Identification of a Vesicular-Arbuscular Mycorrhizal Fungus by Using Monoclonal Antibodies in an Enzyme-Linked Immunosorbent Assay

Sara F. Wright, Joseph B. Morton and Janis E. Sworobuk Appl. Environ. Microbiol. 1987, 53(9):2222.

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APPLIED

AND

ENVIRONMENTAL MICROBIOLOGY, Sept. 1987, p. 2222-2225

Vol. 53, No. 9

0099-2240/87/092222-04$02.00/0 Copyright 1987, American Society for Microbiology

Identification of a Vesicular-Arbuscular Mycorrhizal Fungus by Using Monoclonal Antibodies in an Enzyme-Linked Immunosorbent Assayt
SARA F. WRIGHT,'* JOSEPH B. MORTON,2 AND JANIS E. SWOROBUK1 Appalachian Soil and Water Conservation Research Laboratory, Agricultuiral Research Service, U.S. Department of

Agriculture, Beckley, West Virginia 25802-0867,1 and Division of Plant and Soil Sciences, West Virginia University, Morgantown, West Virginia 26506-60572
Received 10 April 1987/Accepted 9 June 1987

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Spore morphology is currently used to identify species of vesicular-arbuscular mycorrhizal fungi. We report the first use of a highly specific immunological method for identification of a vesicular-arbuscular mycorrhizal fungus. Two monoclonal antibodies were produced against Glomus occultum. Monoclonal antibodies reacted strongly with both spores and hyphae in an indirect enzyme-linked immunosorbent assay. All other mycorrhizal (29 species) and nonmycorrhizal (5 species) fungi tested were nonreactive with the monoclonal antibodies. A single spore of G. occultum was detectable in the presence of high numbers of spores of other vesicular-arbuscular mycorrhizal fungi. Variation in the reaction of G. occultum isolates from West Virginia, Florida, and Colombia suggests that monoclonal antibodies may differentiate strains.

Vesicular-arbuscular mycorrhizal (VAM) fungi are ubiquitous and form obligate symbiotic associations with a majority of land plants. However, species and strains differ in their ability to infect and colonize roots, to promote plant growth (5), and to tolerate dissimilar soil environments (4). Most field soils contain spores of more than one VAM fungus (1), resulting in roots being colonized by a mixture of species. VAM fungi associated with field-grown plants have been identified solely by morphology of asexual reproductive spores or sporocarps (14). Identification is becoming more difficult as new species are described which differ only slightly in morphology. The distribution and habitat requirements of most VAM fungi remain unknown because individual species cannot easily be identified in roots of infected plants. With only a few exceptions (1), the morphology of mycorrhizal structures (e.g., hyphae, arbuscules, and vesicles) is similar among species. The discovery of several atypical VAM fungi (6, 9, 12) which fail to stain in mycorrhizal roots with available methods (3, 11) also prevents detection in mixed infections, unless spores are produced in sufficient numbers (8). Immunofluorescent (7, 16) and immunochemical (2) techniques have been attempted for identification of hyphae of individual VAM fungi using polyclonal antisera which were elicited by crushed spore or hyphal antigen preparations. Low antiserum titers and strong cross-reactivity among species, however, have hampered further development of these methods. Enzyme staining of electrophoretic protein profiles of spores has been used to characterize Glomus species (13). This method is applicable to taxonomic studies but is not suitable for use when large numbers of samples are to be examined. We report the development of a highly specific indirect enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies (MAbs) for detection and identification of propagules of a VAM fungus, Glomus occuiltlum Walker (8). G. occultuim is a common and abundant endophyte in highly
* Corresponding author. t Scientific article no. 2061 of the West Virginia Agriculture and Forestry Experiment Station.

acidic West Virginia soils, particularly in soils disturbed by surface mining of coal (8). This fungus was chosen because its hyaline spores are difficult to identify when mixed with other VAM fungal spores which are either immature or hyaline at maturity. Also, G. occuiltum produced large quantities of spores in greenhouse pot culture (9), which facilitated antigen preparation.
MATERIALS AND METHODS Antigen for immunization of mice. Spores of G. occultum Walker (isolate 419) were extracted from overburden material at an abandoned coal mine site near Osage, W.V. The soil was predominantly black shale with a pH of 3.6 and contained 8.6 mg of bicarbonate-extractable P per kg; 0.1, 0.24, and 0.1 cmol(+) of K, Mg, and Ca, respectively, per kg of soil; and 2.8 cmol(+) of Al per kg of soil. Enough spores were mixed thoroughly with a sterilized soil-sand mixture (1:2, vol/vol) so that the final inoculum density was 10 spores per cm of soil. This mixture was added to 6-in. (ca. 18-cm) plastic pots and seeded with Sorghum sudanense (Piper) Staph. at a rate of 30 to 40 seeds per pot. Soil and roots from pot cultures were harvested after 4 months and stored at 4C until needed. Spores were extracted by forcing soil through 150- and 38-gm-mesh sieves with water, centrifuging the fraction on the 38-tm-mesh sieve in a sucrose gradient (15 ml of 20% sucrose overlaid with 15 ml of 60% sucrose) at 950 x g for 4 min, and then washing spores collected in the 20% sucrose fraction in sterile distilled water. Spores were crushed and lysed in 3 ml of water with a 0.5-ml Potter Elvehjem tissue homogenizer, diluted in physiological saline to a concentration (optical density at 450 nm [OD450]) of 1.27 (approximately 50,000 spores per ml), and stored frozen. Immunization, fusion, and ELISA protocols. Two 6-weekold female BALB/c mice were injected intraperitoneally with 1.0 ml of the spore suspension on days 0, 17, and 37 and with 0.5 ml on day 55. Spleen cells were harvested on day 58 and fused with murine myeloma cell line P3/NS1/1-Ag4-1 (10). Hybridomas were cultured in 96-well microtiter plates. All 196 wells plated contained from five to eight fused cells per
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IDENTIFICATION OF VA MYCORRHIZAE

2223

well. After 10 days of growth, the hybridoma supernatant was examined by indirect ELISA (15) with 50 of crushed spore suspension (OD45() = 0.089; approximately 400 spores), dispensed in 96-well polyvinyl chloride plates (Costar), and allowed to dry overnight at 35C. Goat antimouse immunoglobulin G labeled with horseradish peroxidase was used as the immunoconjugate, and hydrogen peroxide and 2,2'-azino-di-(3-ethyl-benzthiazoloine sulfonate) were used as the substrate and chromogen, respectively (17). Absorbance of hydrolyzed substrate was measured at 405 nm with an EIA Reader (Bio-tek Instruments, Inc.: model E1307). The ELISA reactivity of blanks (antigen reacted with all reagents except MAbs) was subtracted from test absorbance values. Supernatants from wells testing positive to G. occultitin were tested for cross-reactivity a against Glomuliis itiaplialnlillm (50 pd: OD450 = 0.095)l VAM fungus with similar spore characteristics (8). MAbs. Cell lines that were not cross-reactive with G. dtiaplhaniumtz were cloned twice, and supernatants were collected from subsequent cultures which had been allowed to overgrow. MAbs were characterized as to subclass by double immunodiffusion in agarose on glass microslides. Antigens tested for cross-reactivity. Hyphae of G. occultiml were collected by spreading infected sudangrass roots over a 250-p.m-mesh sieve. Hyphae were washed from roots onto a 106-p.m-mesh sieve with a forced water spray. Sievings were decanted with water into a glass petri dish, and hyphae were removed from root fragments. Care also was taken to remove attached spores, which often were enmeshed in hyphal aggregates. The hyphal mass was washed, blotted dry, weighed, and crushed in a tissue homogenizer in 200 pL. of water. Hyphae (with associated spores) of nonmycorrhizal fungi were collected from potato dextrose agar plates and processed similarly. Nonmycorrhizal fungi tested were Trichlodermna sp., Moutier>ellai candelabrum var. Tiegh and LeMann isolate 1056, Altenariatii solani (Ellis and Martin) Sorauer isolate 1521, Enidotliiai parasitica (Murrill) Anderson (from W. L. MacDonald, West Virginia University), and Phytoplithora infestains (Montagne) de Bary (from R. J. Young, West Virginia University). Spores of Glomtuzis (ldbiclidam Walker and Rhodes isolate 623, Morton and Walker isolate 388, G. clarmmnz G. Nicolson and Schenck isolate 559, G. claroidleum Schenck and Smith (from H. D. Skipper, Clemson University), G. conlstrictitun Trappe isolate 597, G. etuni(caltm Becker and Gerdemann Isolate 579, G. intraradicices Schenck and Smith (from N. C. Schenck, University of Florida), G. geosporumn (Nicolson and Gerdemann) Walker isolate 546, G. mnacrocarpuln Tulasne and Tulasne isolate 638, '"G. spurcum Pfeiffer and Walker nom. ined. isolate 567, G. versifo)rne (Karsten) Berch isolate 135, G. leptotichItimn Schenck and Smith, "Glonitus sp. OSI'' 386. "GlomIiius sp. BO1'' 596, ''"Glomuis sp. JL1'' 591. Acaulospora bireticulata Rothwell and Trappe isolate 631, A. dlelicata Walker. Pfeiffer, and Bloss isolate 566, A. cdiltatat Morton isolate 492, A. lacuniosa Morton isolate 461, A. i-lugos(i Morton

[1I

extracted from greenhouse pot cultures by the same method used for G. occultumn. Thirty spores of each fungus were collected, crushed in 300 .I of water, and dispensed in 50-p.l samples to each of six wells. Sensitivity of the ELISA. The sensitivity of the ELISA to detect spores of G. occultium in a mixture with other VAM fungi was examined by crushing different numbers of spores of G. occmultum alone or in combination with spores of other VAM fungi in 200 pL. of water and adding 50 .I of homogenate to each of four wells. Spores from 15 isolates that were morphologically consistent with G. occultuim (8) were tested by ELISA. Spores of isolates from West Virginia were collected from various plants in 10 locations. Spores of isolates from Florida (courtesy of N. C. Schenck) and Colombia (courtesy of E. Sieverding) were from pot cultures of the original samples. Autoclaved Lily soil (Fine-loamy, siliceous, mesic Typic Hapludults) in "D-pots'' (6 by 25 cm) (McConkey and Co., Sumner, Wash.) were inoculated with spores which were mixed in the upper 6 cm of soil. Pots were seeded with sudangrass (6 to 8 seeds per pot) which was grown for 12 weeks at 22 to 28C under artificial light (combined fluorescent and incandescent lamps at 163 p.E/m2 per s for 12 h per day). Spores were extracted as described above. Characterization of epitope. Crushed spores were exposed to temperatures ranging from 25 to 100C, 4% (wt/vol) sodium dodecyl sulfate, and 5% (vol/vol) Formalin and then tested by ELISA. Water-soluble and washed spore wall fragments fractionated from crushed spores were tested by ELISA.
RESULTS AND DISCUSSION The fusion yielded two cell lines (B5 and F8) which produced antibodies specific for G. occultuma. Both antibodies were subclass immunoglobulin G2b. The antibody titer of supernatant from each hybridoma cell line, using five crushed spores of G. occultumn per well, was 1:512 and 1:1,024 for B5 and F8, respectively. For further ELISA tests, B5 and F8 were diluted 1:16 and 1:32, respectively. MAbs produced from cell lines B5 and F8 were highly specific against spores and hyphae of G. occultumn (Table 1). Only spores of G. gigainitea reacted with an OD405 of >0.20 against both MAbs. However, the average volume of eaich G. gigainitea spore was 216 times that of an average G. occmdltamn spore. Mean diameters of spores of G. occi(dtum, G. etmunicatum, A. rugosa, and G. giganlEtea are 65, 120, 92. and 390 p.m, respectively, giving spherical volumes of 143.720, 904,320, 407,513, and 31,043,610 p.m3, respectively. Crushed spores of other VAM fungi also produced significant reactions (OD1405 > 0.20) when spore numbers were increased to a biomass similar to that of G. gigainite(a (Table 2). Single spores of G. occultitun were still detectable in spore mixtures by using a maximum background biomass control with an OD405 of 0.30 (Table 2). Reactivity of G. occultiumn with other VAM fungi was additive or enhanced above that of G. occultumn spores alone. Spore contents of the other VAM fungi may have enhanced the attachment of the target antigen to well surfaces. These results suggest that as bioassays are developed to detect G. occultiu)n in soil or roots, mixtures of VAM fungi will not interfere with the sensitivity of the tests. We have preliminary evidence to suggest that MAbs against G. occultumn also may be able to differentiate strains. Ten isolates of the fungus from different locations in West Virginia were compared. All reacted positively to both

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di(apilzanaiomn

isolate 593. 'Acaulospora sp. BD1'' 417, ''Acaulospora sp. JL5'' 455, "Acaulospora sp. MN4' 575. Gigaosporal gigaitnteti (Nicolson and Gerdemann) Gerdemann and Trappe

isolate 588, G. martiJ-gar-itai Becker and Hall isolate 204, Scutellospora hleteirogaminal (Gerdemann and Trappe) Walker and Sanders isolate 402, S. pellicida (Nicolson and Schenck) Walker and Sanders isolate 556, "S. dipurpurescens" Morton and Koske nom. ined. isolate 557. Entrophospora colomnbiana Spain and Schenck isolate 571. and E. infr qmns Ames and Schneider isolate 621 were e

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WRIGHT ET AL.

APPL. ENVIRON. MICROBIOL.

TABLE 1. Absorbance values for ELISA reactions of G. occultum MAbs B5 and F8 with crushed spores and hyphae of mycorrhizal and nonmycorrhizal fungi
Antigen
B5

OD40_, of MAb"
F8

Glomus occultum Crushed spores (5)b Hyphae (0.25 mg)

Crushed spores (5) of': Glomus albidum G. diaphanum G. clarum G. claroideum G. constrictum G. etunicatum G. intraradices
G.
G.

0.763 0.732

geosporum macrocarpum

spurcum" versiforme G. leptotichum "Glomus sp. OS1" "Glomus sp. B01" "Glomus sp. JL1" Acaulospora bireticulata A. delicata A. dilatata A. lacunosa
"G. G.

A.

rugosa

"Acaulospora sp. BD1" "Acaulospora sp. JLS" "Acaulospora sp. MN4" Gigaspora gigantea G. margarita Scutellospora heterogama S. pellucida "S. dipurpurescens" Enterospora colombiana E. infrequens
Hypha and spore mixture (0.25 mg) from: Glomus diaphanum Trichomderma sp. Mortierella candelabrum Alternaria solani Endothia parasitica Phytophthora infestans

0.070 0.079 0.036 0.053 0.055 0.069 0.049 0.038 0.026 0.013 0.039 0.027 0.096 0.051 0.042 0.062 0.021 0.018 0.031 0.042 0.035 0.056 0.017 0.224 0.088 0.078 0.092 0.104 0.074 0.052

0.075 0.062 0.063 0.071 0.096 0.082 0.079 0.032 0.035 0.016 0.027 0.033 0.020 0.119 0.058 0.041 0.053 0.049 0.040 0.056 0.026 0.092 0.049 0.218 0.039 0.083 0.122 0.075 0.120 0.032

with cell membrane or other particulate fractions in spores and hyphae of G. occultum. Using immunofluorescence, other workers found that less specific polyclonal antiserum against crushed spores or hyphae of particular VAM fungi also reacted with antigens associated with inner cell walls or cell membranes (7, 16). Reactions of both MAbs were consistently the same. Both lost activity against crushed spores exposed to temperatures above 80C, 4% sodium dodecyl sulfate, or 5% Formalin. Characterization of the epitope will require careful manipulation to prevent loss of antigenicity. Moreover, these results indicate that spores preserved in Formalin are nonreactive. Based upon results of this study, MAbs overcame the obstacle of cross-reactivity among species of VAM fungi. Only isolates with morphology consistent with that of G. occultum exhibited ELISA reactivity, and ELISA reactivity was always >0.30 (Table 1). This establishes a firm taxonomic basis for distinguishing G. occultum from other VAM fungal species with similar spore characteristics. Application of the indirect ELISA to detect and quantify mycorrhizae and external hyphae development of G. occultum in the
TABLE 2. Absorbance values for ELISA reactions with MAb F8 and G. occultum spores alone or with spores of other VAM fungi"
no. of spores"

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VAM fungus,

OD405
Mean Range

Glomus occultum 1 2 3 4 5 10 20 40 Glomus etunicatum 5 40 80

0.365 0.358 0.616 0.657 0.723 1.164 1.637 >2.000 0.082 0.248 0.276

0.331-0.392 0.334-0.426 0.522-0.678 0.546-0.713

0.641-0.874
0.987-1.372 1.493-1.724 1.784->2.000 0.059-0.102 0.232-0.285 0.241-0.322 0.018-0.036 0.064-0.215 0.143-0.291 0.156-0.328 0.167-0.334

0.087 0.026 0.021 0.104 0.079 0.060

0.119 0.041 0.032 0.084 0.102 0.091

Gigaspora gigantea
1 3 5 10 20

0.028 0.169 0.218 0.292 0.277

Acaulospora rugosa
5 10 40 80 G. etunicatum plus G. occultum 40 + 1 40 + 3 40 + 5 G. gigantea plus G. occultum 1 + 1 1 + 5 5 + 1 5 + 5 A. rugosa plus G. occultum 5 + 1 5 + 5 40 + 1 40 + 3 40 + 5

"Values are means of three replicate assays. Numbers in parentheses are the number of spores or the weight of hyphae and hypha-spore mixtures in 50 ,ul of water used to charge each replicate
ELISA plate well. See the text for authorities of cultures.
"

0.056 0.041 0.134 0.218

0.597 0.892 1.094
0.789 1.129 1.273 1.778

0.032-0.093
0.026-0.104

0.064-0.188 0.177-0.252
0.524-0.638 0.7614.967 0.917-1.142
0.702-0.826 1.003-1.214 1.141-1.355 1.642-1.830

MAbs, but OD values differed significantly (P = 0.01) among isolates (Fig. 1). Comparison among spores of isolates from Florida and Colombia, which were produced under a uniform host-soil environment, suggests that the MAbs are able to differentiate among strains. Either concentration of the epitope(s) in spores or affinity between MAb and epitope varied among these isolates of G. occultum. Variation among the West Virginia isolates must be evaluated for differences in reactivity associated with host and soil factors. Water-soluble and washed spore wall fragments fractionated from crushed spores were both reactive. Absorbance was stronger for the soluble fraction (OD405 1.060) than cell wall fragments (OD405 = 0.663). This evidence suggests that the epitope may be a soluble protein associated either
=

0.649 1.118 0.527 0.901 1.260

0.610-0.707 1.103-1.239 0.421-0.638 0.818-1.012 1.143-0.316

" Values greater than 0.30 are considered positive. Values are means of at least four replicate assays. b Spores were crushed in 50 ,l of water and placed into ELISA plate wells.

VOL. 53, 1987

IDENTIFICATION OF VA MYCORRHIZAE

2225

ct
*1.0

06-

0.4-

0.2-

West Virginia lofIs

LFlorida j olates

LCotombiaj
Isolates

FIG. 1. ELISA reactions with G. occultitin MAbs B5 and F8 with crushed spores of G. occiultuin isolates from different soils in West Virginia and spores of isolates from Florida and Colombia produced in pot cultures with uniform soil and plant conditions. Each value is the mean of six replicate wells (eight spores per well) for each MAb, with absorbance readings for both MAbs pooled. The isolate used to produce the MAbs is marked with an asterisk. Differences among means were significant at P = 0.01. The standard error of the mean assay value for each isolate did not exceed the standard error bar.
presence of competing VAM fungi appears promising, as does use of the ELISA to differentiate strains of G. occultitin. Identification of G. o(ccultIlon in intact roots and hyphae with fluorescent antibody techniques will be feasible if MAbs react with internal epitopes localized near the cut ends of hyphae (7, 16).

ACKNOWLEDGMENT This research was partially supported with funds appropriated under the Hatch Act.

LITERATURE CITED 1. Abbott, L. K. 1982. Comparative anatomy of vesiculararbuscular mycorrhizas formed on subterranean clover. Aust. J. Bot. 30:485-499. 2. Aldwell, F. E. B., I. R. Hall, and J. M. B. Smith. 1983. Enzyme-linked immunosorbent assay (ELISA) to identify endomycorrhizal fungi. Soil Biol. Biochem. 15:377-378. 3. Bundrett, M., Y. Piche, and R. L. Peterson. 1984. A new method for observing the morphology of vesicular-arbuscular mycor-

rhizae. Can. J. Bot. 62:2128-2134. 4. Gildon, A., and P. B. Tinker. 1983. Interactions of vesiculararbuscular mycorrhizal infection and heavy metals. I. The effects of heavy metals on the development of vesiculararbuscular mycorrhizas. New Phytol. 95:247-261. 5. Haas, J. H., and J. Krikun. 1985. Efficacy of endomycorrhizal fungus isolates and inoculum quantities required for growth response. New Phytol. 100:613-621. 6. Hetrick, B. A., J. Bloom, and S. M. Feyerherm. 1985. Root colonization of Glottiius epigaeium in nine host species. Mycologia 77:825-828. 7. Kough, J., N. Malajczuk, and R. G. Linderman. 1983. Use of indirect immunofluorescent technique to study the vesiculararbuscular fungus Glornis epigaelutn and other Gloinus species. New Phytol. 94:57-62. 8. Morton, J. B. 1985. Variation in mycorrhizal and spore morphology of Glotiuiis occ(Iultum and Gloinis diaplantzuin as influenced by plant host and soil environment. Mycologia 77: 192-204. 9. Morton, J. B. 1985. Underestimation of most probable numbers of vesicular-arbuscular endophytes because of nonstaining mycorrhizae. Soil Biol. Biochem. 17:383-384. 10. Oi, V. T., and L. A. Herzenberg. 1980. Immunoglobulinproducing hybrid cell lines, p. 351-372. In B. B. Mishell and S. M. Shiigi (ed.), Selected methods in immunology. W. H. Freeman & Co.. San Francisco. 11. Phillips, J. M., and D. S. Hayman. 1970. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans. Br. Mycol. Soc. 55:158-160. 12. Schenck, N. C., and G. S. Smith. 1982. Additional new and unreported species of mycorrhizal fungi (Endogonaceae) from Florida. Mycologia 74:77-92. 13. Sen, R., and C. M. Hepper. 1986. Characterization of vesiculararbuscular mycorrhizal fungi (Glomiis spp) by selective enzyme staining following polyacrylamide gel electrophoresis. Soil Biol. Biochem. 18:29-34. 14. Trappe, J. M. 1982. Synoptic keys to the genera and species of zygomycetous mycorrhizal fungi. Phytopathology 72:11021108. 15. Voller, A., D. Bidwell, and A. Bartlett. 1980. Enzyme-linked immunosorbent assay, p. 359-371. In N. R. Rose and H. Friedman (ed.), Manual of clinical immunology, 2nd ed. American Society for Microbiology, Washington, D.C. 16. Wilson, J. M., M. J. Trinick, and C. A. Parker. 1983. The identification of vesicular-arbuscular mycorrhizal fungi using immunofluorescence. Soil Biol. Biochem. 15:439-445. 17. Wright, S. F., J. G. Foster, and 0. L. Bennett. 1986. Production and use of monoclonal antibodies for identification of strains of Rhliiobiium t-if lii. Appl. Environ. Microbiol. 52:119-123.

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