Plant and Soil 226: 171177, 2000. 2000 Kluwer Academic Publishers. Printed in the Netherlands.

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A uorescent antibody assay for hyphae and glomalin from arbuscular mycorrhizal fungi
S. F. Wright
Soil Microbial Systems Laboratory, United States Department of AgricultureAgricultural Research Service, Beltsville, MD 20705, USA
Received 31 March 1999. Accepted in revised form 22 November 1999

Key words: glycoprotein, immunouorescence, monoclonal antibody, soil biology

Abstract Studies on the role of arbuscular mycorrhizal (AM) fungi in soil have been aided by the use of a monoclonal antibody that detects a molecule common to all isolates of these fungi studied to date. The molecule, glomalin, is a glycoprotein that forms on hyphae, but apparently sloughs off and adheres to soil particles or imbedded plastic mesh. An indirect immunouorescence (IF) assay is described for detection of glomalin on hyphae attached to roots, in roots, on hyphae traps and on the surface of soil aggregates. Small sieves are used to process hyphae attached to roots and soil aggregates. Glomalin on hyphae and glomalin attached to plastic or nylon are assayed on a 1 cm2 section of meshes. Examples of IF assay results are shown and discussed.

Introduction Antibodies have been used to study arbuscular mycorrhizal (AM) fungi at the level of host-symbiont interactions (Bonfante-Fasolo et al., 1991; Perotto et al., 1994), as taxonomy aids (Aldwell et al., 1985; Hahn et al., 1993; Sanders et al., 1992; Wright et al., 1987) and in ecological investigations of hyphae in soil (Aldwell and Hall, 1986; Fries and Allen, 1991; Kough and Linderman, 1986; Wilson et al., 1983). Three of these studies employed monoclonal antibodies (Hahn et al., 1993; Perotto et al., 1994; Wright et al., 1987). Monoclonal antibodies are highly specic antibodies generated by hybridoma technology (Khler and Milstein, 1975). A monoclonal antibody (MAb) is a single antibody type that detects a few amino acids, nucleic acids or monosaccharides. As a taxonomy aid, a hybridoma may be chosen because the MAb is reactive with one genus and species (Hahn et al., 1993; Wright et al., 1987) or with a broad range of genera
FAX No: (301) 504-8370. TEL No: (301) 504-8156. E-mail: swright@asrr.arsusda.gov 1 The U.S. Governments right to retain a non-exclusive royalty free licence in and to any copyright is acknowledged.

within a taxonomic group. Because hybridomas can be preserved and regrown, they provide a consistent and plentiful supply of the antibody. During an attempt to produce a MAb that would react specically with Glomus intraradices Schenck and Smith as a taxonomy aid, MAb32B11 was produced. This MAb was generated using freshly collected spores as the immunogen. The reactive molecule was detected by indirect immunouorescence on the surface of fresh, young spores of G. intraradices, on hyphae of this species and on hyphae of isolates from all other culturable AM fungal genera (Wright et al., 1996). Low cross-reactivity with the pathogenic fungus Leptosphaeria korrea J C Walker and A M Smith was not considered to be a signicant impediment in the use of MAb32B11 to study the target molecule on AM fungi. MAb32B11 is an IgM antibody. This class of antibody has ve pairs of heavy and light chains giving a total of 10 potential reactive sites to attach to the target molecule. This is in contrast to the two pairs of heavy and light chains of an IgG antibody, the predominant antibody class in polyclonal antisera. Generally, only one or two of the reactive sites of an IgM antibody attach to an immobilized target molecule. However,

172 the 10 heavy chains provide numerous sites for attachment of the FITC-tagged secondary antibody used to reveal the MAb resulting in a strong uorescent signal (Goding, 1986). The rst use of the immunouorescence assay was to assess the build up and decline of a sheath-like coating on hyphae from early colonization to plant senescence (Wright et al., 1996). After initially being revealed by MAb32B11 in immunouorescence assay, the antibody-reactive molecule was extracted from hyphae and studied further (Wright et al., 1996). Harshness of conditions necessary to extract the molecule suggested that it was very insoluble and stable. This led to use of the extraction procedure on soil and assessment of the role of the molecule in soil stability (Wright and Upadhyaya, 1996, 1998). Also, because of the uniqueness and abundance of the molecule in soil and apparent production by members of the fungal order Glomales, the name glomalin was ascribed to it (Wright and Upadhyaya, 1996). The presence of glomalin on hyphae and glomalin sloughed from hyphae onto plastic was detected by immunouorescence which led to use of hyphae traps to estimate activity of hyphae and glomalin production over time periods of 1214 weeks (Wright and Upadhyaya, 1999). Extracted glomalin was also subjected to capillary electrophoresis analysis which demonstrated that the antibody-reactive compound is a glycoprotein (Wright et al., 1998). This paper describes several variations of the immunouorescence assay developed to assess glomalin production on hyphae in the rhizosphere, monitor hyphal growth and production of glomalin during plant growth, and to reveal glomalin on the surface of water stable soil aggregates. genera of AM fungi. The antibody was produced in bulk by overgrowth of the hybridoma in tissue culture medium. Antibodies in cell-free supernatant were stored at 4 C as a sterile solution. Batches of MAb 32B11 were produced as needed and tested for reactivity against known amounts of extracted glomalin (Wright et al., 1996). Staining sieves Small sieves to hold root and soil samples during incubations with antibodies and in washing steps were made from 10 mm ID polyvinyl chloride tubing or other plastic plumbing tubing. Tubing was cut into 7 mm sections. A ne nylon mesh (40 m openings) was glued to one end of each section of tubing with epoxy glue, and after the glue dried, the mesh was cut to t the outer diameter. Sieves t into wells of a 12-well tissue culture plate. Samples Root pieces with attached hyphae AM fungi were cultured in sand or a sand-coal mixture on sudangrass (Sorghum sudanese (Piper) Staph). Glomalin, naturally present in small amounts on sand, was extracted before the sand was used. This was accomplished by ooding large batches of sand with 50 mM citrate and autoclaving for 1 h. The sand was rinsed thoroughly to remove the citrate and then dried. Dried sand and coal were sterilized by autoclaving for 1 h. Inoculated plants were grown for 34 months. At harvest, several pieces of root (ca. 3 cm lengths) were placed in a sieve. Root pieces were examined under a microscope to aid in choosing sections to assay, but they were not allowed to dry before processing. Immunoreactive material remained on hyphae for a minimum of several weeks if root pieces were kept in water. Intraradical colonization Roots to be examined for internal colonization were dried before processing. To clear roots, boiling hot KOH (10% w/v) was poured over root pieces of ca. 1 cm length and incubated at room temperature (RT) for 15 min. The material was transferred to a small sieve, KOH was neutralized with 3N HCl for 5 min followed by 35 min washes in PBS.

Materials and methods Monoclonal antibody Monoclonal antibody (MAb) 32B11 was produced against fresh spores of Glomus intraradices FL208 (Dazzo and Wright, 1996). Briey, a BALB/c mouse was immunized with four intraperitoneal injections of 5000 spores crushed in 0.5 mL physiological solution at 3-week intervals. Hybridomas were produced and hybridoma 32B11 was selected based on positive enzyme-linked immunosorbent assay reactions with ve spores of the targeted isolate, other morphologically similar AM fungi and isolates of other

173 Hyphae traps Hyphae traps made of plastic horticultural mesh (Wright and Upadhyaya, 1999) were placed in pot cultures to monitor growth and production of extraradical hyphae in a root-free zone. Nylon mesh (40 m) is used in pot cultures to conne roots. Traps were placed against the inner wall of the pot at the time seeds were planted and inoculated. At biweekly or monthly intervals, or at the termination of the experiment, strips were carefully removed using a spatula to push sand away from the trap and prevent the sand from abrading material on the trap as it was pulled out of the pot. The top 2 cm of a strip was cut away and discarded because this area is often contaminated with algae. Several 1 cm2 sections were cut out of the remaining material and placed directly into a well of a 12-well plate. Soil aggregates Aggregates measuring 12 mm or 0.251 mm were sieved from air-dry soils and placed in the staining sieves in amounts that covered the bottom of the sieve. Water stability of the batch from which aggregates were sampled for the immunoassay was determined on other samples using the method of Kemper and Rosenau (1986). Stability 70% is required for aggregates to maintain integrity during processing. Indirect immunouorescence assay All incubation and washing steps were performed at RT on a rotating platform (5075 rpm). Care was taken to add sufcient volumes of solutions to cover samples. Samples were blocked to prevent nonspecic adsorption of antibodies using 2% (w/v) non-fat dry milk in phosphate buffered saline (PBS) (10 mM Na2 HPO4 , 138 mM NaCl, 2.7 mM KCl, pH 7.4) and incubated for 15 min. Sieves or hyphae traps were removed from wells with tweezers and placed on a paper towel to blot off excess milk, wells were emptied, and sieves and hyphae traps were replaced in wells. A 1:2 mixture of MAb32B11 and PBS was added to wells, and samples were incubated for 1 h for all except for cleared roots which were incubated for 2 h. Samples were washed for 35 min in PBS with 0.05% Tween 20. A commercially available goat antimouse IgM antibody (-chain reactive) tagged with uorescein isothiocyanate (FITC) was diluted as recommended by the supplier in PBS with 0.1% bovine serum albumin. The FITC-labeled antibody was added to wells and incubated for 1 h. Samples were washed three times as described above. PBS without Tween 20 was used for the fourth and nal wash. For controls, an anti-Rhizobium IgM MAb was used in place of MAb32B11. Autouorescence of roots can be quenched by erichrome black (Bolhool, 1987). The staining solution consists of 9 mg of dye dissolved in 1 mL of dimethyl sulfoxide (DMSO) and 5 mL of chelating solution (DMSO 50 mL; deionized water 20 mL; 0.1 M aluminum chloride 10 mL; 1.0 M acetic acid 10 mL). The dye and chelating solution mixture is adjusted to pH 5.2 and then diluted to 100 mL with deionized water. After antibody-tagging, samples were ooded with erichrome black until the root was colored pink to dark red. Excess stain was removed by washing in PBS. Aggregates were viewed in a hanging drop slide. A Pasteur pipette with the tip broken off at the neck to provide a bore larger than the aggregates was used to transfer aggregates. Aggregates in PBS were carefully drawn into the pipette and transferred to the well of a slide. Excess PBS was removed by blotting, mounting medium was added to cover the aggregates and a cover slip was placed over the well. Samples were mounted in a medium formulated to prevent rapid photobleaching that can be prepared (Davidson and Goodwin, 1983) or obtained commercially. A coverslip was used. Samples, except aggregates, mounted in commercially available mounting medium (Vectashield, Vector Laboratories, Burlingame, CA, USA) can be preserved by freezing at 20 C. Aggregates break apart after a few hours in the mounting medium and thus cannot be preserved this way. After adding the mounting medium, but before placing the cover slip over roots to be examined for intraradical colonization, the stele was removed and pieces of cortex were torn away so that a single layer of cells could be examined for immunouorescence. This was done with the aid of a dissecting microscope. An epiuorescence microscope was used with a band pass combination BP450-BP490 exciter lter, a dichroic chromatic beam splitter FT-510 lter and a longwave pass LP-520 barrier lter. A digital camera attached to the microscope and computer with a frame grabber were used to capture and save images.

Results Glomalin was detected on hyphae attached to roots, hyphae from the root-free zone of a pot culture, intraradical structures, on hyphae traps placed in the

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Figure 1. Results of MAb32Bll immunouorescence assays for glomalin. (A) Hyphae of an isolate of Acaulospora mellea on a piece of horticultural plastic. The square-shaped light areas are pores in the plastic mesh (20). (B) A closer view hyphae of A. mellea on horticultural mesh shows bright uorescence and pieces of glomalin attached to the plastic (125). (C) Sudangrass colonized by an isolate of Glomus mosseae after 4 months of growth (125). (D) Immunouorescence of an arbuscule in crimson clover (Trifolium incarnatum L.) root colonized by Glomus etunicatum BR220 (400). (E) A piece of nylon mesh with glomalin from an isolate of Glomus viscosum attached (180). (F and G) Aggregates from a silt loam soil coated with glomalin. (F) Shown are 0.251 mm size aggregates (63) and (G) is a 12 mm size aggregate (63). (H) Root hairs of sudangrass (Sorghum sudanese (Piper) Staph) (non-colonized) showing auto uorescence (400).

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Figure 1. Continued

root-free zone and on 40 m nylon mesh enclosing colonized roots. A hyphae trap for an isolate of Acaulospora mellea Spain and Schenck showed large masses of hyphae (Figure 1A) and, at a higher magnication of the same sample, hyphae and sloughed glomalin (Figure 1B). Sudangrass roots, 4 months after inoculation with an isolate of Glomus mosseae (Nichol. and Gerd.) Gerdemann and Trappe, had abundant hyphae and glomalin on the root surface (Figure 1C). The assay also revealed intraradical structures in cleared roots of clover inoculated with Glomus etunicatum Becker and Gerdemann BR220 (Figure 1D). A piece of 40 m nylon mesh used to inclose roots in sand-cultured sudangrass inoculated with an isolate of Glomus viscosum Nicholson had large amounts of glomalin deposited on the mesh (Figure 1E). Visualizations of copious deposits of glomalin were indirect evidence of the major role this molecule might play in soil stabilization. Glomalin was detected by immunouorescence on the surface of small and large water-stable aggregates of a soil sieved from a native grassland near Lincoln, Nebraska (Figure 1F,G). Higher magnication sometimes revealed immunouorescent hyphae attached to aggregates (Wright and Upadhyaya, 1998). Controls for all types of samples, using the antiRhizobium MAb in place of MAb32B11, showed no reactivity. Root hairs of non-colonized sudangrass showed smooth green autouorescence (Figure 1H), but this type of uorescence was easily distinguished from aky, globular or raised glomalin (Figure 1A C,E). Corn (Zea mays L.) root hairs are less autouor-

escence than those of sudangrass. Glomalin on hyphae generally had the appearance of an uneven sheath and was patchy and rough when deposited on surfaces. In rare cases, sand that adhered to hyphae traps also showed green autouorescence. This was easily distinguished from glomalin because of the size and smooth surface of sand.

Discussion Immunouorescence using MAb32Bll is a useful tool to monitor glomalin deposition on AM hyphae. Colonization can be assessed on hyphae traps without cutting or coring roots. Hyphae can be seen in a more natural state on root surfaces than after conventional methods for staining that require clearing of roots. As hyphae age, glomalin either sloughs off or becomes non-immunoreactive as indicated by immunouorescence assays during time-course studies of colonization (Wright et al., 1996) and enzyme-linked immunoassay of extracts of glomalin (Wright and Upadhyaya, 1996). Therefore, probably only young hyphae and freshly deposited glomalin are immunoreactive. MAb32B11 is a sensitive probe for freshly deposited glomalin, on hyphae and reveals the loss of glomalin from hyphae and attachment to other surfaces. Results of several variations of the immunouorescence assay show that this approach can help dene the inuence of AM fungi on soil stabilization. Large quantities of polysaccharides in the form of the glycoprotein on water stable soil aggregates need to be put into perspective with historical and current reports of the role of polysaccharides in soil stabil-

176 ization. The contribution of polysaccharides to soil stabilization and the link with AM fungi are discussed by Tisdall and Oades (1982) and by Tisdall (1991, 1995). They propose that polysaccharides are involved in stabilization of aggregates, as measured by differences in water stable particles of soil before and after oxidation. Hyphae of AM fungi and extracellular polysaccharides are thought to be temporary agents that bind microaggregates (0.02-0.25 mm diameter) into stable macroaggregates (>0.25 mm). Oxidation of polysaccharides on soil aggregates by exposure to periodate for 6 h, presumably at RT (Tisdall and Oades, 1979) compares with the exposure required (8 h at 37 C) to eliminate immunouorescence of glomalin on hyphae (unpublished). Therefore, periodate oxidation probably affects glomalin as the polysaccharide-containing molecule on aggregates (Tisdall and Oades, 1979). With the antibody against glomalin for detection, it is now possible to identify a specic polysaccharide-containing compound from AM hyphae associated with soil aggregates. We propose that glomalin may play a role as a transported, insoluble biolm that is uniquely involved in the soil aggregation process. When glomalin is sloughed from hyphae, other organic matter and microbes can become entrapped and then attached to nearby soil particles by the protective glomalin coating. A localized environment is created for bacteria to degrade organic matter and to produce extracellular polysaccharides, which, in turn, further stabilizes aggregates. There is much to explore in the relationship between aggregate stability and glomalin because of the above-mentioned characteristics of the molecule, i.e. abundant production, sloughing, attachment to soil particles and apparent recalcitrance. In addition to these characteristics, glomalin also contains iron, and iron can attach to glomalin in solution (unpublished data). Also, the turnover rate of glomalin, as determined by a carbon dating technique, is at least decadal (unpublished data). Recent work indicated that glomalin is a part of the fulvic acid fraction of soils, but methods to extract fulvic acid do not extract the total amount of glomalin present in soils (manuscript in prep). We currently are investigating the binding and incorporation of iron in the molecule and complete analysis of oligosaccharides and amino acids. Acknowledgements The author would like to thank K. Nichols for technical assistance. References
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