Influence of mycorrhizal fungi on fate of E. coli O157:H7 and Salmonella in soil and internalization into Romaine lettuce plants.

The objectives of this study were to determine the influence of a symbiotic arbuscular mycorrhizal (AM) fungus on persistence of Salmonella and enterohemorrhagic Escherichia coli O157:H7 (EHEC) within soil, and survival within Romaine lettuce. Romaine seedlings were grown with or without AM fungi. Soil surrounding plants was inoculated with ca. 8 log CFU/plant of either Salmonella enterica or E. coli EHEC composites. Samples (soil, root, and shoot) were analyzed on days 1, 8, 15 and 22 for Salmonella and EHEC by direct plating and selective enrichment. Twenty-four hours after inoculation, populations of Salmonella and EHEC, respectively, were 4.20 and 3.24 log CFU/root, 2.52 and 1.17 log CFU/shoot, and 5.46 and 5.17 log CFU/g soil. By selective enrichment, samples tested positive for Salmonella or EHEC at day 22 at rates of 94 and 68% (shoot), 97 and 56% (root), and 100 and 75% (soil), respectively, suggesting that Salmonella has a greater propensity for survival than EHEC. Salmonella populations in soil remained as high as 4.35 log CFU/g by day 22, while EHEC populations dropped to 1.12 log CFU/g in the same amount of time. Ninety-two percent of all Romaine leaves in our study were positive for internalized Salmonella from days 8 to 22 and remained as high as 1.26 log CFU/shoot on day 22 in AM fungi+Romaine plants. There were no differences (P>0.05) between the survival of either pathogen based on the presence or absence of mycorrhizal fungi. Results of this study suggest that AM fungi do not affect the internalization and/or survival of either S. enterica or E. coli O157:H7 in Romaine lettuce seedlings. Our results should provide Romaine lettuce farmers confidence that the presence and/or application of AM fungi to crop soil is not a contributing factor to the internalization and survival of Salmonella or E. coli O157:H7 within Romaine lettuce plants.

Inactivation of E. coli O157:H7 in cultivable soil by fast and slow pyrolysis-generated biochar.

An exploratory study was performed to determine the influence of fast pyrolysis (FP) and slow pyrolysis (SP) biochars on enterohemorrhagic Escherichia coli O157:H7 (EHEC) in soil. Soil + EHEC (inoculated at 7 log colony-forming units [CFU]/g of soil) + 1 of 12 types of biochar (10% total weight:weight in soil) was stored at 22°C and sampled for 8 weeks. FP switchgrass and FP horse litter biochars inactivated 2.8 and 2.1 log CFU/g more EHEC than no-biochar soils by day 14. EHEC was undetectable by surface plating at weeks 4 and 5 in standard FP switchgrass, FP oak, and FP switchgrass pellet biochars. Conversely, EHEC populations in no-biochar control samples remained as high as 5.8 and 4.0 log CFU/g at weeks 4 and 5, respectively. Additionally, three more SP hardwood pellet biochars (generated at 500°C for 1 h, or 2 h, or generated at 700°C for 30 min) inactivated greater numbers of EHEC than did the no-biochar control samples during weeks 4 and 5. These results suggest that biochar can inactivate E. coli O157:H7 in cultivable soil, which might mitigate risks associated with EHEC contamination on fresh produce.

Salmonella and Escherichia coli O157:H7 survival in soil and translocation into leeks (Allium porrum) as influenced by an arbuscular mycorrhizal fungus (Glomus intraradices).

A study was conducted to determine the influence of arbuscular mycorrhizal (AM) fungi on Salmonella and enterohemorrhagic Escherichia coli O157:H7 (EHEC) in autoclaved soil and translocation into leek plants. Six-week-old leek plants (with [Myc+] or without [Myc-] AM fungi) were inoculated with composite suspensions of Salmonella or EHEC at ca. 8.2 log CFU/plant into soil. Soil, root, and shoot samples were analyzed for pathogens on days 1, 8, 15, and 22 postinoculation. Initial populations (day 1) were ca. 3.1 and 2.1 log CFU/root, ca. 2.0 and 1.5 log CFU/shoot, and ca. 5.5 and 5.1 CFU/g of soil for Salmonella and EHEC, respectively. Enrichments indicated that at days 8 and 22, only 31% of root samples were positive for EHEC, versus 73% positive for Salmonella. The mean Salmonella level in soil was 3.4 log CFU/g at day 22, while EHEC populations dropped to ≤ 0.75 log CFU/g by day 15. Overall, Salmonella survived in a greater number of shoot, root, and soil samples, compared with the survival of EHEC. EHEC was not present in Myc- shoots after day 8 (0/16 samples positive); however, EHEC persisted in higher numbers (P = 0.05) in Myc+ shoots (4/16 positive) at days 15 and 22. Salmonella, likewise, survived in statistically higher numbers of Myc+ shoot samples (8/8) at day 8, compared with survival in Myc- shoots (i.e., only 4/8). These results suggest that AM fungi may potentially enhance the survival of E. coli O157:H7 and Salmonella in the stems of growing leek plants.

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.

Mid-Infrared and near-infrared spectral properties of mycorrhizal and non-mycorrhizal root cultures.

We investigated the Fourier-transformed mid-infrared (MIR) and near-infrared (NIR) spectroscopic properties of mycorrhizal (M) and non-mycorrhizal (NM) carrot roots with the goal of finding infrared markers for colonization by arbuscular mycorrhizal (AM) fungi. The roots were cultured with or without the AM fungus Glomus intraradices under laboratory conditions. A total of 50 M and NM samples were produced after pooling subsamples. The roots were dried, ground, and scanned separately for the NIR and MIR analyses. The root samples were analyzed for fatty acid composition in order to confirm mycorrhizal infection and to determine the presence of fatty acid markers. Besides the roots, fatty acid standards, pure cultures of saprophytic fungi, and chitin were also scanned in order to identify spectral bands likely to be found in M samples. Principal components analysis (PCA) was used to illustrate spectral differences between the M and NM root samples. The NIR analysis achieved good resolution with the raw spectral data and no pretreatment was needed to obtain good resolution in the PCA analysis of the NIR data. Standard normal variate and detrending pretreatment improved the resolution between M and NM in the MIR range. The PCA loadings and/or the spectral subtraction of selected samples showed that M roots are characterized by absorbances at or close to 400 cm(-1), 1100-1170 cm(-1), 1690 cm(-1), 2928 cm(-1), and 5032 cm(-1). The NM samples had characteristic absorbances at or near 1734 cm(-1), 3500 cm(-1), 4000 cm(-1), 4389 cm(-1), and 4730 cm(-1). Some of the bands that differentiate M from NM roots are prominent in the spectra of pure fungal cultures, chitin, and fatty acids. Our results show that mycorrhizal and nonmycorrhizal root tissues can be differentiated via MIR and NIR spectra with the advantage that the same samples can then be used for other analyses.

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.

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.

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.

The fungus does not transfer carbon to or between roots in an arbuscular mycorrhizal symbiosis.

• Carbon transfer from fungus to plant in the arbuscular mycorrhizal (AM) symbiosis has been reported, but its significance and even its existence have been called into question and the issue remains controversial. We investigated carbon movement from fungus to plant and from one mycorrhizal root system to another via a common AM fungal network in monoxenic cultures to avoid limitations of some previous studies. • 13 C and 14 C labeled substrates were supplied to functioning in vitro AM mycorrhizas between Ri T-DNA transformed carrot (Daucus carota) roots and Glomus intraradices to follow carbon movement into and between host and fungal metabolite pools. • Fungal triacylglycerol and trehalose were labeled when permeant substrates were supplied to the extraradical mycelium (ERM), but host-specific compounds in the roots did not become labeled. When labeled glucose was provided to a donor root system, label moved to recipient roots via a common AM fungal network but remained in fungal compounds. • We conclude that carbon flow in the AM symbiosis is normally unidirectional from plant to fungus and that while carbon is translocated by the fungus from one metabolically active root system to another, it remains within the intraradical mycelium (IRM).

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.

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 (13)C(1) 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 (13)C-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.

Increased spore production by Glomus intraradices in the split-plate monoxenic culture system by repeated harvest, gel replacement, and resupply of glucose to the mycorrhiza.

Monoxenic culture of Glomus intraradices Schenck and Smith with Ri T-DNA transformed roots in two-compartment Petri dishes is a very useful technique for physiological studies and the production of clean fungal tissues. Experiments were conducted to increase the efficiency of this method for the production of arbuscular mycorrhizal fungus spores. Approximately 20,000 spores could be harvested every 2 months from the distal (fungus only) compartment of a 9-cm-diameter divided Petri dish. The method requires replacement of the gelled media in the distal compartment and resupply of 200 mg glucose to the proximal (root) compartment coincident with harvest of spores. These modifications resulted in an approximate threefold increase in spore production per unit time over the standard split-plate culture technique.