FEMS Microbiology Letters 212 (2002) 171^175
A molecular marker diagnostic of a speci¢c isolate of an arbuscular mycorrhizal fungus, Gigaspora margarita Kazuhira Yokoyama
, Takahiro Tateishi b , Takuya Marumoto a , Masanori Saito
Department of Biological Science, Faculty of Agriculture, Yamaguchi University, 1677-1, Yoshida, Yamaguchi 753-8515, Japan b Bio-oriented Technology Research Advancement Institution, 1-40-2, Nisshin-cho, Saitama, Saitama 331-8537, Japan Laboratory of Soil Ecology, Department of Grassland Ecology, National Institute of Livestock and Grassland Science, Nishi-nasuno, Tochigi 329-2793, Japan Received 11 March 2002 ; received in revised form 19 April 2002; accepted 19 April 2002 First published online 28 May 2002
Abstract To investigate the auto-ecology of a strain of Gigaspora margarita in a commercial inoculum, we found a pair of PCR primers amplifying a sequence of 235 bp diagnostic of the isolate. We designed an oligonucleotide probe based on the DNA sequence. The combination of PCR and the probing successfully detected the diagnostic sequence from both DNA preparations of single spores and colonized roots. This protocol enabled us to distinguish the isolate among several isolates from Japan, Nepal and the USA. 4 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Gigaspora margarita ; Isolate-speci¢c identi¢cation ; Diagnostic sequence ; Oligonucleotide probe
1. Introduction Arbuscular-mycorrhizal fungi (AMF) form symbiosis with a wide range of plant species. These promote plant growth by improving water status and nutrient uptake from bulk soil  and, thus, they have been inoculated into soil with lower fertility in agricultural practice. Recently, AMF are expected to be very bene¢cial in enhancing vegetational development in degraded and barren land . Problems such as how the inoculated AMF survived in soil under the competition with native AMF or how they a¡ected the reforestation have remained unclear. It is necessary to trace the fate of the ¢eld-inoculated AMF strain to obtain fundamental information for its better use. We have employed a commercially available inoculum containing the spores of Gigaspora margarita isolate CK in reforestation along the bare slopes of a volcano, Mt. Fugendake, in Nagasaki Prefecture, Japan, that had been covered by a pyroclastic £ow in 1992 [3,4]. Along with reforestation, we are focusing on the fate of the AMF strain that was introduced after lethal destruction of the native ecosystem. Because morphological discrimination
* Corresponding author. Tel. : +81 (83) 933-5837; Fax : +81 (83) 933-5837. E-mail address : [email protected]
among G. margarita strains was impossible, we attempted to ¢nd a molecular marker of the strain to analyze its auto-ecology. Many e¡orts have been made to identify Gigaspora at genus and species levels [5^7]. In AMF, the genetic heterogeneity of thousands of nuclei in a single spore often poses a di⁄culty for the identi¢cation of a speci¢c isolate [6,8,9]. Since the sequences of the internal transcribed spacer (ITS) region of the rRNA gene have been widely used for the identi¢cation of fungi, we examined the possibility of employing it to distinguish some Japanese isolates and isolate BEG34 of G. margarita. However, because of the sequence diversity of glomalean fungi [6,9], the distribution of their ITS clones overlapped each other in the phylogenetic tree of the ITS sequences . Therefore, the DNA sequences of the ITS region could not serve as a molecular marker for the identi¢cation of G. margarita isolates. The M13 mini-satellite primer was also tested because it had been found to be e¡ective for the RAPD analysis and produced a diagnostic band (1180 bp) from the sporal DNA of G. margarita BEG34 . The PCR displayed a polymorphism with a major product of about 500 bp among the Japanese isolates tested. The pattern of minor bands was inconsistent when the concentration of the template was varied. Therefore, the resolution by the single use of the primer was insu⁄cient for our purpose. The M13 mini-satellite primer was expected, however, to
0378-1097 / 02 / $22.00 4 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 0 3 7 8 - 1 0 9 7 ( 0 2 ) 0 0 7 1 4 - 0
FEMSLE 10511 26-6-02
K. Yokoyama et al. / FEMS Microbiology Letters 212 (2002) 171^175
anneal to the genome of G. margarita isolates. The present study revealed that we found a PCR primer to produce a diagnostic sequence for the isolate CK of G. margarita in combination with the M13 mini-satellite primer.
2. Materials and methods 2.1. Mycorrhizal fungi and plants The isolates of G. margarita and the related species tested are listed in Table 1. All voucher specimens of the spores used were deposited in the Laboratory of Soil Ecology, National Institute of Livestock and Grassland Science (NILGS). Mycorrhizal plants were raised as described elsewhere . The following mycorrhizal plants were used: onion with MAFF520054, mixed culture of bahia grass and white clover either with MAFF520052, with Ni-A, or with G. rosea MAFF520062. 2.2. DNA extraction Spores were extracted by wet-sieving from the suspensions of the potting medium or the commercial inoculum under a dissecting microscope and sonicated for 1 min in sterilized water. Then spores were transferred in 0.1% SDS and heated at 50‡C for 10 min followed by sonication again. After rinsing ¢ve times in sterilized water by vortexing, a single spore was transferred in 20 Wl of Instagene matrix (Bio-Rad) and crushed under a dissecting microscope. Genomic DNA was extracted according to the manufacturer’s instructions. The DNA from a single spore was diluted to 20 Wl and, after 10-fold dilution, 1 Wl was
Table 1 Isolates of G. margarita and G. rosea used in this study Isolate G. margarita CKa MAFF520052b MAFF520054b;c Ni-Ab WV205Ad SM478d G. rosea MAFF520056b a
Saitama, Japan Iwate, Japan Saitama, Japan Niigata, Japan West Virginia, USA Daman, Nepal
Central Glass Co. Ltd. Saito Saito Saito Isobe Isobe
Spores of isolate CK were recovered from the commercial inoculum ‘Cerakinkong’ produced by Central Glass Co. Ltd. b These isolates have been maintained in pot culture in the National Institute of Livestock and Grassland Science. MAFF numbers are the accession numbers of these isolates registered in Gene Bank, MAFF, Japan (http://www.gene.a¡rc.go.jp/micro/index.html). c The isolate MAFF520054 was originally provided by Central Glass Co. Ltd., and has been maintained in successive pot cultures for more than 8 years at the Soil Ecology Laboratory, National Institute of Livestock and Grassland Science (previously called the National Grassland Research Institute). d These spores were the kind gift of Prof. K. Isobe, Nihon University.
subjected to PCR of a 10 Wl reaction mixture. The roots of the mycorrhizal plants were washed and frozen until use. The roots, equivalent to 12 cm for onion or 50 cm for bahia grass and white clover mixture, were subjected to each batch of DNA extraction. The root samples did not contain AMF spores when examined under a dissecting microscope. Sections of the onion leaves (1 cm long) were also included in the experiment as a negative control. Plant materials were soaked in 0.5% Tween 20 solution and sonicated for 2 min, then rinsed ¢ve times in cold sterilized water by vortexing. The materials were cut in 0.5^2-mm lengths with a pair of sterilized scissors. DNeasy plant mini kit (Qiagen) was employed for the DNA extraction procedure. Finally, DNA was extracted twice with 25 Wl of the extraction bu¡er. 2.3. PCR To ascertain the performance of the DNA preparations as templates, we ran a PCR with the primers pair of ITS1 and ITS4  before the preparations were subjected to PCR with other primers. A DNA sequence speci¢c for AMF was ampli¢ed by semi-nested PCR with the primer sets VANS1 and AM1-2 followed by VANS1 and NS21  from the root DNA preparations. The primer AM1-2 (5P-GTT TCC CGT AAG CGC CGA A-3P) was designed by modifying AM1  to match better with the DNA sequence of Gigaspora sp. The PCR conditions were identical to the universal program, as described below, in the ¢rst series of the PCR but the annealing temperature was elevated to 58‡C in the semi-nested PCR. The preparations giving a 547-bp band were subjected to PCR with M13 mini-satellite and 639R primers. The genomic DNA fragments of G. margarita MAFF520054 were randomly ampli¢ed by RAPD with commercially available 12-mer oligonucleotides (BEX). Five sequences with more than 500 bp were taken into consideration. We designed a pair of oligonucleotide primers for each sequence. These primers were expected to anneal with genomic DNA of G. margarita. In addition to them, ITS1, ITS4 and (GACA)4  primers were randomly combined with each other or with the M13 mini-satellite primer. We employed a universal program: 94‡C for 1 min, 48‡C for 1 min and 72‡C for 1 min in 32 cycles after 3-min runs of each step three times. The ¢nal elongation step was extended for another 4 min at 72‡C. PCR products were electrophoresed with 1^3% agarose gels to obtain a suitable resolution. 2.4. DNA sequencing Ampli¢ed DNA were extracted from agarose gel (Seakem GTG, Takara) slices by either Easy Trap (Takara) or the L-agarase method. Puri¢ed DNA was treated with Taq polymerase (Takara) to add an adenosine overhang on its termini then ligated to pCR 2.1 with TA cloning kit
FEMSLE 10511 26-6-02
K. Yokoyama et al. / FEMS Microbiology Letters 212 (2002) 171^175
Fig. 1. The speci¢c detection of G. margarita MAFF520054 and CK. Lane 1, PX174 HaeIII fragments; lane 2, MAFF520054; lanes 3^5, CK ; lane 6, Ni-A ; lane 7, MAFF520052. a: PCR with primers, M13 mini-satellite and 639R. b: Southern hybridization with 230PBC. That one of the DNA preparations from G. margarita CK (lane 4) did not act as the template for PCR was con¢rmed with the ITS1 and ITS4 primer set.
(Amersham Pharmacia Biotech). DNA sequences were determined with ABI Prism Big Dye Terminator cycle sequencing ready reaction kit and ABI 310 DNA sequencer (PE Biosystems) in the Center for Gene Research of Yamaguchi University. 2.5. Southern hybridization The oligonucleotide was labeled by ECL 3P-oligo labeling kit (Amersham Pharmacia Biotech). After electrophoresis, PCR products were transferred to a piece of Hybond Nþ membrane by capillary action. Detection of positive signals was according to ECL 3P-oligo detection kit. The probe was hybridized at 56‡C for 2^5 h, then washed twice in 0.1USSC/0.1% SDS at 60‡C for 20 min. Other conditions were according to the supplier’s manual. The DIG oligonucleotide tailing kit and DIG luminescent detection kit (Roche Diagnostics), respectively, were used in place of the probing and detecting kit in the experiments on DNA preparations from plant roots.
3. Results and discussion Of 13 PCR primers tested, 639R (5P-TAC CTA ATG
CCA AGA TGA C-3P) produced a 235-bp band speci¢c for G. margarita MAFF520054 and CK in combination with the M13 mini-satellite primer (Fig. 1a). This primer set did not produce bands from DNA preparations other than these isolates. Other primer sets tested did not amplify such a product (data not shown). The results were reproducible with the DNA preparations of di¡erent spores of each isolate. The DNA sequence diagnostic for G. margarita MAFF520054 and CK was deposited in the GenBank/EMBL/DDBJ databases (accession number AB052750). The sequence of the diagnostic product was searched for homology by the FASTA program in the EMBL/GenBank/DDBJ databases. We chose an area that had rarely matched with the published sequences for designing an oligonucleotide probe, 230PBC (5P-CTA ACC ATT TAT CGC ATT TTT CGC AGT AGC GAG TGT ATT GCC AAG-3P). The probe, 230PBC, hybridized only with the PCR products from both G. margarita MAFF520054 and CK (Fig. 1b). No PCR product positively reacting with the probe was obtained from the DNA of the other isolates tested even in di¡erent sizes. The same was true for the other fungi tested (data not shown). We tested the e⁄ciency of the protocol to detect the hyphal DNA of G. margarita MAFF520054 in plant roots. The semi-nested PCR clearly detected the AMF colonizing in
Fig. 2. The isolate-speci¢c detection of G. margarita MAFF520054 in AMF-colonized plant roots. a: AMF DNA in the preparations from plant roots was detected by the semi-nested PCR with the primer set of VANS1 and AM1-2 followed by VANS1 and NS21. b: A portion of each preparation shown in panel a was subjected to ampli¢cation with the primer set of M13 mini-satellite and 639R followed by probing with 230PBC. Lane 1, PX174/ HaeIII digest; lane 2, negative control; lane 3, G. margarita MAFF520054 sporal DNA; lanes 4, 5, onion roots colonized by G. margarita MAFF520054; lanes 6, 7, onion leaves colonized by G. margarita MAFF520054; lanes 8, 9, bahia grass and white clover roots colonized by G. margarita sp. Ni-A; lanes 10, 11, bahia grass and white clover roots colonized by G. margarita MAFF520052; lanes 12, 13, bahia grass and white clover roots colonized by G. rosea MAFF520062; lane 14, PX174/HaeIII digest.
FEMSLE 10511 26-6-02
K. Yokoyama et al. / FEMS Microbiology Letters 212 (2002) 171^175
Fig. 3. The distribution of the loci homologous to eucaryotic satellite sequences.
roots (Fig. 2a). Among the preparations containing fungal DNA, the primer set ampli¢ed the diagnostic sequence only from onion roots colonized by G. margarita MAFF520054 (Fig. 2b). This is the ¢rst study on the isolate-speci¢c identi¢cation of G. margarita by using a single DNA sequence as a diagnostic molecular marker. By focusing on the sequence, we could deal with not only spores (Fig. 1) but also plant roots colonized by the AMF of interest (Fig. 2). This may have a great advantage in ecological studies of AMF introduced into the environment compared to methods based on the polymorphism of PCR products. Recently, the isolate-speci¢c identi¢cation of Glomus sp. from plant roots was carried out by means of single-stranded conformation polymorphisms of PCR products from the D2 region of the large subunit rRNA gene . As in the case of ITSs, the intra-isolate variation of the D2 sequences lowered the speci¢city of the sequence as a molecular marker for a Gl. coronatus isolate . For tracing the fate of G. margarita CK in environments, in contrast, the detection of the diagnostic sequence is a quite simple and reliable method. We examined the relationship of the sequence to the highly repeated satellite sequence (SC1) of Scutellospora castanea . The homology of the two sequences was lower than 50%. The speci¢c primers for SC1, SC1-1 and SC1-2 , did not amplify any sequence from G. margarita MAFF520054 (data not shown). Therefore, it does not seem to correlate to SC1. The homology search of the DNA sequence of the diagnostic product from G. margarita MAFF520054 showed that three loci up to 33 bp were highly homologous to the sequences contained in repetitive elements such as Alu and L1 families. Locus 1 or 3 contains trinucleotide repeats, CAA or CAT, respectively (Fig. 3). This suggested that the 235-bp product was ampli¢ed from one of the satellite sequences. The stability of the sequence should be considered because repetitive sequences like satellites, especially inverted ones, have been known to act as ‘hot spots’ for genome rearrangement [18^20]. If the diagnostic sequence found in this study were located in a ‘hot spot’, such an ‘unstable’ genetic marker might disappear after several cycles of DNA
replication. Ze¤ze¤ et al.  showed genetic variation in M13 mini-satellite-primed RAPD among spores after a cycle of single-spore cultures of G. margarita BEG34. This could be explained by two putative mechanisms: DNA rearrangement and karyotype shift as predicted by Lanfranco et al. . The present diagnostic sequence was detected from both CK and MAFF520054 (Fig. 1) that were cultured independently for 8 years (see footnote to Table 1). The persistence of the sequence was also evident even in the hyphal DNA in the symbiotic phase (Fig. 2). These facts provide strong evidence for the stability of the sequence at a practically acceptable level. The sequence may be conserved in the majority of nuclei. In conclusion, the diagnostic sequence serves as a powerful molecular marker to identify the genetic lineage of G. margarita MAFF520054 and CK regardless of generation and sampling location. We have begun to apply the method, with morphological and cell-physiological identi¢cation techniques for Gigaspora spores (unpublished data), to the ecological study of the isolate in the AMF-inoculated area of Mt. Fugendake.
Acknowledgements This study was funded in part by a grant from the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), Bio-oriented Technology Research Advancement Institution, Japan. The authors are grateful to the sta¡ members of the Center for Gene Research of Yamaguchi University for DNA sequencing. We also thank Dr. Wu Chi-Guang who allowed us to use his isolate SM478 through the kindness of Dr. T. Isobe. The commercial inoculum ‘Cerakinkong’ was kindly donated by Central Glass Co. Ltd.
References  Smith, S.E. and Read, D.J. (1997) Mineral nutrition, heavy metal accumulation and water relations of VA mycorrhizal plants. In: Mycorrhizal Symbiosis, 2nd edn. (Smith, S.E. and Read, D.J., Eds.), pp. 126^160. Academic Press, San Diego, CA.  Miller, R.M. and Jastrow, J.D. (1992) The application of VA mycorrhizae to ecosystem restoration and reclamation. In: Mycorrhizal Functioning: An Integrative Plant-Fungal Process (Allen, M.F., Ed.), pp. 438^467. Chapman and Hall, New York.  Marumoto, T., Kohno, N., Ezaki, T. and Okabe, H. (1999) Reforestation of volcanic devastated land using the symbiosis with mycorrhizal fungi (in Japanese with English summary). Soil Microorganisms 55, 81^90.  Marumoto, T., Okabe, H., Ezaki, T., Nishiyama, M. and Yamamoto, K. (1996) Application of symbiotic microorganisms to soil conservation and reforestation. BioJapan ’96 Symposium Proceedings, pp. 242^250.  Bago, B., Bentivenga, S.P., Brenac, V., Dodd, J.C., Piche, Y. and Simon, L. (1998) Molecular analysis of Gigaspora (Glomales, Gigasporaceae). New Phytol. 139, 581^588.
FEMSLE 10511 26-6-02
K. Yokoyama et al. / FEMS Microbiology Letters 212 (2002) 171^175  Lanfranco, L., Delpero, M. and Bonfante, P. (1999) Intrasporal variability of ribosomal sequences in the endomycorrhizal fungus Gigaspora margarita. Mol. Ecol. 8, 37^45.  Ze¤ze¤, A., Sulstyowati, E., Ophel-Keller, K., Barker, S. and Smity, S. (1997) Intersporal genetic variation of Gigaspora margarita, a vesicular arbuscular mycorrizal fungus, revealed by M13 minisatelliteprimed PCR. Appl. Environ. Microbiol. 63, 676^678.  Clapp, J.P., Rodriquez, A. and Dodd, J.C. (2001) Inter- and intraisolate rRNA large subunit variation in Glomus coronatum spores. New Phytol. 149, 539^554.  Schu«ssler, A. (1999) Glomales SSU rRNA gene diversity. New Phytol. 144, 205^207.  Saito, M. (2000) Use of VA mycorrhizal fungi. In: Biseibutsu no shizaika: Kenkyu no saizensen (Microorganisms Resources: Its Characterization and Utilization, Suzui, T. et al., Eds.), pp 55^70. Soft Science, Tokyo (in Japanese).  Saito, M. (1995) Enzyme activites of the internal hyphae and germinated spores of an arbuscular mycorrhizal fungus, Gigaspora margarita Becker and Hall. New Phytol. 129, 425^431.  White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Ampli¢cation and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols. A Guide to Methods and Applications (Innis, M.A. et al., Eds.), pp. 315^322. Academic Press, San Diego, CA.  Simon, L., Lalonde, M. and Bruns, T.D. (1992) Speci¢c ampli¢cation of 18S fungal ribosomal genes from vesicular-arbuscular endomycor-
rhizal fungi colonizing roots. Appl. Environ. Microbiol. 58, 291^ 295. Helgason, T., Daniell, T.J., Husband, R., Fitter, A.H. and Young, J.P.W. (1998) Ploughing up the wood-wide web? Nature 394, 431. Hering, O. and Nirenberg, H. (1995) Di¡erentiation of Fusarium sambucinum Fuckel sensu lato and related species by RAPD PCR. Mycopathologia 129, 159^164. Ze¤ze¤, A., Hosny, M., Gianinazzi-Pearson, V. and Dulieu, H. (1996) Characterization of a highly repeated DNA sequence (SC1) from the arbuscular mycorrhizal fungus Scutellospora castanea and its detection in plant. Appl. Environ. Microbiol. 62, 2443^2448. KjZller, R. and Rosendahl, S. (2000) Detection of arbuscular mycorrhizal fungi (Glomales) in roots by nested PCR and SSCP (single stranded conformation polymorphism). Plant Soil 226, 189^196. Field, D. and Wills, C. (1998) Abundant microsatellite polymorphism in Saccharomyces cerevisiae, and di¡erent distributions of microsatellites in eight prokaryotes and S. cerevisiae, result from strong mutation pressures and a variety of selective forces. Proc. Natl. Acad. Sci. USA 95, 1647^1652. Gordenin, D.A., Lovachev, K.S., Degtyareva, N.P., Malkova, A.L., Perkins, E. and Resnick, M.A. (1993) Inverted DNA repeats: a source of eukaryotic genomic instability. Mol. Cell. Biol. 13, 5315^ 5322. Van Belkum, A., Scherer, S., Van Alphen, L. and Verbrugh, H. (1998) Short-sequence DNA repeats in prokaryotic genomes. Microbiol. Mol. Biol. Rev. 62, 275^293.
FEMSLE 10511 26-6-02