Detection of Vesicular-Arbuscular Mycorrhizal Fungus Colonization of Roots by Using a Dot-Immunoblot Assay

An immunoblotting procedure was developed to overcome the difficulty in identifying root colonization by a vesicular-arbuscular mycorrhizal fungus. The procedure utilized a murine monoclonal antibody that reacts with a protein in spores and hyphae of Glomus occultum, a fungus characterized by abundant production of hyaline spores and nonstaining intraradical infection. Minimally disturbed whole roots were squashed on nitrocellulose membranes. After inactivation of endogenous peroxidase, an indirect enzyme-linked immunosorbent assay was performed on the nitrocellulose with peroxidase-conjugated anti-mouse antibody as the second antibody. Antigen from G. occultum, revealed by a precipitating stain, was seen as purple dots on the nitrocellulose, which also retained the impression of the root.     

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.     

Occurrence of nitrogen-fixing Azospirillum in vesicular-arbuscular mycorrhizal fungi

Nitrogenase activity measured by acetylene reduction, was detected when surface-sterilized spores of vesicular-arbuscular (VA) mycorrhizal fungi (Glomus fasciculatum, G. intraradices, G. scientillans, G. mosseae, Gigaspora gilmorei andEndogone dusii) were inoculated into nitrogen-free liquid medium containing malic acid and incubated under microaerophilic conditions (99% N₂+1% O₂) at 30°C.Azospirillum species were isolated from the nitrogenase-active cultures.     

Effect of liming on spore germination,germ tube growth and root colonization by vesicular-arbuscular mycorrhizal fungi

Summary The effect of soil acidity on spore germination, germ tube growth and root colonization of vesicular-arbuscular mycorrhizal (VAM) fungi was examined using a Florida Ultisol. Soil samples were treated with 0, 4, 8 and 12 meq Ca/MgCO₃/100 g soil and each lime level received 0, 240, and 720 ppm P as superphosphate. Corn (Zea mays L.) was planted in the soil treatments, inoculated with eitherGlomus mosseae orGigaspora margarita spores and grown for 31 days. Acid soil inhibits mycorrhizal formation byG. mosseae through its strong fungistatic effect against the spores. The dolomitic lime increased mycorrhizal formation by both fungal species.G. margarita is much less sensitive to acidic conditions thanG. mosseae. Al ions are a very important component of the fungistatic property against the VAM symbiosis. VAM fungus adaptation may be important for plants growing on infertile acid soils if soil inoculation with these fungi is to contribute significantly to low-input technology for tropical agricultural systems.     

Production of Polyclonal and Monoclonal Antibodies Against Hyphae from Arbuscular Mycorrhizal Fungi

Abstract

Polyclonal and monoclonal antibodies were produced against hyphae of the arbuscular mycorrhizal fungus Glomus monosporum. The polyclonal antibodies (pAbs) were raised in a rabbit by immunizing with hyphae. They were tested for their specificity by a dot-immunoblot assay (DIBA). After the third immunization, a distinct difference in the signal strength was observed between the antisera and the preimmune serum. The pAbs showed cross-reactions to a number of fungal species, both mycorrhizal and other. For the production of monoclonal antibodies (mAbs), mice were immunized intraperitoneally with hyphae. The resulting hybridoma cell culture supernatants were tested by an indirect immunolabeling procedure. For this purpose the hyphae were immobilized on silane-coated microscopic slides. The mAb 8A7 reacted with hyphae from all Glomus isolates tested so far. Cross-reactivities were not observed with hyphae from fungi of the family Acaulosporaceae, phytopathogenic fungi tested so far, or from spores from Glomus species.     

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.     

Production of monoclonal antibodies against surface antigens of spores from arbuscular mycorrhizal fungi by an improved immunization and screening procedure

Monoclonal antibodies (mAbs) were produced against surface antigens of chlamydospores of arbuscular mycorrhizal (AM) fungi by immunizing mice with crushed or complete spores. The intrasplenic approach proved to be superior to the intraperitoneal method of immunization with regard to the amount of antigen required for the immune response. The hybridoma technology was combined with an improved screening procedure, applying an immunogold-silver staining technique to semi-thin sections of spores. In this way, mAbs to surface antigens on the outer wall could be selected. Two mAbs were raised against Glomus etunicatum and G. scintillans spores. Cross-reactivities of the antibodies to other structures of the fungus, to other species of Glomus and to other soil-borne fungi were tested with indirect immunofluorescent labelling. The mAbs did not react with non-AM fungi. One mAb (A5B1) selectively recognized G. etunicatum, another (D12F11) exhibited limited interspecies cross-reactivities. One further mAb (H8F7), which reacted with spores of all AM fungi but not with other fungi, was shown to be specific for Bacillus mycoides. The implications are discussed.     

Vesicular-arbuscular mycorrhizal spore populations in sugar maple (Acer saccharum marsh. L.) forests

 The numbers and types of spores of vesicular-arbuscular mycorrhizal fungi occurring in the top 15 cm of the soil in three maple forests in Eastern Canada were investigated using traditional wet-sieving/decanting methods. In the most acid site, at St. Hippolyte, Québec, where the soil had been amended with base cations, after 1 year there was no effect on the numbers of spores present. Vesicular-arbuscular mycorrhizal spores present at St. Hippolyte consisted of Glomus rubiforme, other Glomus spp. and Acaulospora spp. Although the sporocarpic species, G. aggregatum, G. macrocarpum and G. rubiforme occurred at St. Hippolyte, they were not found at the two less acid sites (Waterloo, Ontario and Lacolle, Québec) . Spores of Acaulospora spp. were found at all three sites, but were most abundant at St. Hippolyte. At St. Hippolyte the total number of spores was much higher than at the other two sites; at Waterloo numbers were an order of magnitude lower than at St. Hippolyte. It is suggested that G. rubiforme and Acaulospora species may be adapted to acid conditions. Seasonal patterns of spore abundance suggested that Acaulospora spp. may sporulate during the spring, whereas G. rubiforme may sporulate during the fall. Accepted: 6 September 1995     

Interaction of vascular plants and vesicular-arbuscular mycorrhizal fungi across a soil moisture-nutrient gradient

Summary Abundance and distribution of vascular plants and vesicular-arbuscular mycorrhizal (VAM) fungi across a soil moisture-nutrient gradient were studied at a single site. Vegetation on the site varied from a dry mesic paririe dominated by little bluestem (Schizachyrium scoparium) to emergent aquatic vegetation dominated by cattail (Typha latifolia) and water smartweed (Polygonum hydropiperoides). Plant cover, VAM spore abundance, plant species richness, and number of VAM fungi represented as spores, had significant positive correlations with each other and with percent organic matter. The plant and VAM spore variables had significant negative correlations with soil pH and available Ca, Mg, P and gravimetric soil moisture. Using stepwise multiple regression, Ca was found to be the best predictor of spore abundance. Test for association between plant species and VAM fungal spores indicated that the spores of Glomus caledonium are associated with plants from dry, nutrient poor sites and spores of gigaspora gigantea are positively associated with plants occurring on the wet, relatively nutrient rich sites. Glomus fasciculatum was the most abundant and widely distributed VAM fungus and it had more positive associations with endophyte hosts than the other VAM fungi. We found no relationship between beta niche breadth of plant species and the presence or absence of mycorrhizal infection. However, our data suggest that some plant species may vary with respect to their infection status depending upon soil moisture conditions that may fluctuate seasonally or annually to favor or hinder VAM associations.     

Production of Vesicular-Arbuscular Mycorrhizal Fungus Inoculum in Aeroponic Culture

Bahia grass (Paspalum notatum) and industrial sweet potato (Ipomoea batatas) colonized by Glomus deserticola, G. etunicatum, and G. intraradices were grown in aeroponic cultures. After 12 to 14 weeks, all roots were colonized by the inoculated vesicular-arbuscular mycorrhizal fungi. Abundant vesicles and arbuscules formed in the roots, and profuse sporulation was detected intra-and extraradically. Within each fungal species, industrial sweet potato contained significantly more roots and spores per plant than bahia grass did, although the percent root colonization was similar for both hosts. Mean percent root colonization and sporulation per centimeter of colonized root generally increased with time, although with some treatments colonization declined by week 14. Spore production ranged from 4 spores per cm of colonized root for G. etunicatum to 51 spores per cm for G. intraradices. Infectivity trials with root inocula resulted in a mean of 38, 45, and 28% of bahia grass roots colonized by G. deserticola, G. etunicatum, and G. intraradices, respectively. The germination rate of G. etunicatum spores produced in soil was significantly higher than that produced in aeroponic cultures (64% versus 46%) after a 2-week incubation at 28°C. However, infectivity studies comparing G. etunicatum spores from soil and aeroponic culture indicated no biological differences between the spore sources. Aeroponically produced G. deserticola and G. etunicatum inocula retained their infectivity after cold storage (4°C) in either sterile water or moist vermiculite for at least 4 and 9 months, respectively.     

大豆毛状根-VA菌根真菌双重培养体系的建立

以大豆毛状根为宿主,接种VA菌根真菌珠状巨孢囊霉（Gigaspora margarita）,经过3.5个月的双重培养,观察到VA菌根真菌珠状巨孢囊霉对大豆毛状根的侵染,辅助细胞形成,并获得VA菌根真菌成熟孢子,在无菌条件下建立了大豆毛状根-VA菌根真菌双重培养体系,为研究菌根真菌侵染大豆根部形成共生体系及相关分子机制提供了一种有效的研究方法。     

Dual axenic culture of sheared-root inocula of vesicular-arbuscular mycorrhizal fungi associated with tomato roots

Surface-sterilized sheared-root inocula of two vesicular-arbuscular mycorrhizal (VAM) fungi (Glomus intraradices and G. versiforme) from pot cultures associated with excised tomato roots showed significant sporulation and the production of an extensive hyphal biomass. As many as 10²–10³ axenic mature spores were recovered in Petri dishes during 3 months incubation in the dark. Propagules of both species were able to complete their vegetative life cycle in vitro and efficiently colonize Acacia albida roots after 1 month under greenhouse conditions. The effectiveness of 0.5 cm pieces of VAM roots as starter inocula indicates the high inoculum potential of intravesicle propagules.     

ROLE OF MYCORRHIZAL FUNGUS ORIGIN IN GROWTH AND NUTRIENT UPTAKE BY GERANIUM ROBERTIANUM

Prior field studies have shown that populations of forest herbs on relatively nutrient poor soils have higher vesicular-arbuscular mycorrhizal (VAM) infection intensity than plants on rich soils. However, the growth responses and ability to take up P against the soil nutrient gradient are often not linearly related to infection intensity. To determine if intraspecific differences among populations of the common VAM fungus Glomus occultum could differentially affect growth and nutrient uptake, Geranium robertianum seedlings were inoculated with Glomus occultum isolated from four forest types along a gradient of soil fertility, and grown in a greenhouse at P levels typical of the extremes of that gradient. Plants given inoculum from relatively infertile forest sites generally produced greater root, shoot, and total mass than plants given inoculum from fertile sites or uninoculated plants, especially at the low P supply rate. Total P uptake and both P and N uptake efficiency were also highest in plants given inocula from low fertility sites. These results indicate that local adaptation and intraspecific variations in the ability of VAM fungi to induce growth and nutrient uptake effects on host plants may be as important as interspecific differences among VAM fungus species.     

Controls for rhizosphere microorganisms to study effects of vesicular-arbuscular mycorrhizae on Artemisia tridentata

Seven treatments were set up to test the effects of vesicular-arbuscular (VA) mycorrhizal fungi and other rhizosphere microorganisms on the growth of Artemisia tridentata ssp. tridentata. Soil sievings had no significant effect on root or shoot mass. Spores and surface-sterile spores were a poor inoculum source, but roots and fresh soil caused 45–75% mycorrhizal infection. Whereas root-inoculated plants still had low growth responses by the end of the experiment, fresh soil inoculum caused the greatest response, and partial fresh inoculum caused a lesser response. These results suggest that fresh soil is an appropriate inoculum for this plant-fungal-soil system, and that the major effect on plant growth of the fresh soil inoculum is from the mycorrhizal fungi and not from the other microorganisms, because the sievings had no effect on plant growth. In addition, soil dilution plating of saprophytic fungi showed 85% species similarity between sterile and fresh soil inoculum by the end of the experiment. Since the effects of non-VA microorganisms are complex and varied, we suggest that researchers work out the type of mycorrhizal controls that best suit their system.     

Glomalean mycorrhizal fungi from tropical Australia

 A comparison of different methods for isolation of vesicular-arbuscular mycorrhizal (VAM) fungi into open-pot cultures was undertaken as part of a study of the diversity of these fungi. Four different isolation techniques using spores separated from soil, soil trap cultures, root samples, or transplanted seedlings grown in intact soil cores were used to obtain as many fungi as possible from each site. Isolation methods were compared using paired samples from the same locations within natural (savanna, rocky hill, wetland, rainforest) and disturbed (minesite) habitats in a seasonally dry tropical region in the Northern Territory of Australia. There were large differences in (i) the efficiency (rate of increase in mycorrhizal colonisation), (ii) the proportion of successful cultures, (iii) fungal diversity (number of fungal species in each culture) and (iv) specificity (identity of species isolated) between these four procedures. However, the less-efficient procedures generally resulted in a higher proportion of cultures of one fungus, which could be used without further isolation steps. Most species of Scutellospora, Acaulospora and Gigaspora were obtained primarily from field-collected spores, but only 50% of these culture attempts were successful. Spores from these initial cultures produced mycorrhizas much more rapidly and successfully when used to start second-generation cultures. Several species of fungi, rarely recovered as living spores from field soils, were dominant in many trap cultures started from soil or roots. Most of these fungi were Glomus species, that were first distinguished by colonisation patterns in roots and eventually identified after sporulation in second- or third-generation trap cultures. These experiments demonstrated that glomalean fungi in the habitats sampled belonged to two functional categories, based on whether or not spores were important propagules. The "non-sporulating" fungi were dominant in many trap cultures, which suggests that these fungi had higher total inoculum levels in soils than other fungi. Pot-culturing methods provided additional information on fungal diversity which complemented spore occurrence data obtained using the same soil samples and provided valuable new information about the biology of these fungi. Accepted: 26 December 1998     

Extensive In Vitro Hyphal Growth of Vesicular-Arbuscular Mycorrhizal Fungi in the Presence of CO(2) 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% CO(2), 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 muM) 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.     

Identification of arbuscular mycorrhizal fungi in soils and roots of plants colonizing zinc wastes in southern Poland

 Analysis of the community of arbuscular mycorrhizal (AM) fungi in roots of Fragaria vesca growing in a heavy metal contaminated site was carried out on a Zn waste site near Chrzanow (southern Poland). The waste substratum was characterized by high contents of Pb, Zn, Cd, Cu and As, and by low levels of N, P and organic matter. Spores of Glomales were isolated by wet sieving and DNA was isolated from individual spores. Nested polymerase chain reaction (PCR) with taxon-specific primers was used to identify the species Glomus mosseae, Glomus intraradices and Glomus claroideum. Spores of other fungi were morphologically characterized and new taxon-discriminating molecular probes were developed for two of them (Glomus sp. HM-CL4 and HM-CL5) based on variations in the large ribosomal subunit (25S rDNA). High sequence similarities were found between Glomus sp. HM-CL4 and Glomus gerdemanii, and between Glomus sp. HM-CL5 and Glomus occultum. The designed primers were used to characterize the population of AM fungi colonizing the roots of F. vesca collected from the Zn waste site. The analysis, carried out on roots stained with trypan blue, showed that the most effective colonizer was closely related to G. gerdemannii. G. claroideum and the G. occultum-like fungus were slightly less common whilst frequencies of G. intraradices and G. mosseae in roots were much lower. The analysis of mycorrhiza stained with rhodizoniate to localize heavy metal accumulation showed that the stain does not influence the PCR reaction. Seventy percent of the root samples containing positively stained fungal hyphae were found to be colonized by G. mosseae. The data obtained demonstrate the usefulness of nested PCR for studies carried out in polluted areas. It will enable selection of AM fungi which are able to colonize plant roots under heavy metal stress conditions, as well as the identification of fungi showing high in situ accumulation of potentially toxic elements. Accepted: 7 July 2000     

Mycorrhizal status of epiphytes in Malaysian oil palm plantations

Epiphytic plants were collected from four oil palm plantations in Peninsular Malaysia and their mycorrhizal status determined. Conspecific plants with a terrestrial habit (16 species) and rhizosphere soils were also examined for mycorrhizal colonization and glomalean fungi, respectively. Twelve species of glomalean fungi were recovered from the four oil palm plantation soils. Of the 29 epiphytic species in 16 families belonging to the bryophytes, pteridophytes and angiosperms, only four species of angiosperms that were facultative epiphytes and a hemiepiphyte growing within 0.4 m of ground level had vesicular-arbuscular mycorrhizal (VAM) fungi. Bioassays of organic debris from oil palm trunks did not produce vesicular-arbuscular mycorrhizas on maize. Six epiphytic species grown in the greenhouse in pots containing oil palm rhizosphere soils rooted and had VAM fungi and thus may be facultative epiphytes. Five other epiphyte species failed to grow in pots and are probably obligate epiphytes. Seven epiphyte species that established themselves in pots failed to form vesicular-arbuscular mycorrhizas.     

Absence of VA colonization in Oxalis pes-caprae inoculated with Glomus mosseae

Although members of the Oxalidaceae family have been described as host plants of vesicular-arbuscular mycorrhizal fungi, Oxalis pes-caprae did not become colonized by Glomus mosseae. Extracts of Ox. pes-caprae root inhibited the germination of G. mosseae spores. However, the presence of G. mosseae in the rhizosphere of Ox. pes-caprae produced browning of the roots, which was interpreted as a hypersensitivity response of the plant to the presence of VA fungus.     

Nontarget effects of the herbicide tebuthiuron on mycorrhizal fungi in sagebrush semidesert

The effects of the herbicide tebuthiuron (0.36, 0.6, and 1.01 kg/ha in pellet form) on nontarget organisms, vesicular-arbuscular mycorrhizal fungi, were observed in sagebrush semidesert in central Utah. Only the highest level of tebuthiuron application showed any significant effects on mycorrhizal fungi compared to the untreated control. The introduced annual Bromus tectorum L. had both a reduced percent mycorrhizal root infection and reduced spore density in its rhizosphere with the highest herbicide level. The herbicide did not significantly affect mycorrhizal root infection of Sitanion hystrix, a short-lived perennial grass, at any level of application. There was no significant effect of any level of tebuthiuron on germination of mycorrhizal spores collected 6 months after herbicide application.Published with the approval of the Director, Utah Agricultural Experiment Station, as Journal Paper No. 3777