A procedure for the establishment of Glomus mosseae in dual culture with Ri T-DNA-transformed carrot roots

 A stepwise procedure was investigated to determine the optimal conditions for the establishment of Glomus mosseae (Nicol. & Gerd.) Gerdemann & Trappe in dual in vitro culture with Ri T-DNA-transformed roots of Daucus carota L. Glomus mosseae spores germinated best in 10 mm Tris or MES-buffered medium at pH values just above neutral. Growth of hyphae from germinated spores was much greater in the presence of Tris than MES, eg. 8 mm versus 4 mm per spore for Tris and MES, respectively, at pH 7.2. Roots exhibited a broad pH optimum for growth of 6.0–7.0 in both MES and Tris, but did not grow well above pH 7.5. In addition, purified gelling agent, gellan gum, was utilized to lower the P concentration of media. With these factors combined, mycorrhizas were successfully established in 14% of dual cultures. Accepted: 5 March 1997     

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.     

Increased Sporulation of Vesicular-Arbuscular Mycorrhizal Fungi by Manipulation of Nutrient Regimens

Adjustment of pot culture nutrient solutions increased root colonization and sporulation of vesicular-arbuscular mycorrhizal (VAM) fungi. Paspalum notatum Flugge and VAM fungi were grown in a sandy soil low in N and available P. Hoagland nutrient solution without P enhanced sporulation in soil and root colonization of Acaulospora longula, Scutellospora heterogama, Gigaspora margarita, and a wide range of other VAM fungi over levels produced by a tap water control or nutrient solutions containing P. However, Glomus intraradices produced significantly more spores in plant roots in the tap water control treatment. The effect of the nutrient solutions was not due solely to N nutrition, because the addition of NH₄NO₃ decreased both colonization and sporulation by G. margarita relative to levels produced by Hoagland solution without P.     

The uptake, metabolism, transport and transfer of nitrogen in an arbuscular mycorrhizal symbiosis

Nitrogen (N) is known to be transferred from fungus to plant in the arbuscular mycorrhizal (AM) symbiosis, yet its metabolism, storage and transport are poorly understood. In vitro mycorrhizas of Glomus intra-radices and Ri T-DNA-transformed carrot roots were grown in two-compartment Petri dishes. (15)N- and/or (13)C-labeled substrates were supplied to either the fungal compartment or to separate dishes containing uncolonized roots. The levels and labeling of free amino acids (AAs) in the extra-radical mycelium (ERM) in mycorrhizal roots and in uncolonized roots were measured by gas chromatography/mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). Arginine (Arg) was the predominant free AA in the ERM, and almost all Arg molecules became labeled within 3 wk of supplying (15)NH(4) (+) to the fungal compartment. Labeling in Arg represented > 90% of the total (15)N in the free AAs of the ERM. [Guanido-2-(15)N]Arg taken up by the ERM and transported to the intra-radical mycelium (IRM) gave rise to (15)N-labeled AAs. [U-(13)C]Arg added to the fungal compartment did not produce any (13)C labeling of other AAs in the mycorrhizal root. Arg is the major form of N synthesized and stored in the ERM and transported to the IRM. However, NH(4) (+) is the most likely form of N transferred to host cells following its generation from Arg breakdown.     

Monitoring the decline in AM fungus populations and efficacy during a long term bare fallow

Producing nonmycorrhizal plants in the field is a challenge due to the ubiquitous distribution of arbuscular mycorrhizal [AM] fungi and impacts of chemical treatments upon nontarget organisms. A field plot was covered with ground cover fabric to prohibit plant growth and take advantage of the obligate symbiotic nature of AM fungi to selectively starve and remove them from the soil microbiological community. The decline in the AM fungus population was monitored through spore counts and most probable number bioassays. Response to inoculation experiments were conducted to contrast the response of Allium porrum L. to inoculation with in vitro produced spores of Glomus intraradices Schenck and Smith when plants were grown in the AM fungus-depleted soil vs. soil from an adjacent, cropped plot. Data indicated a strongly diminished, yet still viable population of AM fungi after 39 months of bare fallow. Plants grown in cropped soil showed no growth response nor increase in percentage root length colonized as a result of inoculation, while the response to inoculation of plants grown in the covered soil increased as the population of AM fungi declined below 1 propagule cm^?3.     

Extensive In Vitro Hyphal Growth of Vesicular-Arbuscular Mycorrhizal Fungi in the Presence of CO2 and Flavonols

Various flavonoids were tested for their ability to stimulate in vitro growth of germinated spores of vesicular-arbuscular mycorrhizal fungi. Experiments were performed in the presence of 2% CO₂, previously demonstrated to be required for growth of Gigaspora margarita (G. Bécard and Y. Piché, Appl. Environ. Microbiol. 55:2320-2325, 1989). Only the flavonols stimulated fungal growth. The flavones, flavanones, and isoflavones tested were generally inhibitory. Quercetin (10 μM) prolonged hyphal growth from germinated spores of G. margarita from 10 to 42 days. An average of more than 500 mm of hyphal growth and 13 auxiliary cells per spore were obtained. Quercetin also stimulated the growth of Glomus etunicatum. The glycosides of quercetin, rutin, and quercitrin were not stimulatory. The axenic growth of G. margarita achieved here under rigorously defined conditions is the most ever reported for a vesicular-arbuscular mycorrhizal fungus.     

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.