Characterisation and identification of arbuscular mycorrhizal fungi
species by PCR/RFLP analysis of the rDNA internal transcribed spacer
Annals of Microbiology, 58 (2) 341-344 (2008)
Adália Cavalcanti do Espírito Santo MERGULHÃO1*, Márcia Vanusa da SILVA2, Márcia do Vale Barreto FIGUEIREDO1,
Hélio Almeida BURITY1, Leonor Costa MAIA2
1Instituto Agronômico de Pernanbuco - IPA, Laboratórios de Genoma e Biologia do Solo, Av. General San Martin, 1371,
Bongi, 50761-000, Recife, PE; 2Universidade Federal de Pernambuco - UFPE, Departamentos de Bioquímica e Micologia,
Av. Prof. Nelson Chaves s/n, Cidade Universitária, 50670-420, Recife, PE, Brasil
Received 4 September 2007 / Accepted 29 February 2008
Abstract - The polymerase chain reaction coupled with restriction fragment length polymorphism analysis (PCR/RFLP)
was applied to distinguish arbuscular mycorrhizal fungi (AMF) species from an impacted semiarid soil. The ITS1-5.8S-
ITS2 region was amplified with the primers ITS1 and ITS4 and the products of amplification were digested with the
restriction enzymes HinfI, MboI and AluI. The obtained banding pattern, except for the AluI, allowed the distinction at
the molecular level of the AMF species: Paraglomus occultum, Glomus mosseae, Glomus intraradices and Glomus etuni-
catum. The results showed that this technique has a potential to be used as a marker to differentiate AMF species with
high phylogenetic affinity.
Key words: AM fungi, ITS, Glomus and Paraglomus species.
Mycorrhizal fungi occur in highly diverse communi-
ties, representing the widest association between
plants and fungi found in nature (Dickie and
FitzJohn, 2007). Mycorrhized plants show higher
capacity of nutrient uptake, and resistance to biotic
and abiotic stresses than non mycorrhized plants
(Smith and Read, 1997; Renker et al., 2003). The
identification and characterisation of these fungi are
the first steps for study of this association. The mor-
phological identification of arbuscular mycorrhizal
fungi (AMF) species relies mainly on the characteri-
stics of soil-borne spores and it also difficult in envi-
ronmental samples due to developmental variation
or parasitism of spores. Because factors controlling
sporulation and morphological differentiation of AMF
taxa are poorly understood, and occurrence of taxa
in the spore community and in roots do not neces-
sarily coincide, community studies based solely on
spores appear to be of little ecological relevance
(Gamper and Leuchtmann, 2007).
Sporocarp production is only loosely related to
belowground community patterns and many fungi
produce cryptic and/or hypogeous sporocarps.
Because of these difficulties, there has been an
increasing reliance on molecular methods for identi-
fying species from belowground structures (Horton
and Bruns, 2001; Dickie and FitzJohn, 2007).
Many molecular techniques have been used to study
the AMF, improving the knowledge on phylogenetic,
cytogenetic, functional and ecological aspects and
the selection of one or more techniques is a function
of the research objective (Renker et al., 2003,
2006; Dickie and FitzJohn, 2007; Gamper and
Approaches polymerase chain reaction (PCR) -
based on seem to offer the best current prospects
for detecting most of the AM fungi present in an
ecosystem. However, the results of PCR-based ana-
lyses may be based by differential amplification (or
non-amplification) of target DNA at different con-
centrations, bearing the risk that PCR analyses
focusing exclusively on spores, roots or extra-radi-
cal mycelium, could will fail to detect some impor-
tant components of the AM community that are
weakly represented in the sampled material
(Hempel et al., 2007).
PCR/RFLP has been applied in mycorrhizal rese-
arch to identify strains of introduced or naturally
occurring mycorrhizal fungi or of economically
important species and also to differentiate and iden-
tify mycorrhizal symbionts unambiguously. The
PCR/RFLP polymorphism of the ITS region is gene-
rally regarded as appropriate to differentiate AMF at
* Corresponding author. E-mail: email@example.com
A. Cavalcanti do Espírito Santo MERGULHÃO et al.
the species level (Renker et al., 2003, 2006;
Hempel et al., 2007).
The fragment of rDNA which will be amplified for
analysis of intra-specific diversity or between
groups of AMF isolates with high phylogenetic affini-
ty should include the internally transcribed spacer
(ITS1, 5.8S, ITS2), a region that presents high
variability in both, the composition of bases and in
the fragment size (Grifoni et al., 1995; Renker et
al., 2003, 2006; Gamper and Leuchtmann, 2007).
Semiarid soils have a high diversity of plant and
AMF but the identification of AMF which based on
morphological character of spores is difficult, main-
ly when spores are in low amounts as occurs in
impacted areas. Thus alternative techniques should
be employed to allow identification. Further studies
with AMF can be useful for helping establishment of
plants and revegetation of impacted semiarid areas.
The aim of this study was to differentiate betwe-
en AMF species with high phylogenetic affinity by
comparison of their ITS (ITS1, 5.8S, ITS2) restric-
MATERIALS AND METHODS
Soil samples were collected from an impacted gyp-
site mining area in Araripina, Pernambuco State.
Spores of AMF, present in the samples, were multi-
plied in successive cycles of greenhouse pot, cultu-
ring in soil with sorghum and peanut as hosts. After
three months, spores of AMF, extracted from soil by
wet sieving (Gerdemann and Nicolson, 1963) and
sucrose centrifugation (Jenkins, 1964) were selec-
ted on a stereomicroscope. Similar spores were
grouped, mounted its glass slides with PVLG or
Melzer reagent + PVLG (1:1). The species were iden-
tificated with the help of light microscope (Schenck and
Pérez, 1990; http://invam.caf.wvu.edu).
After identification, spores of four species
(Paraglomus occultum Morton & Redecker, Glomus
mosseae Gerdemann & Trappe, Glomus etunicatum
Becker & Gerdemann and Glomus intraradices
Schenck & Smith) were separated and the DNA was
extracted using the method described by Lanfranco
et al. (2001). Approximately 150 to 200 spores of
each AMF species were sonicated (3 to 4 cycles of
30 s) and broken with the aid of a micropestle in 50
µL of 1X reaction buffer (LABTRAD do Brasil LTDA).
With 10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.1 mM
MgCl2, they were incubated at 95 ºC for 15 min and
centrifuged at 10.000 x g for 5 min. The superna-
tant was stored at -20 ºC.
The PCR reaction consisted of 3 µL of total DNA
added to the mix to receive a final volume of 25 µL,
containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.1
mM MgCl2, 10 mM of each dNTPs, 25 ng of each pri-
mer ITS1 (5’TCCGTAGGTGAACCTGCGG3’) and ITS4
(5’TCCTCCGCTTATTGATATC3’) (White et al., 1990)
and 2 units of Taq DNA polymerase (Invitrogen).
The amplification reactions were carried out in ter-
mocycler M.J. Research, Inc. (USA) PCR, model PTC
100, using a program with initial denaturation at 95
ºC 3 min, forty cycles of 95 ºC 45 s, 50 ºC 45 s, 72
ºC 1 min and a final extension at 72 ºC for 5 min.
After the thermal cycling, an aliquot of the PCR pro-
ducts was digested with the restriction enzymes
MboI, HinfI and AluI, according to the recommenda-
tion of the manufacturer (Invitrogen). The restric-
tion products were separated by electrophoresis, at
3V/cm for 2 h, in 1.5% agarose gel, in TBE buffer
which was stained with Sybr Gold. The banding pat-
terns generated with the restriction of the ITS
region were observed and photographed in a photo-
documentation system with UV light. The 100 bp
DNA Ladder (Invitrogen) was used as the fragment
RESULTS AND DISCUSSION
Using the primers ITS1 and ITS4, the amplified pro-
duct of the locus ITS1-5.8S-ITS2 of the rDNA pre-
sented fragments of about 600 bp for G. mosseae
and G. etunicatum and 700 bp for P. occultum and
G. intraradices (Fig. 1). Similar results were obser-
ved by Redecker et al. (1997), who mentioned that
the amplification products generated for species of
Glomus presented approximately 600 bp. The
amplified products of the ITS1 and ITS2 regions
from the rDNA, using the ITS1 and ITS4 primers,
also generated fragments of about 600 bp for
Gigaspora margarita Becker & Hall, Gigaspora deci-
piensHall & Abbott and Scutellospora sp.
FIG. 1 - PCR amplification of the ITS regions of AMF isola-
tes using primers ITS1/ITS4. M: 100 bp DNA lad-
der, 1: Paraglomus occultum, 2: Glomus mosseae,
3: Glomus intraradices, 4: Glomus etunicatum.
Ann. Microbiol., 58 (2) 341-344 (2008)343
(Lanfranco et al., 1999). Our results showed two
polymorphic patterns: one comprising P. occultum
and G. intraradices and the other including the spe-
cies G. mosseae and G. etunicatum (Fig. 1). By
polymerase chain reaction and sequencing of the
ITS region members of seven different families and
species, groups within Glomeromycota were identi-
fied. The revealed data showed remarkable diffe-
rences in the composition of AMF taxa (Hempel et
The amplification of the ITS region allowed the
distinction of three AMF species at the generic level:
Acaulospora laevis Gerdemann & Trappe and
Glomus deserticola Trappe; Bloss & Menge using the
primer pairs ITS1/ITS2 and ITS1/ITS Harney et al.
(1997). The spores of these species have enough
variation in the ITS region, allowing their genetic
distinction in field samples (Harney et al., 1997).
Within the rDNA, the non-coding ITS displays a
higher polymorphism than the SSU and LSU (White
et al., 1990). It is routinely used to identify ectomy-
corrhizas (Buscot et al., 2000) and was demonstra-
ted to be also adequate for AMF identification
(Redecker et al., 1997, 2000; Renker et al., 2003).
In most of the studies of ITS on AM spores
ITS1/ITS4 were used (Antoniolli et al., 2000;
Hildebrandt et al., 2001; Renker et al., 2003; Dickie
and FitzJohn, 2007).
In our work the ITS1/ITS4 amplification pro-
ducts did not differentiate all the AMF at the species
level. But with the help of the restriction enzymes
HinfI and MboI digestion (180 to 590 bp) fragments
(Table 1) differentiate between the four AMF species
(Fig. 2 and 3). The restriction banding patterns sho-
wed 2 to 3 fragments per restriction enzyme, with 5
profile for the enzyme HinfI and 6 for MboI (Fig. 2
and 3). No restriction sites were observed for the
enzyme AluI (Table 1). Consequently, no variability
among these four AMF species was detected with
this enzyme. Conversely, the combination of the
restriction patterns of the other enzymes allowed
the molecular distinction of all AMF species, mainly
the profiles obtained with endonuclease MboI. The
analysis of the restriction patterns of the amplified
Walker & Sanders,
TABLE 1 - ITS length and fragment after restriction of ITS
Arbuscular mycorrhizal fungi ITS - length class
HinfI MboI AluI
Glomus etunicatum600 500
Glomus intraradices 700 320
Paraglomus occultum 700-
FIG. 2 - HinfI restriction profile of the ITS fragments ampli-
fied from AMF spores DNA. M: 100 bp DNA ladder,
1: Paraglomus occultum, 2: Glomus mosseae, 3:
Glomus intraradices, 4: Glomus etunicatum.
FIG. 3 - MboI restriction profile of the ITS fragments ampli-
fied from AMF spores DNA. M: 100 bp DNA ladder, 1:
Paraglomus occultum, 2: Glomus mosseae, 3:
Glomus intraradices, 4: Glomus etunicatum.
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A. Cavalcanti do Espírito Santo MERGULHÃO et al.
rDNA internally transcribed spacer allowed the dif-
ferentiation of the strains at the intraspecific level
due to the easily analysable and reproducible ban-
ding patterns generated from ITS region.
Different restriction patterns detected genetically
between species, confirm different species.
However, if differences are not observed, the spe-
cies may or may not be identical that depends on
the specific restriction site of the restriction enzyme
used in the fragment analysis. The lack of restriction
sites may have occurred with all AMF digested with
the AluI. Redecker et al. (1997) also observed a
lack of restriction sites for the AluI endonuclease in
a great variety of Glomus species. Glomus sp. strain
S328 is an exception. Analysis of the ITS sequence
of G. mosseae BEG12 (GenBank access nº X84232)
reported by Sanders et al. (1995) confirmed the
absence of the AluI site. A search among ITS sequences
available in GenBank (http://www.ncbi.nlm.nih.gov/)
indicated that for most
Glomeromycota, the ITS displays contair no restric-
tion site for AluI (Renker et al., 2003).
The digestion of the ITS fragment with HinfI and
MboI was effective for the inter- and intra-specific
separation of AMF, constituting an additional techni-
que to study these fungi, and may be used to resol-
ve controversies in the taxonomy of the group. The
molecular markers are as important as the morpho-
logical and physiological methods. Molecular
methods contribute to increase the knowledge of
identity and variability of the AMF. The results con-
firm that the PCR/RFLP technique, using the MboI
and HinfI enzyme, has a potential for utilization as
a marker to differentiate between species of AMF
with high phylogenetic affinity.
members of the
Thanks to Dra. Neiva Tinti de Oliveira and Dra.
Elaine Malosso for the helpfull suggestions and to
the anonymous referee by the changes suggested.
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