Specific Polymerase Chain Reaction-Based Assay for the Identification of the Arbuscular Mycorrhizal Fungus Glomus intraradices
Specific primers and Polymerase Chain Reaction (PCR)
assays that identify Arbuscular Mycorrhizal (AM) fungi Glomus intraradices
were developed. Monoxenic cultures of fungi G. intraradices and
Gigaspora gigantea in association with Ri T-DNA transformed carrot
roots were established in order to obtain fungal DNA free of host and
others contaminants. RAPD analysis using 10 AM fungi from genera Glomus,
Gigaspora and Acaulospora allowed the determination of two
amplified fragments that were specific to G. intraradices. The
DNA fragments were cloned, sequenced and subsequently used to design SCAR
species-specific primers. A set of primers, GIN630F and GIN630R,
drove the amplification of a 630 bp fragment specific for G. intraradices,
which was absent when DNA of other AM fungi or plants were used as templates.
The assay allowed the detection of G. intraradices in colonized
roots of carrot. The SCAR-based protocol described here may be a tool
of great value in studies of Glomeromycota`s molecular systematic and
The Phylum Glomeromycota represents an interesting biological group
because all its members fall in one of two types of symbiotic mutualistic
associations. One of them is constituted for the monotypic family Geosiphonaceae
whose only member Geosiphon pyriformis forms a peculiar symbiosis
named endocytobiosis with cyanobacteria belonging to the genus Nostoc
(Gehrig et al., 1996). The other symbiotic type is the very common
arbuscular mycorrhiza, which is formed by the rest of the fungal species.
Recent data establish that AM is associated to the roots of 80% of plant
species, which indicates the complexity in origin, evolution and diversification
of this group (Wang and Qui, 2006).
Traditionally, the taxonomic identification of AM fungi has been based
on the morphological features of spores. Undoubtedly these structures
contain important taxonomic information (Sieverding and Oehl, 2006) and
the morphological approach to the identification of species although useful
to estimate biodiversity it is also limited and controversial. The spores
are persistence structures basically formed under unfavorable environmental
conditions; their presence may be resultant of past events and not to
show prevalent conditions during the sampled period (Lovelock and Ewel,
2005). Moreover, because most of the spores are produced outside the roots
it could lead to an underestimation of the biodiversity of AM fungi on
field samples. In order to solve these problems recent research has focused
on the selection and implementation of tools to facilitate an accurate
and reproducible identification of AM fungus (Reddy et al., 2005).
Molecular markers have been specifically developed for the detection
and identification of pathogens with impressive accuracy. Furthermore,
they have been successfully used in detecting fungi (Nazar et al.,
1991; Simon et al., 1992; Berbee and Taylor, 1995). Nevertheless,
the sequences used to design the assays have been targeted to ribosomal
genes and consequently, they are universal and in some cases no species-specific
(Schübler et al., 2001a), but genera or superior taxonomic
ranks. Studies on Glomus intraradices have proven that the ITS
region of the ribosomal genes is variable; therefore this fact compromises
the specificity of the test (Jansa et al., 2002; Reddy et al.,
An interesting strategy to develop species-specific molecular markers
is based on the isolation, further sequencing of DNA fragments amplified
by RAPD PCR and the use of designed primers to specific target sequences
that may be unique for a species. These molecular markers are known as
Sequence Characterized Amplified Region or SCARs (Paran and Michelmore,
1993). This approach has been applied to the identification of different
species of fungi, including some ectomycorrhizal (Gandeboeuf et al.,
1997). In contrast, this is not an easy task for MA fungi due to the mutualistic
symbiosis with the plant, which makes difficult the isolation of uncontaminated
DNA. The use of in vitro root-organ cultures (Bécard and
Piché, 1992) may overcome this obstacle; however, there have been
very few species on which this system has been successful obtained. The
objective of this research was to establish monoxenic cultures of the
fungi G. intraradices and Gigaspora gigantea to obtain pure
DNA to further develop a specific test designed to identify the species
by the use of a SCAR marker. This method would help to determinate accurately
the identity of these species in natural ecosystems; moreover, it may
contribute to the estimation of the relative abundance of the fungus into
MATERIALS AND METHODS
Fungal material and strains: The AM fungi Glomus intraradices
Schenck and Smith strain 0046TLX03, G. intraradices strain BEG
144, G. caledonium (Nicol. and Gerd.) Trappe and Gerd. strain BEG
20, G. claroideum Schenck and Smith strain 0003TLX01, G. etunicatum
Becker and Gerdemann strain 0004MOR01, G. fasciculatum (Thaxter)
Gerd. and Trappe emend. Walker and Koske, G. mosseae (Nicol. and
Gerd.), Gigaspora gigantea (Nicol. and Gerd.) Gerd and Trappe strain
0033TLX06, G. margarita Becker and Hall strain 0036TLX06, Scutellospora
dipurpurasens Morton and Koske strain 0020TLX06, S. pellucida
(Nicol. and Schenck) Walker and Sanders strain 0018TLX06, Acaulospora
lacunosa Morton strain BEG 78, A. laevis Gerdemann and Trappe
strain BEG 13, A. longula Spain and Schenck strain BEG 8 and A.
spinosa Walker and Trappe strain 0039TLX01 were obtained in pure pot
culture from AM fungi collections of the CICB from the Universidad Autónoma
de Tlaxcala (TLX and MOR codes) and the European Bank of Glomales (BEG
codes) and kept in soil at 4 °C.
Production of hairy roots and monoxenic cultures: The strains
of Agrobacterium rhizogenes LBA 9402 and agropine type AR12 were
used for DNA transformation of carrot (Daucus carota L.) to which
the binary vector pBI121 was inserted. Both strains harbored the wild
type Ri plasmid. Bacterial cells were grown at 28 °C in YEB (10 g
L-1 yeast extract, 5 g L-1 beef extract, 5 g L-1
peptone, 5 g L-1 saccharose, 0.49 g L-1 MgSO4.7H2O
supplemented with 50 mg L-1 of riphampycin plus 100 mg L-1
of kanamycin) to an OD 600 of 0.4. The infection process was performed
on transversal disks of carrot co-cultivated with 0.5 mL of the bacterial
suspension (OD 600 = 1) for 24 h in the darkness. Tissues were transferred
to medium MS (Murashige and Skoog, 1962) supplemented with 500 mg L-1
of cephotaxyme and incubated at 25 °C. The bacteria-free hairy roots
were removed and transferred to minimum media (M) (Bécard and Piché,
1992) and maintained as clones. Spores of G. intraradices and G.
gigantea were extracted by wet sieving and surface sterilized by treatment
with 2% Chloramine T plus 0.1% Tween 20 for 15 min at 4 °C, vacuum
was applied and the procedure was repeated twice. The spores were transferred
to an antibiotic solution (100 mg L-1 of gentamycin sulphate,
2000 mg L-1 streptomycin sulphate) for 24 h at 4 °C. After
this treatment, the spores were rinsed with double distilled water and
gently distributed over a plate containing M medium and incubated at 25 °C for 3-4 days. Germinated spores were then transferred to the proximity
of the transformed Ri T-DNA carrot roots having active growth to stimulate
the colonization. In order to obtain a massive production of spores, the
dual system developed by St-Arnaud et al. (1996) was used.
PCR analysis of transformed roots: Genomic DNA of the different
root clones was extracted according to Doyle and Doyle (1990). Twenty
nanogram of genomic DNA was used in a reaction volume of 25 μL containing
15 mM Tris-HCl (pH 8), 0.1% Triton X-100, 50 mM KCl, 1.5 mM MgCl2,
100 μM of each dNTP, 2.5 U de Taq DNA polymerase (Promega, Madison,
WI) and 10 pmol of each of the primers ROLB1 (5´ATG GAT CCC AAA TTG CTA
TTC CTT CCA CGA), ROLB2 (5´ TTA GGC TTC TTT CTT CAG GTT TAC TGC AGC),
VIRD1 (5´ATG TCG CAA GGA CGT AAG CCCA) and VIRD2 (5´GGA GTC TTT CAG CAT
GGA GCA A) (Hamill et al., 1991). These primers drove the amplification
of 780 bp and 450 bp fragments from the genes rol B and vir D1
of Agrobacterium, respectively. The amplification was performed using
an MJ Research PT-100 thermocycler (MJ Research, Watertown, MA) using
the following profile: an initial denaturation step of 94 °C/3 min,
followed by a 25 cycles of 94 °C/1 min, 55 °C/1 min, 72 °C/1.5
min and a final extension step of 72 °C/7 min then held at 4 °C.
The amplified products were fractionated in 1.2% agarose gels.
Fungal DNA extraction and RAPD analysis: Spores of G. intraradices
and G. gigantea were obtained from the distal part of the monoxenic
culture by dissolving Phytagel in 10 mM sodium citrate and crushed in
40 μL of TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA) and heated
to 95 °C for 20 min in 40 μL of 30% w/v Chelex-100 resin (BioRad).
Genomic DNA was separated from cellular debris by centrifugation at 14,000
rpm for 1 min; the resulting extract was diluted and used immediately
for use in the PCR assays. Spores of the other AM fungi species were isolated
from the soil of propagation pots using the wet sieving and decantation
process (Gerdemann and Nicolson, 1963) and were later surface sterilized
by treatment with 2% Chloramine T and antibiotics solutions. DNA extraction
of these fungal species was conducted according to Lee and Taylor (1991).
RAPD reactions were performed in a total volume of 50 μL containing
10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.1% Triton
X-100, 100 μm each of dNTP (Invitrogen, Carslbad, CA), 0.5 μM
each random 10-mer (Bio-Synthesis Co., Lewisville, TX), 20 ng of genomic
DNA and 2 units of Taq DNA polymerase (Promega, Madison, WI). Thermal
Cycler MJ Research PTC-100 was programmed for an initial denaturation
at 94 °C/3 min, followed by 35 cycles of 94 °C/1 min, 36 °C/1
min, 72 °C/2 min and a final extension at 72 °C/7 min. Amplification
products were separated in 1.4% agarose gels and stained with ethidium
bromide; the DNA bands were visualized under UV light and photographed.
Every experiment was performed by duplicate.
Cloning and sequencing of RAPD markers: Polymorphic RAPD fragments
amplified from G. intraradices and G. gigantea were purified
using the Wizard PCR Preps kit (Promega, Madison, WI) and cloned into
the pGEM-T Easy vector (Promega, Madison, WI) as recommended by the manufacturer.
The recombinant vectors were used to transform competent Escherichia
coli cells DH5α. The selection of recombinants was performed
by PCR using white colonies directly as source of template DNA that was
amplified utilizing the RAPD primers employed for the first amplification.
Plasmid DNA from recombinant colonies was purified using the High Pure
Plasmid Isolation kit (Boehringer Ingelheim, GmbH, Germany). Insert size
was verified by EcoR I digestions, followed by 1.5% agarose gel
fractionation. The complete sequence of each cloned fragment was obtained
by the use of an automated sequencing robot ABI PRISM 377 (Applied Biosystems,
Foster City, CA). DNA sequences were compared by alignment by the DNASIS
V 2.0 program for the Macintosh system. DNA sequences for G. intraradices
were deposited to the GenBank databases.
Design of SCAR primers and PCR conditions: The search for DNA
similarities was performed using the BLAST and BLASTX programs from the
NCBI network service (Altschul et al., 1997). For each RAPD fragment
several SCAR primers were designed using the complete DNA sequence for
the non-occurrence of secondary structures. The absence of cross hybridization
was checked using the PrimerSelect 3.11 software For Windows (DNAStar,
Lasergene, Madison, WI). Primers were designed with GC content of 50-60%
and synthesized by Invitrogen. The PCR specific assays were carried out
in a total volume of 25 μL containing approximately 5 ng DNA, 1X
reaction buffer (Promega), 200 μM of each dNTP (Invitrogen), 1.5
mM MgCl2, 20 pmol SCAR primers, 2.5 U Taq polymerase (Promega).
Amplifications with SCAR primers were performed in the MJ-Research PTC-100
as follows: initial denaturation at 94 °C/2 min, followed by 30 cycles
at 94 °C/1 min, 53-58 °C/1 min, 72 °C/1 min and then a final
extension at 72 °C/7 min. As proper controls, DNA of every species
was amplified by using the universal ITS primers ITS1 and ITS4 as described
by White et al. (1990). Amplification products were fractionated
by gel electrophoresis in 1.4% agarose gels in 1X TAE buffer (40 mM Tris-acetate,
1 mM EDTA, pH 8), stained with ethidium bromide and visualized under UV
RESULTS AND DISCUSSION
Carrot roots transformation and monoxenic culture: Spores are
the only differentiated biologically structures of AM fungi that could
be studied outside the host. Even though spores need to be disinfected
to remove contaminants, the presence of multiple nuclei makes the interpretation
of the RAPD analysis complex. In order to avoid that, we established monoxenic
cultures of G. intraradices and G. gigantea. The culture
system requires the maintenance of roots in an autonomous and undefined
stage of continuous growth under controlled conditions. In this research,
we used two types of A. rhizogenes strains such as LBA9402 and
AR12 for the production of transformed roots (Fig. 1A).
Both strains transfer their T-DNA and induced hairy roots free of bacterial
cells. Consequently, roots obtained with strain LBA9402 agropine-manopine
type resulted in better vigor and branching growth compared with roots
Monoxenic culture of Glomus
intraradices with transformed roots. (A) Ri T-DNA transformed
roots growth on carrot disc. (B) Dual culture of carrot root and
fungus in a two compartment Petri dish: –S, M medium minus
saccharose; +S, plus saccharose. (C) Massive production of spores
by G. intraradices grown in M medium and (D) Details of vegetative
spores of G. intraradices
strain AR12. This may be due to a higher accumulation of auxins in roots
(Nin et al., 1997). On the other hand, the disinfection method
of spores reduced contamination to a 10% with a germination percentage
of 70% of both species in agar-water. There are reports about several
factors influence the in vitro germination rates of the spores,
such as radical exudates, flavonoids, pH conditions, presence of CO2,
low temperature storage and physiological status of the spore (Bécard
et al., 1992; Chabot et al., 1992; Poulin et al.,
1993; Juge et al., 2002). In our study germination was successful
probably due to the optimal physiological situation of the spores or the
disinfection process itself without need to add external components. Four
weeks later of the in vitro infection initiation, a massive development
of mycelium was observed on the medium accompanied by formation of Branched
Absorbing Structures (BAS) as described by Bago et al. (1998).
The spore formation under the dual system (Fig. 1B)
as described by St-Arnaud et al. (1996) started four months posterior
to the inoculation of G. intraradices with an average of 800 spores
per dish (Fig. 1C, D), whereas G.
gigantea only produced 40 spores per dish during the same period.
RAPD analysis and PCR detection: Only three of the twenty five
primers produced consistent amplification patterns. Primer 5´-TGCAGCACCG
(Bio-synthesis 70-09), which has a 70% content of G+C, drove the amplification
of two specific fragments of 950 and 650 bp for G. intraradices
(Fig. 2). These fragments were cloned and sequenced
(GenBank accession numbers AY244447 and AY244448). The terminal ends of
the sequenced fragments perfectly matched the sequence of the 10-mer used
for the PCR amplification. From all stock of AM fungi species used in
this study, only G. claroideum, G. fasciculatum, G. gigantea
and G. margarita could be amplified, all other produce no satisfactory
results, which reinforce that AM fungi DNA extraction and amplification
is a difficult task for almost all species, specially if these species
are not been propagated monoxenically.
Based on the disclosed DNA sequences, several set primers were designed,
synthesized and used to optimize PCR assay and the amplification of a
unique fragment to accurately identify G. intraradices. Primers
GIN930F (5` TGC AGC ACC GCC TCC ACC) and GIN930R
(5` TGC AGC ACC GTC GCT TGT TA) drove the amplification of an expected
930 bp fragment. This method encouraged the detection of G. intraradices
strain 0046TLX03 in fragments of infected in vitro roots of
D. carota and in vivo roots of Sorghum sp. colonized
with G. intraradices strain BEG144 (data not shown).
In addition, consistent results were obtained with primer set GIN630F
(5` GCA CCG CAA GTT AAG TAC
Random amplified polymorphic DNA
(RAPD) fingerprints using genomic DNA of mycorrhizal arbuscular
fungi. Lanes 1-2 Glomus claroideum, 3-4 G. fasciculatum,
5-6 G. intraradices 0046TLX03, 7-8 Gigaspora gigantea,
9-10 G. margarita. M 100 bp DNA ladder. Arrows indicate specie-specific
and reproducible RAPD bands of 950 and 650 bp that were converted
into SCAR markers
(A) Agarose gel electrophoresis
of PCR products obtained using the Glomus intraradices-specific
primers GIN630F/R designed in this study. (B) Agarose
gel electrophoresis of ITS 1/4 primers used as internal controls.
M 100 bp DNA ladder. Lanes (1) genomic DNA from Glomus intraradices
0046TLX03, (2) carrot root colonized with G. intraradices
BEG144, (3) G. fasciculatum, (4) G. claroideum, (5)
G. mosseae, (6) Acaulospora laevis, (7) Daucus
CCA AC) and GIN630R (5` CCG TGA TCA TGA TGT CTC AGG TT). Annealing
temperatures of 54 °C were used to produce the expected fragment of
630 bp. Lower temperatures allowed the amplification of an unspecific
fragment of 800 bp. The test proved to be highly specific to G. intraradices
and no amplified DNA was observed when genomic DNA from others AM fungi
was used as template (Fig. 3A). Controls DNA of every
species were successfully amplified by using the universal ITS primers
There are reports of PCR-based tests that have been presented as specific
for several fungi of the order Glomerales (cited as Glomales by Simon,
1996). Many of them have been developed by using ribosomal DNA as template
such as the VANS1 primer set proposed by Simon et al. (1993). Recently,
it has been demonstrated that these primers are not specific and that
primer homologous sequences are absent in at least 88 of the MA fungi
analyzed (Lloyd-MacGilp et al., 1996; Schübler et al.,
2001b; Sanders, 2003). Moreover, for G. mosseae alone, at least
23 sequences with homology varying from 66 to 98% have been reported by
Antoniolli et al. (2000).
The nucleotide sequences corresponding to SCAR fragments reported here
may be of vital importance for the development of quantitative PCR assays
for the study of G. intraradices. The distribution of the AM fungus
in the host roots could be now elucidated and consequently a better understanding
of spore abundance in the soil and infection process.
This research has successfully demonstrated the use of monoxenic cultivation
of two AM fungi and its further use to analyze RAPD patterns in order
to develop specific PCR tests based on the selection of unique SCAR sequences
for different species. Furthermore, the use of this approach contributed
to the accurate detection and identification of G. intraradices
on in vitro and in vivo tests. The test offers promising
use for further studies on the ecology of the mycorrhiza-plant interaction
This study was supported by funds from Consejo Nacional de Ciencia
y Tecnología (CONACyT) grant number 26357 B. We thank Juan Carlos
Ochoa-Sánchez, Magali Hernandez-Valencia and Juan Enrique Cortés-Valle
for his technical support.
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