Gerald Nagahashi

The differential effects of cell wall-associated phenolics, cell walls, and cytosolic phenolics of host and non-host roots on the growth of two species of AM fungi

Experiments were conducted to test the hypothesis that cellular compounds, especially wall-associated compounds, released during emergence of secondary roots, stimulate the growth of arbuscular mycorrhizal (AM) fungi. Purified cell walls, crude cell-wall extracts, crude cytoplasmic extracts, and phenolic compounds previously identified as cell wall-associated, from Ri T-DNA-transformed roots of host (Daucus carota L.) and non-host (Beta vulgaris L.) were incorporated into growth medium and tested for their effects upon growth of the AM fungi Gigaspora gigantea (Nicol. & Gerd.) Gerdemann and Trappe and Gigaspora margarita Becker and Hall. Purified cell walls of both plants had little effect on G. gigantea but non-host cell walls inhibited the growth of G. margarita. Ferulic acid, a major constituent of non-host root, depressed the growth of both fungi. Nothing tested which was unique to the non-host root affected hyphal growth to the point that contact would be prohibited. Caffeic acid, found in D. carota cytoplasm, also depressed growth of both fungi. Para-hydroxybenzoic acid, a constituent of D. carota roots, stimulated growth of G. margarita hyphae, but did not affect hyphal growth of G. gigantea. Vanillic acid, unique to D. carota root cell-wall extracts, stimulated hyphal growth and branching of both fungi, and should increase the probability of contact between fungus and host root.

Root exudate of pmi tomato mutant M161 reduces AM fungal proliferation in vitro

Soluble factors released from roots of the pre-mycorrhizal infection (pmi) myc(-) tomato mutant M161 were analyzed and compared with normal wild-type released factors. Aseptic whole exudates from the M161 mutant retarded the proliferation of Glomus intraradices in vitro. When the whole exudate was further fractionated on a C18 SEPAK cartridge, the 50/70% methanol fraction showed an activity against hyphal tip growth of Gigaspora gigantea and Gl. intraradices. Preliminary characterization of the exudate suggests that the inhibitory moieties are heat labile, bind to PVPP (polyvinyl polypyrrolidone), and are not volatile. This is the first reported instance of the inhibition by a myc(-) plant being ascribed to inhibitory component(s) released in root exudate.

Root exudates stimulate the uptake and metabolism of organic carbon in germinating spores of Glomus intraradices

* Root exudates play a key role during the presymbiotic growth phase and have been shown to stimulate hyphal branching and the catabolic metabolism of arbuscular mycorrhizal (AM) fungal spores. * Here, the effect of root exudates on presymbiotic growth, uptake of exogenous carbon and transcript levels for genes putatively involved in the carbon metabolism of germinating spores were determined. * Crude root exudates led to a slight acceleration of spore germination, increased germ tube branching and stimulated uptake and catabolic metabolism of acetate, and to a greater extent of glucose, but had no effect on gene expression. By contrast, partially purified root exudates increased the transcript levels of acyl-CoA dehydrogenase (ss-oxidation of fatty acids to acetyl-CoA), malate synthase (glyoxylate cycle) and glutamine-fructose-6-phosphate aminotransferase (chitin biosynthesis), but did not differ from crude root exudates in their effect on substrate uptake and respiration. The expression of glycogen synthase (glycogen biosynthesis), glucose-6-phosphate dehydrogenase (pentose phosphate pathway) and neutral trehalase (hydrolysis of trehalose) were only marginally or not affected by root exudates. * Root exudates have an effect on both membrane activity and gene expression and the results are discussed in relation to the catabolic and anabolic metabolism of spores during presymbiotic growth.

Isolated root caps, border cells, and mucilage from host roots stimulate hyphal branching of the arbuscular mycorrhizal fungus, Gigaspora gigantea

Unlike previous reports that have shown that water soluble and volatile compounds from roots or root exudates play an important role in precolonization events during arbuscular mycorrhizal (AM) fungus-host root interactions (Bécard & Piché 1989, Giovannetti et al. 1993), the results shown here deal with particulate and viscous fractions isolated from host roots. Root caps and a slow sedimenting particulate fraction (SSPF) were rapidly isolated and separated from Ri T-DNA transformed carrot roots (D. carota) grown in liquid culture. In addition, border cells (BC) and mucilage were isolated from aseptically grown corn seedlings (Zea mays). Root caps, SSPF (composed mainly of small root cap fragments and some BCs), BCs, and mucilage all had an associated AM fungus hyphal branching stimulator. Root caps stored for 5 d at 4 degrees C appeared to either synthesize or slowly release the branching stimulator. Also, isolated root caps from roots grown in the absence of P contained more branch stimulating activity than those isolated from roots grown in the presence of P. Although the branching stimulation activity in particulate fractions was low compared to that of the exudate, the particulate fractions can stick to the root surface at considerable distances from the root tip. This may be significant during the infection and colonization of host roots at sites far removed from the primary location of exudation.

Action spectrum for the induction of hyphal branches of an arbuscular mycorrhizal fungus : exposure sites versus branching sites#

The first action spectrum for a photo-induced response of an arbuscular mycorrhizal fungus is reported. At low light intensity, the responsive wavelengths for light-induced hyphal branching of the primary germ tube of Gigaspora gigantea were determined to be in the blue to uv-A range. The action spectrum showed the greatest stimulation of branching occurred around 390 nm although a shoulder was observed between 360-370 nm. A second major peak of light-induced branching occurred at 430 nm. The exposure of specific areas of the germ tube to high intensity blue light for a short period led to several interesting observations. By exposing 2 mm segments (0-2, 2-4, 4-6, etc.) or 3 mm segments away from the tip, it was determined that photoinduction of hyphal branches could occur anywhere along the axis of a growing germ tube except in the apical 2 mm. When 3 mm segments were exposed at greater distances from the tip (6-9, 9-12, and up to 33-36 mm), branches frequently formed in areas not directly exposed to light. The branches were usually in clusters which were spaced approximately 3 or 6 mm apart. Since light scattering could be ruled out, these results indicated that the exposure sites and sites of hyphal branching did not necessarily coincide and suggested the probable involvement of a second messenger during this blue light-induced event.

Germinating spores of Glomus intraradices can use internal and exogenous nitrogen sources for de novo biosynthesis of amino acids

* Here, nitrogen (N) uptake and metabolism, and related gene expression, were analyzed in germinating spores of Glomus intraradices to examine the mechanisms and the regulation of N handling during presymbiotic growth. * The uptake and incorporation of organic and inorganic N sources into free amino acids were analyzed using stable and radioactive isotope labeling followed by high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and liquid scintillation counting and the fungal gene expression was measured by quantitative polymerase chain reaction (Q-PCR). * Quiescent spores store Asp, Ala and Arg and can use these internal N resources during germination. Although not required for presymbiotic growth, exogenous N can also be utilized for the de novo biosynthesis of amino acids. Ammonium and urea are more rapidly assimilated than nitrate and amino acids. Root exudates do not stimulate the uptake and utilization of exogenous ammonium, but the expression of genes encoding a putative glutamate dehydrogenase (GDH), a urease accessory protein (UAP) and an ornithine aminotransferase (OAT) were stimulated by root exudates. The transcript levels of an ammonium transporter (AMT) and a glutamine synthetase (GS) were not affected. * Germinating spores can make effective use of different N sources and the ability to synthesize amino acids does not limit presymbiotic growth of arbuscular mycorrhizal (AM) spores.

Inoculation of Strawberries with AM Fungi Produced On-Farm Increased Yield

ABSTRACT Inoculation of plants with arbuscular mycorrhizal [AM] fungi has the potential to increase or maintain yields and allow for reduced fertilizer and pesticide application, thereby enhancing agricultural sustainability. Strawberry plants (Fragaria x ananassa Duch. cv. Chandler) were inoculated prior to outplanting with a mixed species inoculum of AM fungi. The inoculum was produced on-the-farm in 2003 in a mixture of compost and vermiculite with bahiagrass (Paspalum notatum Flugge) as host plants. Plants were outplanted into raised black plastic beds on 30 June 2004 and harvested 6–25 June 2005. Inoculation with AM fungi increased yield 17% over uninoculated controls, 5.5 vs. 4.7 kg per ten plant sampling unit, respectively. Inoculation had no significant effect on whole season mean fruit weight, indicating an average increase of 3.6 fruit per plant for inoculated plants over uninoculated plants. Utilization of AM fungus inoculum produced on-farm as an amendment to horticultural potting media for the production of seedlings later outplanted has the potential for significant increases in crop yields.

Functional Categories of Root Exudate Compounds and their Relevance to AM Fungal Growth

It is well established that plants grown under limited phosphorus (Pi) conditions are more readily colonized by arbuscular mycorrhizal (AM) fungi. It is also known that certain components of host root exudates can stimulate hyphal growth and branching of AM fungi and these compounds are elevated when the host is grown under Pi stress. To obtain a more general picture of the types of compounds exuded by host roots that effect the growth of AM fungi, a global analysis was performed on crude exudates of Ri TDNA-transformed carrot roots grown in the presence and absence of Pi. The results show that there is a distinct population of exudate compounds that are elevated in the absence versus the presence of Pi. Putative identifications were made for some of these compounds from data obtained by Fourier Transform Ion Cyclotron Mass Spectrometry (FTMS). The results were then compared to components of biologically active fractions purified by two dimensional thin layer chromatography (2D TLC). The data selection was restricted to compounds that were initially identified to be elevated in the crude – Pi exudate. The categories of compounds, such as plant growth regulators, phenols, flavonoids, and sesquiterpenoids were selected that had the most relevance to AM fungal/host interactions. The previous results with some of these compounds will be discussed in relation to new results obtained from FTMS and their involvement in presymbiotic growth of AM fungi. None of the reported strigolactones which stimulate AM fungi hyphal were found although several sesquiterpene lactones were identified. A number of hydroxy fatty acids were also found, and they were elevated in the – Pi crude exudate and found to be present in 2D TLC fractions. One hydroxy fatty acid, 2-hydroxytetradecanoic acid, stimulated lateral branching of the primary germ tube of Gigaspora gigantea, when applied to Petri dishes in amounts as low as 1–10 ng.

Analytical and preparative HPLC of carbohydrates: inositols and oligosaccharides derived from cellulose and pectin

Abstract New methods are given for the production of cellodextrins by the TFA-catalyzed hydrolysis of cellulose and for the subsequent analytical and preparative high performance liquid chromatography of these useful oligosaccharides. In addition, recent methods developed in this laboratory for the analytical and preparative HPLC of inositols and pectin oligosaccharides are reviewed.

Frequent cultivation prior to planting to prevent weed competition results in an opportunity for the use of arbuscular mycorrhizal fungus inoculum

Inoculation with arbuscular mycorrhizal (AM) fungi is a potentially useful tool in agricultural systems with limited options regarding use of synthetic chemicals for fertility and pest control. We tested the response of Allium porrum cv. Lancelot to inoculation with AM fungi in a field high in available P (169 μg g −1 soil) that had been repeatedly cultivated to control weeds. Seedlings were inoculated during the greenhouse production period with a mixed species inoculum produced on-farm in a compost and vermiculite medium with Paspalum notatum Flugge as a nurse host. Inoculated and uninoculated seedlings were the same size at outplanting. Inoculated seedlings were over 2.5-fold greater in shoot weight and shoot P content than uninoculated seedlings at harvest. These results demonstrate the potential yield benefits from inoculation with AM fungi in situations where farm management practices may negatively impact on indigenous populations of AM fungi.