APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1995, p. 2809–2810 0099-2240/95/$04.0010 Copyright q 1995, American Society for Microbiology
Vol. 61, No. 7
Detection of an Arbuscular Mycorrhizal Fungus in Roots of Different Plant Species with the PCR† RITA DI BONITO,1 M. L. ELLIOTT,2*
AND
E. A. DES JARDIN2
INN-BIOAG, ENEA CRE Casaccia, 00060 S. Maria di Galeria, Rome, Italy,1 and Fort Lauderdale Research and Education Center, University of Florida, Fort Lauderdale, Florida 333142 Received 27 January 1995/Accepted 24 April 1995
PCR was used with the primer pair VANS1-NS21 to detect the arbuscular mycorrhizal fungus Glomus intraradices (commercial inoculum source) on roots of lettuce, zinnia, leek, pepper, and endive plants. The appropriate amplification product was obtained directly from roots without DNA extraction and purification. HCl, pH 8.5) for 15 min. DNA template from roots was also obtained by adding Chelex 100 resin (Bio-Rad Laboratories, Hercules, Calif.), to a final concentration of 5%, to the extraction buffer prior to boiling. The resin was removed by centrifugation at 12,000 3 g for 20 s before the DNA template was used for PCR. The processed samples were either immediately used for PCR or frozen at 2208C. Serial dilutions of the DNA template made with water were added at the ratio 1:1 (vol/vol) to 50-ml final reaction mixtures containing 50 pmol each of VANS1 and NS21 primers (9), 200 mM each deoxynucleoside triphosphate, 20 mM Tris-HCl (pH 8.4), 100 mM KCl, 3 mM MgCl2, 0.02% gelatin, and 1.25 U of Taq DNA polymerase (AmpliTaq; Perkin-Elmer Cetus, Norwalk, Conn.). Forty cycles of amplification were carried out in a TempCycler II instrument, model 110 (COY Corporation, Grass Lake, Mich.), with the following parameters: denaturation for 60 s at 948C, annealing for 45 s at 508C, extension for 60 s at 728C, and a final extension for 10 min at 728C (10). For some samples, 1 ml of the reaction product was diluted in 49 ml of a fresh reaction mixture for a second amplification round of 40 cycles. The reaction products were analyzed by electrophoresis through 3% agarose gels (Low electroendosmosis; Fisher Scientific, Pittsburgh, Pa.). DNA in gels was visualized by UV transillumination after being stained with ethidium bromide and photographed. In this study, the 550-bp rDNA amplification product characteristic of AM fungi (9) was consistently amplified from samples of G. intraradices inoculum (Fig. 1) and roots of five plant species colonized by this fungus. This AM fungus was detected in the first few weeks of growth on roots of different species and age when the colonization was at least 30% (Table 1). The extent of root colonization and not the harvest date appeared to be critical for detection. Figure 2 shows typical PCR results obtained with noncolonized and colonized roots. The 550-bp product was never detected from uninoculated, noncolonized control plants, even after two amplification rounds of 40 cycles each. Nonspecific products, however, were routinely amplified in all the inoculum and root samples, including those from control plants. The presence of these DNA products from the control plants indicated that, under the conditions used, the primer pair was not taxon specific and would amplify nonmycorrhizal DNA. None of the DNA template samples used for PCR in this study were purified, and all the material had been exposed to a normal greenhouse environment. Even the G. intraradices inoculum included plant root tissues. Since the nonspecific products did not interfere with detection of the 550-bp AM fungal product, no attempts were made to alter the PCR parameters (e.g., to increase the annealing temperature).
The symbiotic association between arbuscular mycorrhizal (AM) fungi and plant roots is widespread in nature (8). The positive effects of AM fungus inoculation on agricultural cropping systems have been investigated and include improved uptake of phosphorus and other nutrients, increased stress tolerance, and beneficial plant growth regulation (6–8). DNA amplification by PCR has been proposed as a technique for AM fungus identification (11, 13). A portion of the smallsubunit rRNA gene (rDNA) specific for AM fungi can be amplified when a taxon-specific primer (VANS1) is paired with a universal primer (NS21) (9). The studies of PCR application for AM fungi have been carried out with spores or purified DNA from colonized leek roots (9–11, 13). No information concerning the use of this technique for other plant species is available. In this work, the AM taxon-specific primer VANS1 was used for detection of the AM fungus Glomus intraradices Schenck and Smith (‘‘Glomus intraradix’’) on roots of five host species colonized by this fungus. This was accomplished without extraction of DNA from colonized roots by simply boiling root tissue in a buffer (4). The mycorrhizal inoculum used for PCR amplification and root colonization is commercially available and was obtained from the Premier Research Center, Rivie`re du Loup, Quebec, Canada. It contained active propagules of G. intraradices (DAOM 197198) consisting of spores, mycelia, and colonized plant roots. The plant species inoculated were lettuce (Lactuca sativa L.), leek (Allium porrum L.), zinnia (Zinnia elegans Jacq.), endive (Cichorium endivia L.), and pepper (Capsicum annuum L.). The seeds were surface sterilized and planted in pots filled with sterilized soilless potting mix containing the inoculum. Plants were grown in a greenhouse and fertilized weekly with a solution containing only nitrogen and potassium. Control plants without mycorrhizal inoculum were grown under the same conditions. Roots were collected at various times after planting, rinsed thoroughly to remove substrate, and immediately processed for use with PCR. Root colonization was evaluated by the grid line intersect method, which entails evaluation of stained roots with a dissecting microscope (2). Template DNA for PCR was obtained from crushed root samples (8 to 20 mg) or inoculum propagules (10 to 20 mg) by boiling each sample in 800 ml of extraction buffer (1 M Tris* Corresponding author. Mailing address: Fort Lauderdale Research and Education Center, University of Florida, 3205 College Ave., Fort Lauderdale, FL 33314. Phone: (305) 475-8990. Fax: (305) 475-4125. Electronic mail address:
[email protected]. † Florida Agricultural Experiment Station Journal Series no. R-04474. 2809
2810
NOTES
APPL. ENVIRON. MICROBIOL.
FIG. 1. PCR products of G. intraradices inoculum after one amplification round of 40 cycles. The sample was crushed and boiled in buffer before being used as the DNA template. Lane 1, lambda DNA digested with PvuII; lanes 2 to 6, 10-, 100-, 500-, 2,500-, and 5,000-fold dilutions of G. intraradices sample; lane 7, control with no template.
The addition of Chelex resin to the Tris-HCl buffer when the colonized roots were boiled resulted in increased amplification of the 550-bp AM rDNA product from reaction mixtures (Fig. 2). Use of Chelex resin was critical for positive detection of G. intraradices in root samples of pepper and leek plants with low colonization (28 and 32%, respectively). Dilution of the template DNA does appear to be required, and 100- or 500-fold dilutions usually provided positive results. Inhibition of PCR by phenolic substances, polysaccharides, or humic acids has often been reported in association with DNA from plant tissue or environmental samples (1, 5, 12), and the presence of plant phenolics in roots colonized by G. intraradices has been documented (3). Chelex resin combined with dilution of the template has been used previously for DNA amplification from AM fungal spores (13). The method we have described does not require any lengthy DNA extraction procedure, yet it does provide suitable DNA template for PCR. Root samples or inocula were simply boiled with a buffer containing Chelex resin. For the five plant species analyzed, optimal results were obtained when roots were processed for PCR immediately after harvest and DNA samples
TABLE 1. Colonization by G. intraradices and PCR amplification results for inoculum and colonized roots Sample
Plant age (wks)
Colonization (%)
Presence of 550-bp PCR producta
1
G. intraradices (inoculum) Lettuce
3 5 7 8
30 21 35 72
1 1b 1 1
Zinnia
3 5 7
21 50 78
2c 1 1
Endive
8
61
1
Pepper
7
28
1b
11
32
1
Leek
a DNA template was derived from roots and inoculum boiled in buffer with Chelex resin. The primer pair used for PCR was VANS1-NS21. b Product present only after two amplification rounds of 40 cycles. c Product absent even after reamplification.
FIG. 2. PCR products from dilutions of zinnia root samples with 78% colonization by G. intraradices and from noncolonized control roots (100-, 500-, 2,500-, and 5,000-fold dilutions for each sample). Roots were crushed and boiled in buffer, with and without Chelex resin, before being used as the DNA template. Lanes 2 to 5, colonized roots at 100-, 500-, 2,500-, and 5,000-fold dilutions, respectively; lanes 6 to 9, colonized roots boiled with Chelex resin; lane 10, control with no template; lanes 12 to 15, noncolonized roots; lanes 16 to 19, noncolonized roots boiled with Chelex resin; Lanes 1, 11, and 20, lambda DNA digested with PvuII. The arrows indicate the 550-bp product characteristic of endomycorrhizal fungi.
were then used in the PCR assay within a few days. Conversely, storing the roots in the refrigerator or the processed DNA samples in the freezer (2208C) for more than 2 weeks diminished or eliminated amplification of the 550-bp rDNA product (unpublished data). The availability of a simple method to confirm AM fungal colonization on a broad range of species and in the first few weeks of growth would be useful in the application and evaluation of endomycorrhizal inoculants in agriculture. We are grateful to J. Kramer for comments on the methods used in this work. We also thank N. Harrison for valuable discussions and critical reading of the manuscript. REFERENCES 1. Demeke, T., and R. P. Adams. 1992. The effect of plant polysaccharides and buffer additives on PCR. BioTechniques 12:332–333. 2. Giovannetti, M., and B. Mosse. 1980. An evaluation of techniques for measuring vesicular arbuscular mycorrhizal infection in roots. New Phytol. 84:489–500. 3. Grandmaison, J., G. M. Olah, M. R. Van Calsteren, and V. Furlan. 1993. Characterization and localization of plant phenolics likely involved in the pathogen resistance expressed by endomycorrhizal roots. Mycorrhiza 3:155–164. 4. Henson, J. M., T. Goins, W. Grey, D. E. Mathre, and M. L. Elliott. 1993. Use of polymerase chain reaction to detect Gaeumannomyces graminis DNA in plants grown in artificially and naturally infested soil. Phytopathology 83:283–287. 5. John, M. E. 1992. An efficient method for isolation of RNA and DNA from plants containing polyphenolics. Nucleic Acids Res. 20:2381. 6. Millner, P. D. 1991. Characterization and use of vesicular-arbuscular mycorrhizae in agricultural production systems, p. 335–342. In D. L. Keister and P. B. Cregan (ed.), The rhizosphere and plant growth. Kluwer Academic Publishers, Dordrecht, The Netherlands. 7. Pedersen, C. T., G. R. Safir, S. R. Parent, and M. Caron. 1991. Growth of asparagus in a commercial peat mix containing vesicular-arbuscular mycorrhizal (VAM) fungi and the effects of applied phosphorus. Plant Soil 135:75–82. 8. Safir, G. R. 1994. Involvement of cropping systems, plant produced compounds and inoculum production in the functioning of VAM fungi, p. 239– 259. In F. L. Pfleger and R. G. Linderman (ed.), Mycorrhizae and plant health. APS Press, St. Paul, Minn. 9. Simon, L., M. Lalonde, and T. D. Bruns. 1992. Specific amplification of 18S fungal ribosomal genes from vesicular-arbuscular endomycorrhizal fungi colonizing roots. Appl. Environ. Microbiol. 58:291–295. 10. Simon, L., R. C. Le´vesque, and M. Lalonde. 1992. Rapid quantitation by PCR of endomycorrhizal fungi colonizing roots. PCR Methods Applic. 2:76–80. 11. Simon, L., R. C. Le ´vesque, and M. Lalonde. 1993. Identification of endomycorrhizal fungi colonizing roots by fluorescent single-strand conformation polymorphism-polymerase chain reaction. Appl. Environ. Microbiol. 59:4211–4215. 12. Tsai, Y.-L., and B. H. Olson. 1992. Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Appl. Environ. Microbiol. 58:2292–2295. 13. Wyss, P., and P. Bonfante. 1993. Amplification of genomic DNA of arbuscular-mycorrhizal (AM) fungi by PCR using short arbitrary primers. Mycol. Res. 97:1351–1357.
