ORIGINAL PAPER

A method for assessing arbuscular mycorrhizal fungi groupdistribution in tree roots by intergenic transcribedsequence variation

Mervyn Shepherd Æ Linh Nguyen ÆMegan E. Jones Æ J. Doland Nichols ÆF. Lynn Carpenter

Received: 14 August 2006 / Accepted: 1 November 2006 / Published online: 8 December 2006� Springer Science+Business Media B.V. 2006

Abstract We identified five taxonomic groups of

arbuscular mycorrhizal fungi (AMF) inside roots

of young trees of six species of legumes and six

species of non-legumes from a field site in

southern Costa Rica using an AMF group-specific

PCR assay of the intergenic transcribed sequence

and 18S rRNA gene fragment. Assay specificity

was verified by cloning and sequencing represen-

tatives from four of the five AMF groups. We

found no difference in overall AMF diversity

levels between legumes and non-legumes or

between plant species. Some groups of AMF

may associate more frequently with legumes than

others, as Glomus Group A (Glomus mosseae/

intradices group) representatives were detected

more frequently in legumes than non-legumes

relative to Glomus Group B (Glomus etunicatum/

claroideum) representatives.

Keywords Acaulosporaceae � Gigasporaceae �Glomales � AMF diversity

Introduction

Studies of genetic diversity and community

structure of AMF in the rhizosphere until

recently relied upon identifications based on

morphology of spores cultured from soils. This

method is limited because the spore cultures

may not reflect the kinds of AMF that are active

inside the roots (Clapp et al. 1995). Further-

more, some recently identified lineages of

Glomales may go undetected by staining tech-

niques (Redecker et al. 2000). Intensive effort

using molecular approaches to characterise the

entire fungal community that inhabit plant roots

show that in some cases only a small fraction of

the fungal diversity revealed was previously

known from spore counts (Vandenkoornhuyse

et al. 2002).

Increasingly, molecular methods offer suffi-

cient sensitivity and specificity to detect and

identify AMF in roots from the field (e.g., Chelius

and Triplett 1999; Kowalchuk et al. 2002). Here

we apply a polymerase chain reaction (PCR)

assay to explore differences in AMF communities

inside the roots of young trees of six species of

legumes and six species of non-legumes on a field

site in southern Costa Rica.

M. Shepherd (&) � L. Nguyen � M. E. JonesCentre for Plant Conservation Genetics, SouthernCross University, Lismore, NSW 2480, Australiae-mail: Mervyn.Shepherd@scu.edu.au

J. D. NicholsSustainable Forestry Program, School ofEnvironmental Science and Management, SouthernCross University, Lismore, NSW 2480, Australia

F. L. CarpenterDepartment of Ecology and Evolutionary Biology,University of California, Irvine, CA 92697, USA

123

Plant Soil (2007) 290:259–268

DOI 10.1007/s11104-006-9157-5

Our goal was to test whether legume roots

should more consistently yield fungi and show a

broader range of fungal taxa than non-legume

roots. Knowledge of AMF distribution, diversity

and association with different plant types may be

crucial to the success of tropical reforestation

projects on degraded sites and restoration of soil

fertility. Nitrogen-fixing plants, including some

tropical trees, have a high demand for P (Binkley

and Giardina 1997; Israel 1987). Yet tropical soils

often suffer phosphorus (P) fixation in which

phosphate is bound to clay, aluminium and iron in

the soil. This problem affects about 28% of Latin

American soils (Sanchez and Logan 1992). The

resultant reduction of plant-available forms of P

is worsened when soil erosion removes soil

organic matter. Thus, the plants that have prom-

ise for restoring soil N may be limited by soil P.

Studies have shown that most plant species can be

colonized by most AMF species in greenhouse

conditions. However, some studies suggest that

certain kinds of AMF may benefit a given plant

species more than others under field conditions

(Kiers et al. 2000; van der Heijden 1998; van der

Heijden et al. 1998). We propose that legumes

are likely to be more dependent on AMF than

non-legumes in the P-deficient soils of the humid

tropics. If so, then legume trees might have been

selected evolutionarily to benefit from a wider

variety of AMF types than non-legumes. Our data

are intended as a pilot study to validate methods

and as a preliminarily test of this hypothesis.

Methods

Field methods

The experimental site was a restoration project in

25 ha of degraded pasture in southern Costa Rica,

canton Coto Brus, 20 km south of the town of San

Vito, 83�W, 9�N. The site was deforested in the

1950s, planted with coffee, and then converted to

cattle pasture in the 1970s. From 1993 to 2001

cattle were removed sequentially as plots were

planted with native trees.

The site was premontane humid tropical rain-

forest before deforestation. Site elevation ranges

from 1,015 to 1,070 m: annual rainfall averages

4,500 mm. Two rainy seasons occur during the

year, the lengthiest from July through December.

In 1995 the soils across the site were classified

based on five 2 m deep profiles as Typic Haplud-

ults to Humic or Andic Hapludults (R. Mata

et al., unpublished data) under the current USDA

classification system. Before planting, soils in the

experimental plots exhibited low (4.9–5.2) pH,

high aluminium saturation (16.8–45.6%) and low

concentrations of plant-available P (0.4–

3.0 mg kg–1) (Nichols et al. 2001).

Four field experiments were established

between 1994 and 2001 to test the ability of

native tree species to establish in degraded

pasture. The designs of these experiments were

roughly the same, following that of the first assay

in 1994 (Carpenter et al. 2004). Here we take

advantage of these experiments for molecular

analysis of 12 tree species. These species either

improve soil through N-fixation (Fabaceae, six

species) or have commercially valuable wood.

Table 1 lists the tree species and number of

individuals from which we collected samples.

We collected fine roots from selected healthy

individuals of these tree species in August 2002

except where noted in Table 1. We collected

about 2 g each of fine roots from 2 to 6 individuals

per species. Root samples were dried in a low

temperature oven (~45�C) then stored packaged

with silica gel.

Molecular assay for AMF groups

We used a previously described AMF group-

specific PCR assay based on an intergenic tran-

scribed sequence (ITS) and 18S rRNA gene

fragment to detect whether one or more members

of five major groups of AMF were present in total

DNA extracts from root tissue (Redecker 2000).

Primers specific for AMF groups were designed

to distinguish major phylogenetic lineages within

the Glomales and excluded DNA from plants and

the majority of other fungi. The assay potentially

allows discrimination of AMF into five major

groups; Group 1 represents Glomaceae (Glomus

sp. Group A); Group 2 Gigasporaceae (Gigaspora

and Scutellospora spp.); Group 3 Acaulospora-

ceae (Acaulospora and Entrophospora spp.);

Group 4 Glomeraceae (Glomus sp. Group B);

260 Plant Soil (2007) 290:259–268

123

and Group 5 Archaeospora spp. and Paraglomus

spp. (Table 2). The diversity of AMF as detected

by the group-specific PCR, was described as an

index defined as the total number of AMF groups

detected in a root sample.

DNA preparation

Root samples were carefully cleaned of adhering

soil particles in the laboratory. Total genomic

DNA was extracted from approximately 50 mg of

Table 1 Families and species of native trees sampled in 2002 for this study

Family Species Year of field experiment n

Fabaceae Albizia sp. 1998 3Calliandra thyrsifolia 1998 2Diphysa robinioides Benth. 1998 2Inga edulis Mart. 1998 5a

Inga paterno Harms 1996 3a

Schizolobium sp. 1998 2Bignoniaceae Tabebuia ochraceae (Cham.) Standl. 1994, 1998 6Combretaceae Terminalia amazonia (J.F Gmel) Exell 1994, 1998 4Clusiaceae Calophyllum brasiliense Cambess 1994 2Euphorbiaceae Hieronyma oblonga (Tul.) Mull. Arg. 1998 4Meliaceae Cedrela odorata Cedro Hembra 1994 4Vochysiaceae Vochysia guatemalensis Donn. Sm. 1994 2

The total number of tree individuals was 39, and the number of individuals sampled from each species is indicated (n).Planting year of the experiment(s) from which samples were taken is provided to indicate the different ages of trees sampleda Three samples of I. edulis and three of I. paterno were collected in September 2001 for preliminary analysis

Table 2 Glomeromycota classification and the five groups targeted in this study

Groupnumber

Ordera Family Genus Target groupb Primer-pair Productsize(bp)

1 Glomerales Glomeraceae Glomus Glomus group A—Glomusmosseae/intraradicesc

GLOM1310—ITS4 1,012

2 Diversisporales Gigasporaceae Gigaspora Gigasporaceae ITS-1F—GIGA5.8R

305

2 Diversisporales Gigasporaceae Scutellospora Gigasporaceae ITS-1F—GIGA5.8R

305

3 Diversisporales Acaulosporaceae Acaulospora Acaulosporaceae sensustricto

ACAU1660—ITS4 645

3 Diversisporales Acaulosporaceae Entrophospora Acaulosporaceae sensustricto

ACAU1660—ITS4 645

4 Glomerales Glomeraceae Glomus Glomus group B—Glomusetunicatum/claroideum

LETC1670—ITS4 676

5 Paraglomerales Paraglomeraceae Paraglomus A. gerdemannii/A. trappeiand Glomus occultum/G.brasilianum groups

ARCH1311—ITS4 1,052

5 Archaeosporales Archaeosporaceae Archaeospora A. gerdemannii/A. trappeiand Glomus occultum/G.brasilianum groups

ARCH1311—ITS4 1,052

The five groups were defined by Redecker (2000) and are given here with primer-pair and the expected PCR product sizebased on the accession from which they were designeda Glomeromycota classification derived from the NCBI taxonomy database at the time of sequence comparison (September2004)b Groups as defined in Redecker (2000)c A second primer-pair (ITS-1F-GLOM5.8R) targeting Group 1 was used for sequencing and was predicted to produce a194 bp product

Plant Soil (2006) 290:259–268 261

123

root tissues using the DNeasy Plant 96 Kit

(Qiagen PL Victoria, Australia) following the

recommended protocol for frozen tissue but with

modifications described for Araucaria sp. foliage

in Shepherd et al. (2002). Total DNA yields were

quantified by comparison with standard amounts

of DNA following agarose-gel electrophoresis,

staining with ethidium bromide and visualisation

under UV light.

AMF group specific PCR

The PCR assay was nested, with two stages of

PCR to increase assay sensitivity and help over-

come problems with PCR inhibitors co-extracted

with DNA from root samples. The competence of

all DNA preparations for PCR was demonstrated

by the generation of a visible amplification

product of the expected size at either the first or

second (nested) PCR steps.

Stage 1 PCR used the universal ITS primers

NS5 and ITS4 (see Fig. 1 in White et al. 1990).

This stage generated a PCR product of approx-

imately 1,200 bp that included part of the 18S

rRNA gene, the 5.8S rRNA gene, part of the 28S

rRNA gene as well as the two internal transcribed

spacers (ITS1 and ITS2).

For Stage 1, PCR conditions largely followed

those of Redecker (2000) except that we used a

‘‘hot start’’ and touchdown PCR to increase

reaction specificity (Roux 1994, 1995). For the

Stage 1 of PCR all DNA preparations were

diluted 1:100 in TE buffer. An exception was the

preparations from T. amazonia, which were

diluted to 1:500. Samples that failed to produce

a visible product were still included in the next

round of amplification with a group-specific

primer, because a non-visible product on an

agarose gel may still contain enough DNA to be

used as a template in PCR. Each PCR reaction

contained: 1· PCR reaction buffer (Invitrogen,

Carlsbad, CA), 1.5 mM MgCl2, 0.2 mM (total)

dNTPs, 0.15 lM of each primer, 0.5 U Platinum

Taq (Invitrogen, Carlsbad, CA) and 2 ll of

diluted DNA template in a total reaction volume

of 13 ll. Cycling conditions were: denaturation at

95�C for 1 min, annealing at 51�C for 1 min,

extension at 72�C for 1 min for 30 cycles followed

by a final extension stage of 72�C for 5 min.

In the second PCR stage, AMF-specific prim-

ers were coupled with a universal or fungal-

specific primer (ITS-1F, Gardes and Bruns 1993).

Expected PCR product sizes for each group and

primer combination were determined from align-

ments of GenBank accessions containing partial

18S rRNA gene, 5.8S rRNA gene, partial 28S

rRNA gene and the internal transcribed spacers

(ITS1 and ITS2) from various species of Glomales

(Table 2). Each group was targeted individually

with one primer-pair per PCR. PCR reactions

were set up as described for the first stage. Loci

were amplified using a series of touchdown

programs with starting annealing temperatures

(Ta) of 60, 59 or 57�C which decreased by 10�C

over 20 cycles. The cycling segment consisted of

20 cycles with a 95�C hold for 1 min, annealing

temperature for 1 min and 72�C for 1 min. This

was followed by a further 20 cycles at the final

annealing temperature. Cycling was preceded by

a hold at 95�C for 5 min and finished with a final

hold of 3 min at 72�C.

Cloning and sequencing of PCR products

PCR products were cloned using the TA cloning

method (Zhou et al. 1995) using the pGEM-T

Easy vector system (Promega, Madison, WI) as

per the manufacturers recommended protocols

except where detailed below. Sequencing reac-

tions were performed as manufacturer’s recom-

mendations for Big Dye Terminator v3.1

sequencing chemistry (Applied Biosystems,

Foster City, CA). Separation was carried out on

an AB 3730 DNA sequencer at the Southern

Cross Plant Genomics (Lismore, Australia).

Where necessary, cloned fragments were

sequenced in both directions to obtain full-length

sequence using primers T7 and/or SP6 primers.

Following transformation into JM109 (Promega

Madison, WI) and overnight culture at 37�C, two

colonies from each plate were picked and subject

to a PCR with both T7-SP6 and a T7-ITS4

primer-pairs to confirm the presence of an insert

and its direction prior to carrying out sequencing

reactions.

DNA sequence homology was used to infer

the identity of cloned PCR products. Homolo-

gous sequences were identified in GenBank

262 Plant Soil (2007) 290:259–268

123

(non-redundant, all organisms options) using the

BLASTN search algorithm set to default param-

eters (Altschul et al. 1997). A full-length

sequence for each clone was assembled with the

aid of Sequencher V3 software (Gene Codes

Corporation) following the removal of vector

sequence. Sequences obtained in this study were

submitted to GenBank and assigned accession

numbers AY744275–AY744286.

Results

Competence of DNA extracted from tree

roots for PCR

The competence of all DNA preparations for

PCR was demonstrated by the generation of a

visible amplification product of the expected size

at either the first or second (nested) PCR steps

(data not shown). All 39 root samples, except for

one sample from Inga edulis (No. 3522) yielded a

PCR product of the expected size at Stage 1 of

PCR (data not shown). Sample 3522, however,

generated a PCR product of the expected size

with primers for the target Group 3 at Stage 2 of

PCR (Table 3). The lack of a visibly detectable

PCR product for I. edulis sample 3522, therefore,

was apparently due to an amplification yield

below the detectable limit for the assay at Stage

1. DNA yields from approximately 50 mg of tree

roots averaged 0.2 ± SE 0.4 lg.

To achieve, PCR amplification from Termina-

lia amazonia samples, a higher dilution (1:500) of

template DNA was required at Stage 1 than for

the other tree species. DNA preparations from T.

amazonia were highly coloured, suggesting the

presence of compounds such as phenolics that co-

purified with the DNA and inhibited PCR.

Verification of group membership

by sequencing

Twelve AMF group PCR products were cloned

from six root samples (five tree species)

(Table 4). Cloned fragments were obtained from

four of the five AMF group PCR assays. Highly

significant matches were detected in GenBank to

all 12 sequences. The classification of the most

significant (homologous) sequence in all cases

except one (see below), was consistent with the

group targeted by each PCR assay. This was

strong evidence that the PCR assays were detect-

ing members of their target group and validated

the use of this approach to characterise AMF

within roots into major groups.

In one exception, the organism identified by

homology to our cloned PCR fragment was not a

member of the target AMF group. This sequence

(AY744286) was derived from a Group 5 assay,

which targeted the Archeospora and Paraglomus

genera, on roots from Hyeromina oblonga (sam-

ple #3573). In this case a highly significant match

was detected with Leptophaeria bicolour

(AF455415.1), an Ascomycete.

Legumes and non-legumes possessed similar

frequencies and diversity of AMF

Representatives from all five groups of AMF

were detected in the root samples (Table 3).

Group 4 (Glomus Group B) was the most

commonly detected, found in 26 out 39 root

samples tested, followed by Group 1 (Glomus

Group A) (24), Group 5 (Archaeospora) (17),

Group 3 (Acaulospora) (9) and Group 2 (Gigas-

pora & Scutellospora) (8). Detection rates were

highly variable, however, with some samples

having a diversity index of zero (no representa-

tive of any group detected) and others had the

maximum five groups represented. Within a tree

species, the distribution levels were also variable,

with the diversity index for individual samples

again ranging from 0 to 5 (Table 3). The greatest

diversity of AMF was detected in Schizobium sp.,

a legume (Diversity index Av (SD); 4.0 ± 1.4)

whereas Vochysia guatemalensis, a non-legume,

had the lowest diversity index for any species

(0.5 ± 0.71), however, there was no significant

difference in the levels of diversity amongst tree

species (Type III ANOVA, df = 11, F = 1.5,

P-value = 0.185).

Detection of a representative of an AMF group

occurred slightly more frequently in legumes

(45%) than non-legumes (41%) but without

replication it was not clear if this was significant.

The diversity of AMF detected in legumes

was slightly higher than non-legumes, but not

Plant Soil (2006) 290:259–268 263

123

Table 3 AMF groups detected by PCR of ITS in 39 root samples from 12 tree species

Sample identity no. Species Target group

Group 1 Group 2 Group 3 Group 4 Group 5 Diversity indexa

Glomus A Gigaspo. Acaul. Glomus B Arch.

3579 Albizia sp. 1 1 23553 Albizia sp. 1 13556 Albizia sp. 1 1 1 3Mean (SD) 2.0 (1.00)3580 C. thyrsifolia 1 13555 C. thyrsifolia 1 1 1 1 4Mean (SD) 2.5 (2.12)3570 D. robinoides 03593 D. robinoides 1 1 1 1 4Mean (SD) 2.0 (2.83)3520 I. edulis 1 13522 I. edulis 1 13539 I. edulis 03566 I. edulis 1 1 23584 I. edulis 1 1 1 3Mean (SD) 1.4 (1.14)3521 I. paterno 03524 I. paterno 1 1 1 33538 I. paterno 1 1 1 1 1 5Mean (SD) 2.7 (2.52)3548 Schizolobium sp. 1 1 1 1 1 53557 Schizolobium sp. 1 1 1 3Mean (SD) 4.0 (1.41)Legumes sub-total or Av (SD) 11 5 5 9 8 2.24 (1.68)Proportions 0.65 0.29 0.29 0.53 0.47 0.453549 C. brasiliense 1 1 23552 C. brasiliense 1 1 1 1 4Mean (SD) 3.0 (1.41)3591 C. odorata 1 1 1 33581 C. odorata 1 1 1 33565 C. odorata 1 1 23546 C. odorata 1 1 1 1 4Mean (SD) 3.0 (0.82)3573 H. oblonga 1 1 1 33554 H. oblonga 1 1 1 33594 H. oblonga 1 1 1 33563 H. oblonga 1 1 2Mean (SD) 3.0 (0.82)3523 T. ochracea 1 1 1 33588 T. ochracea 1 13592 T. ochracea 1 13590 T. ochracea 1 1 23568 T. ochracea 1 13545 T. ochracea 1 1 1 3Mean (SD) 1.8 (0.98)3550 T. amazonia 1 13558 T. amazonia 1 13583 T. amazonia 03585 T. amazonia 1 1 2Mean (SD) 1.0 (0.82)3569 V. guatemalensis 03544 V. guatemalensis 1 1Mean (SD) 0.5 (0.71)

264 Plant Soil (2007) 290:259–268

123

significantly different (Av (SD) legumes 2.24

(1.68); non-legumes 2.09 (1.23), two-sample,

unequal variance t-test, df = 28, one-sided P-

value = 0.38). The power to test for differences

in diversity, however was low (a = 0.05,

power = 7%), and therefore confidence in reject-

ing the alternative hypothesis was not high.

Legumes and non-legumes had similar propor-

tional representation of AMF groups, showing

similar profiles but generally there were higher

frequencies of detection in legumes (Table 3).

The major difference in the diversity profiles of

the two groups was in the ratio of Group 1 to

Group 4. Legumes had a higher proportion of

Group 1 to Group 4 (i.e. representatives of

Glomus Group A group were more prevalent

than Glomus Group B) whereas in non-legumes,

a lower proportion of Glomus Group A repre-

sentatives was detected than Glomus Group B.

Discussion

We demonstrated the feasibility of characterising

in planta AMF colonising rainforest tree roots

using group specific PCR of ITS. We confirmed

the detection of representatives from four of the

five target AMF groups by cloning and sequence

comparison with reference sequence. As a pilot

study, we have shown the full spectrum of AMF

groups assayed were represented at this field site.

The PCR assay was a relatively quick and

inexpensive molecular method for reliably assess-

ing AMF at the group level. We envisage that in

the future, sequencing would usually only be

needed on a small number of samples to verify

assay specificity and to provide more detailed

characterisation of the group composition.

The group specific assay may be useful in

providing distribution profiles of AMF groups for

ecological or stand management purposes. Our

conclusions at this point, however, can only be

tentative. Our hypothesis predicted that legumes

would be colonized: (1) by a wider diversity of

AMF types than non-legumes, and (2) more

frequently. The data were equivocal in both

cases, the high variance in diversity amongst

individuals within a species provided limited

power to contrast diversity levels of AMF

detected in legumes and non-legumes and the

experiment must be replicated to test whether

there is a significant difference in overall AMF

and AMF group frequencies in the two types of

plants.

For future studies, the large variances in

diversity within a tree species indicate substan-

tially larger sample sizes than in the current study

will be required, and that experimental design

should focus on controlling environmental factors

that may contribute to within-species variance.

One factor that was likely to contribute to this

variation was the extreme differences in site

quality over the experiment. In our study, sam-

pling deliberately targeted poor and good plots to

encompass the full spectrum of environmental

variation. In future studies, variance within spe-

cies may be controlled by selecting either poor or

good plots for analysis or by the application of a

site quality covariate in the analysis to regress out

this factor. In further studies, replication of the

experiment will be required to test whether the

frequency of detection in legumes was higher

than non-legumes and should also use larger

samples of individuals within each species to

increase the power to detect differences between

plant species and plant groups.

The greatest difference between legumes and

non-legumes was in distribution of AMF groups

that were detected their roots. The data tenta-

tively suggested that legume roots contained

Table 3 continued

Sample identity no. Species Target group

Group 1 Group 2 Group 3 Group 4 Group 5 Diversity indexa

Glomus A Gigaspo. Acaul. Glomus B Arch.

Non-legumes sub-total or Av (SD) 13 3 4 17 8 2.09 (1.23)Proportions 0.36 0.14 0.14 0.59 0.23 0.29

a Diversity index was calculated as the total number of AMF groups detected in a sample

Plant Soil (2006) 290:259–268 265

123

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266 Plant Soil (2007) 290:259–268

123

relatively more DNA from Group 1 (Glomus

Group A) than from Group 4 (Glomus Group B),

whereas non-legumes contained a higher propor-

tion of Group 4 than Group 1. If further study

does show that legumes contain relatively more

Group 1 than non-legumes, the explanation may

lie in the functional differences between the

groups. Glomus invermaium, is known to be

effective in assisting 32P uptake (Jakobsen et al.

2001), and belongs to Glomus Group 1 (Redecker

2002). Although this species was not identified by

DNA sequencing in our study, sequencing

revealed Group 1 to be a diverse group with up

to four Glomaceae identified. This may indicate

this species or close relatives with the capacity to

assist P uptake are associating with the roots of

legumes more frequently than non-legumes.

The detection of a non-target organism, an

Ascomycete, by cloning and sequencing of PCR

product was probably due to non-specificity of

the PCR assay for Group 5. Non-specificity of

the Group 5 assay has been reported previously;

a member of both the Ascomycetes and Basid-

iomycetes groups were detected by weak ampli-

fication with the ARCH1311 primer (Redecker

2000). In our study, the Group 5 assay generated

two or three PCR products (distinguishable by

gel separation on agarose) from some root

samples. In the case of the Hyeromina oblonga

(sample # 3573), the smallest of these products

was isolated for cloning and was identified as an

Ascomycete. We suspect that the larger band

represents a member of the target group, Acha-

eosporaceae, as its size was closer to the

expected PCR product size for the group (i.e.

1,052 bp—see Table 2). In the case of Inga

paterno (sample # 3538) multiple PCR products

were also evident from the Group 5 assay. In

this case, the smaller band (~1 kb) was isolated

before cloning and it was highly homologous to a

member of the target group in the Achaeospor-

aceae family (see Table 2). Our data indicate

that interpretation of results for the Group 5

assay required caution as the assay has the

capacity to detect non-target organisms and may

overestimate representation in this group. Pre-

cise determination of PCR product size may help

overcome this problem, as in our case non-target

organisms were distinguishable by PCR product

length. This issue of non-specificity of Group 5

PCR assay may extend to purified isolates, as it

has been shown that Ascomycete fungi can

inhabit AMF spores leading to multiple ITS

sequence types from AMF isolates (Hijri et al.

2002).

Acknowledgments The authors thank Eduer SandiTapia, Rolando Mendez Rodriguez, and Leonel FigueroaVargas for their conscientious assistance in the field work.We thank F. Eliott for technical assistance in thelaboratory. Financial support was provided by Universityof California Research Expeditions Program andRainforest Agrarian International Network.

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