The effects of hydroxy fatty acids on the hyphal branching of germinated spores of AM fungi.

Two hydroxy fatty acids, tentatively identified previously in carrot root exudates, were tested for their effects on hyphal growth of the arbuscular mycorrhizal (AM) fungus, Gigaspora gigantea (Nicol. and Gerd.) Gerdemann and Trappe. Best results were achieved with a long-term bioassay (7-8d) with nanomolar concentrations throughout the Petri dish in contrast to the rapid microinjection bioassay (16-24h) in which nanogram quantities were injected near growing hyphal tips. When 5nM 2-hydroxy fatty acids of various chain length were tested, the length of the hydroxyl fatty acid was significant since only 2-hydroxytetradecanoic acid (2OH-TDA) and to a slightly lesser degree, 2-hydroxydodecanoic acid (2OH-DDA) induced a hyphal growth response while 2-hydroxydecanoic acid (2OH-DA) and 2-hydroxyhexadecanoic (2OH-HDA) acid did not. The position of the hydroxyl group was critical since 5nM 3-hydroxytetradecanoic acid (3OH-TDA) had no effect on hyphal growth. The length of the non-hydroxy containing straight chain fatty acid, per se, did not appear significant since none of these fatty acids had an effect on hyphal growth. The morphological growth response promoted by 2OH-TDA consisted of multiple lateral branches, spaced fairly regularly apart, along the primary germ tubes as well as some lateral branch formation off the major secondary hyphae. This growth response was identical to that observed when germinated spores were allowed to grow towards cultured carrot roots in vitro. This response to 2OH-TDA also was observed with an unidentified Gigaspora species but no morphological response was observed with Glomus intraradices Schenck and Smith. The results indicate that 2-hydroxy fatty acids are another putative category of root exudate signals perceived by Gigaspora species, stimulating an increase in elongated lateral branches.

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.

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.

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.

Separated components of root exudate and cytosol stimulate different morphologically identifiable types of branching responses by arbuscular mycorrhizal fungi.

Two morphologically distinct hyphal branching responses by the AM fungus, Glomus intraradices, were stimulated by separated components of carrot root exudate. Complex branching up to the sixth order was induced by compounds most soluble in 35% methanol, whereas the formation of more lateral branches (second order) was stimulated by compounds most soluble in 70% methanol. This same 70% alcohol soluble fraction also stimulated a completely different type of branching pattern in another fungus, Gigaspora gigantea. This pattern consisted of a very periodic distribution of dense clusters of hyphal branches that had a very high degree of complexity. In contrast to exudate components, separated cytosolic components of carrot roots did not stimulate any of the observed hyphal branching patterns. Alcohol-soluble fractions actually inhibited hyphal tip growth of G. gigantea and induced the formation of "recovery" branches that were identical to those induced by an inhibitor found in the exudate of Chard (Beta vulgaris ssp. cicla), a non-host plant.

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.

Synergism between blue light and root exudate compounds and evidence for a second messenger in the hyphal branching response of Gigaspora gigantea.

Light and chemical components of the host root exudate can induce hyphal growth and branching of arbuscular mycorrhizal fungi. Compounds that induce the same morphogenetic or biochemical response as light are referred to as photo-mimetic compounds (PCs). This is the first report of a synergistic response by Gigaspora gigantea, an arbuscular mycorrhizal fungus, to blue light and naturally occurring photomimetic compounds isolated from the exudate of host roots. The blue light treatment and exposure to photomimetic compounds were effective whether applied sequentially or simultaneously. The number of hyphal branches induced by blue light and photomimetic compounds together was greater than the sum of the branches generated by each separate treatment, and the synergism was greatest at the higher levels or orders of branches. The fact that blue light and PCs, individually, triggered the same hyphal branching response and when given together, they produced a synergistic response, indicated the activation of a second messenger in the induced-branching process. Delaying the application of PCs, after the initial light exposure, showed the second messenger was stable up to 3 h.

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.

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.

A comparative study of phenolic acids associated with cell walls and cytoplasmic extracts of host and non-host roots for AM fungi.

Carrots (Daucus carota L.) are a ubiquitous host for arbuscular mycorrhizal fungi whereas sugar beets (Beta vulgaris L.) are a non-host. Root cultures were used to compare the constitutive phenolic compounds associated with the cell wall or present in the cytoplasm of the host and non-host. Phenolic acids were released from purified cell walls by alkaline hydrolysis and were separated and identified by HPLC, TLC and u.v. absorption spectra analyses. Two phenolic acids unique to carrot root cell walls were identified as p-hydroxybenzoic acid (p-HBA) and vanillic acid. Sugar beet root cell walls had ferulic acid as major constituent and contained several unique phenyl propanoids which were not identified. Caffeic acid was found only in the cytoplasm of carrot roots and was present in the conjugated form (chlorogenic acid). The sugar beet cytoplasm also contained several unidentified hydroxycinnamic acid-type phenolics which were not found in carrot roots.