 1:49 were best for controlled microbial compost, which was high in P, low in N, and moderately high in K. Simple equations were developed which predict the optimal fraction of compost in the mixture for each of the three AM fungi studied (Glomus intraradices, Glomus mosseae, and Gigaspora rosea). Percent N, P, and K and N:P ratio were the significant independent variables. These equations allow a farmer to choose a mixture ratio for the on-farm propagation of AM fungi knowing only the nutrient analysis of the compost to be used.

Cytoplasmic autofluorescence of an arbuscular mycorrhizal fungus Gigaspora gigantea and nondestructive fungal observations in planta

Spores and hyphae of arbuscular mycor- rhizal fungi, grown in vitro with or without Ri T-DNA transformed roots of Daucus carota, were examined via epifluorescence microscopy. Hyphae and root in- fection structures of the arbuscular mycorrhizal fun- gus Gigaspora gigantea exhibited a bright yellowish- green fluorescence when exposed to 450-490 nm light. Structures of G. margarita showed no fluores- cence under the same conditions. The autofluores- cent component of G. gigantea is cytoplasmic and mobile in the cytoplasmic stream, so it can be used as a natural vital marker. This phenomenon makes time-course, nondestructive examination of mycor- rhizal and studies of the competition between G. gi- gantea and other fungi possible for the first time.