mycological research 113 (2009) 11371145

journal homepage: www.elsevier.com/locate/mycres

The ITS region as a taxonomic discriminator between

Fusarium verticillioides and Fusarium proliferatum
I. VISENTINa, G. TAMIETTIa, D. VALENTINOa, E. PORTISb, P. KARLOVSKYc,
A. MORETTId, F. CARDINALEe,*
a

DiVaPRA, Plant Pathology, University of Turin, via L. da Vinci 44, 10095 Grugliasco (TO), Italy
DiVaPRA, Plant Genetics and Breeding, University of Turin, via L. da Vinci 44, 10095 Grugliasco (TO), Italy
c
Molecular Phytopathology and Mycotoxin Research Unit, University of Gottingen, Grisebachstrasse 6, D-37077 Gottingen, Germany
d
ISPA, National Research Council, Via Amendola, 122/O 70126 Bari (BA), Italy
e
Dept. of Arboriculture, University of Turin, via L. da Vinci 44, 10095 Grugliasco (TO), Italy
b

article info

abstract

Article history:

The maize pathogens Fusarium verticillioides (Fv) and Fusarium proliferatum (Fp) are morpho-

Received 6 April 2009

logically very similar to one another, so Fp isolates have been often mistaken as Fusarium

Received in revised form 15 July 2009

moniliforme (the former name of Fv). The only presently accepted morphological discrimina-

Accepted 16 July 2009

tor between these species is the presence/absence of polyphialides. Here, a collection of 100

Available online 23 July 2009

Fusarium strains, isolated from infected maize kernels on plants grown in north-western

Corresponding Editor:

Italy, were assigned as Fv or Fp on the basis of the presence/absence of polyphialides.

Stephen W. Peterson

This classification was tested on a subset of isolates by sexual crosses, ITS and calmodulin
sequencing and AFLP profiling. An ITS-RFLP assay was extended to the full collection and to

Keywords:

a number of Fv and Fp isolates of different geographical origin and hosts. The ITS region is

Fusarium proliferatum

proposed as taxonomically informative for distinguishing between Fp and Fv.

Fusarium verticillioides

2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

ITS
Maize
PCR

Introduction
Fusarium verticillioides (Fv) (sexual stage Gibberella moniliformis)
and Fusarium proliferatum (Fp) [sexual stage Gibberella intermedia] are both common as pathogens of maize worldwide, causing [in conjunction with Fusarium subglutinans] ear, stalk and
root rot (Bottalico 1998; Leslie et al. 1990; Nelson et al. 1981).
They can be particularly aggressive in temperate climates
(Munkvold 2003), and maize produced in northern Italy is often heavily contaminated. Infection can be spread by both
soil- and seed-borne inoculum, but usually invasion of the
growing plant occurs through the silk or the kernels damaged

by insects (Munkvold 2003). Ear rot not only reduces grain

yield, but also grain quality, since both Fv and Fp produce mycotoxins within the infected kernel. In particular, significant
quantities of the class B fumonisins (FB) are frequently
detected in maize grown in northern Italy (Bottalico et al.
1989; Moretti et al. 1995). Over 20 natural analogues of fumonisins are known, but it is fumonisin B1 (FB1) which is the commonest and most dangerous one, and the one which was
associated with severe mycotoxicosis in both domesticate animals and humans (Rheeder et al. 2002). Kernels can become
heavily contaminated long before harvest, so an understanding of the epidemiology of ear, stalk and root rot is necessary

* Corresponding author. Tel.: 39 011 670 8875; fax: 39 011 236 8875.
E-mail address: francesca.cardinale@unito.it
0953-7562/$ see front matter 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.mycres.2009.07.011

1138

I. Visentin et al.

before control strategies can be elaborated. The availability of

a robust and reliable method to taxonomically identify Fv and
Fp (the major two fumonisin-producing species) is an essential requirement for this purpose.
Both Fv and Fp belong to the Gibberella fujikuroi species
complex, in which at least eleven different mating populations (MPs) (i.e. biological species) have been identified (Leslie
& Summerell 2006). Among the species in the G. fujikuroi
complex which are pathogenic on maize, Fv and Fp can be
distinguished by their microconidial chains (not formed by
F. subglutinans), and it is the presence of polyphialides which
differentiates Fp from Fv (Nirenberg & ODonnell 1998). As
sexual crosses are both labour- and time-consuming to generate, species diagnosis relies currently on morphological
traits, supported by the DNA sequence of several genes,
most often, tubulin and calmodulin (ODonnell et al. 1998). Although a number of criteria show Fv and Fp to be true evolutionary species (Taylor et al. 2000), the morphological
distinction between Fp and Fv is rather fine and requires
trained personnel to recognize, while DNA sequencing can

be expensive if large numbers of strains require identification. PCR-based genotyping offers a more cost-effective
means of molecular diagnosis. Primer pairs which should
specifically amplify DNA from Fv have been designed based
on sequence variation within either the intergenic spacer
(IGS) region of the ribosomal locus (VERT1VERT2) (Patino
et al. 2004) or the calmodulin gene (VER1VER2) (Mule et al.
2004). Similarly, it has been possible to design the Fp-specific
primers Fp3FFp4R (IGS) (Jurado et al. 2006) and PRO1PRO2
(calmodulin) (Mule et al. 2004) (Table 1). An important issue
in the context of PCR-based species diagnostics is amplification specificity, especially where the fungal strains to be
tested have been isolated from an environment which was
not sampled during the process of primer design. The intention of the present research was to classify Fusarium strains
in a collection of isolates from maize samples grown in Piedmont (north-western Italy) using morphological, biological
and molecular tools, and to develop robust PCR primers to
distinguish between Fv and Fp as a complement to the
primers already available.

Table 1 Primer sequences used for genotyping Fusarium spp.

Primer pairs

Target sequence

Amplicon size

Specificity

Reference

FUS1
FUS2

Heat shock protein

50 -cttggtcatgggccagtcaagac-30
50 -cacagtcacatagcattgctagcc-30

1600

Fusarium moniliforme

Murillo et al. (1998)

VERT1
VERT2

IGS
50 -gtcagaatccatgccagaacg-30
50 -cacccgcagcaatccatcag-30

800

Fusarium verticillioides

Patino et al. (2004)

Fp3F
Fp4R

IGS
50 -cggccaccagaggatgtg-30
50 -caacacgaatcgcttcctgac-30

230

Fusarium proliferatum

Jurado et al. (2006)

VER1
VER2

Calmodulin
50 -cttcctgcgatgtttctcc-30
50 -aattggccattggtattatatatcta-30

578

F. verticillioides

Mule et al. (2004)

PRO1
PRO2

Calmodulin
50 -ctttccgccaagtttcttc-30
50 -tgtcagtaactcgacgttgttg-30

585

F. proliferatum

Mule et al. (2004)

CL1
CL2A

Calmodulin
50 -gartwcaaggaggccttctc-30
50 -ttttgcatcatgagttggac-30

670

Fusarium spp.

O0 Donnell et al. (1998)

ITS1
ITS4

ITS
50 -tccgtaggtgaacctgcgg-30
50 -tcctccgcttattgatatgc-30

620

All fungal species

White et al. (1990)

verITS-F
ITS4

ITS
50 -aaatcgcgttccccaaattga-30
50 -tcctccgcttattgatatgc-30

172

F. verticillioides

This work, White et al. (1990)

ITS1
proITS-R

ITS
50 -tccgtaggtgaacctgcgg-30
50 -gcttgccgcaagggctcgc-30

390

F. proliferatum

This work, White et al. (1990)

fusALPHArev
fusALPHAfor

MAT1
50 -ggartaracyttagcaatyagggc-30
50 -cgccctctkaaygscttcatg-30

200

Fusaria species with Calonectria,

Gibberella and Nectria teleomorphs

Kerenyi et al. (2004)

fusHMGfor
fusHMGrev

MAT2
50 -tgggcggtactggtartcrgg-30
50 -cgacctcccaaygcytacat-30

260

Fusaria species with Calonectria,

Gibberella and Nectria teleomorphs

Kerenyi et al. (2004)

The ITS region as a taxonomic discriminator

1139

Materials and methods

Fungal isolates
A collection of 100 fungal strains (see Table 2 and Table S1)
was isolated from maize kernels of naturally infected plants
in 25 fields in Piedmont, Italy (north-western Italy, Fig S1).
Monoconidial cultures of all strains were obtained, and cultivated on Komada medium (Komada 1975) to allow a preliminary taxonomical assignment based on colony pigmentation
and the presence/absence of conidial chains (Summerell
et al. 2003). Only those isolates that produced conidial chains
were retained, and the morphology of their conidiophores
when cultured on SNA medium was noted (Nirenberg 1976).
Moreover a strain of Fusarium subglutinans (NOVb1) was identified and retained to be used as outgroup (negative control) in
the phylogenetic analyses. Isolates were stored on malt-agar
at 10  C.

DNA extraction and PCR amplification

Whatman FTA cards were used to extract DNA from freshly
grown mycelium of isolates grown on malt-agar, following
the manufacturers instructions. Five primer pairs were tested
(Table 1): VERT1VERT2 (Patino et al. 2004), Fp3FFp4R (Jurado
et al. 2006), VER1VER2 and PRO1PRO2 (Mule et al. 2004), and
FUS1FUS2 (Murillo et al. 1998). PCRs were performed using
a T-Gradient thermal cycler (Biometra) replicating the

conditions reported by the originators of the primers. The

newly developed primers based on ITS sequence (verITS-F
and proITS-R, see Table 1) were used in combination with
the primer pair ITS1ITS4. Multiplex reactions were carried
out in 25 ml final reaction volume. Each tube contained
0.4 mM ITS1 and 0.4 mM ITS4, 0.5 mM verITS-F and 0.5 mM
proITS-R, 0.625 U GoTaq polymerase (Promega, 5 U ml1),
1 PCR buffer and 0.4 mM dNTP. The amplification conditions
consisted of a denaturation of 94  C/2 min, followed by 35 cycles of 96  C/60 s, 60  C/60 s and 72  C/45 s, and a final extension of 72  C/10 min. PCR products were separated by
agarose gel electrophoresis (1 % w/v in 0.5 TBE) and visualized by ethidium bromide staining.

ITS-RFLP
An aliquot of the ITS DNA amplicon [generated by the ITS1
ITS4 primer pair (White et al. 1990)] from each isolate was
digested overnight at 37  C with one of the 4 bp cutters AluI,
MboI, HaeI or TaqI, or the 5 bp cutter HinfI (all enzymes purchased from Fermentas). The restriction fragments were separated by electrophoresis through 2 % (w/v) agarose gels and
visualized by ethidium bromide staining.

ITS and calmodulin sequencing

DNA from all isolates was amplified with both ITS1ITS4
(White et al. 1990), and the calmodulin gene primers CL1
CL2A (ODonnell et al. 2000) (Table 1). The PCRs consisted

Table 2 Fp and Fv strains isolated from maize grown in north-western Italy. Fp strains identified by the presence of
polyphialides. PCR amplification used extant species-specific primer pairs
Strains

VP2
CH2
CHI1
FR3
SAL4
GE1
PI1
CA2
CM3
SA3
PO1
RT1
BU1
CE1
MATA-1
MATA-2
MATD-1
MATD-2

Morphology

Fusarium
verticillioides
F. verticillioides
F. verticillioides
F. verticillioides
F. verticillioides
F. verticillioides
F. verticillioides
F. verticillioides
F. verticillioides
F. verticillioides
Fusarium
proliferatum
F. proliferatum
F. proliferatum
F. proliferatum
F. verticillioides
F. verticillioides
F. proliferatum
F. proliferatum

MAT
allele/MP

AFLP
group

Calmodulin
group

RFLP
profile

1/NT

2/NT
1/NT
1/NT
1/NT
1/NT
1/A
1/NT
2/A
1/A
1/not fertile

A
A
A
A
A
A
A
A
A
B

2/not fertile
2/D
2/not fertile
1/A
2/A
1/D
2/D

B
B
B
NT
NT
NT
NT

NT: not tested.

a Weak amplification.
b Quantitative analysis for FB1 and FB2.
c FB3 analysis also performed.

Fus1

VERT1

VER1

PRO1

Fp3F

FB
productionb

Fus2

VERT2

VER2

PRO2

Fp4R

A
A
A
A
A
A
A
A
A
B

A
A
A
A
A
A
A
A
A
B





















c

B
B
B
A
A
B
B

B
B
B
A
A
B
B

a

a

a
a









c
c
NT
NT
NT
NT

1140

of a denaturation of 94  C/5 min, followed by 30 cycles of

94  C/30 s, 55  C/60 s and 72  C/60 s, and a final extension of
72  C/10 min. The amplicons were directly sequenced from
both ends by Genelab (ENEA, Rome). Phylogenetic analyses
were carried out using MEGA software (www.megasoftware.net) based on the sequences of 14 Fusarium strains plus mating-type tester strains for Fv and Fp teleomorphs (MATA-1/2
and MATD-1/2; see further on) and one Fusarium subglutinans
strain (NOVb1). Neighbour-joining trees were constructed using Kimuras two-parameter model (Kimura 1980) with 1000
bootstrap replicates. The isolates listed in Table 2 were deposited in the collection of the Institute of Sciences of Food Production (ISPA-CNR, Bari, Italy; http://server.ispa.cnr.it/ITEM/
Collection) and the genomic sequences were deposited in
Genbank. Accession numbers for ISPA and Genbank (ITS and
calmodulin, in this order) for each isolate are as follows: VP2
(10676; EU151483; EU430618); CH2 (10677; EU151473;
EU430621); CHI1 (10678; EU151479; EU430604); FR3 (10679;
EU151474; EU430608); SAL4 (10680; EU151472; EU430614); GE1
(10681; EU151480; EU430612); PI1 (10682; EU151481;
EU430622); CA2 (10683; EU151478; EU430610); CM3 (10684;
EU151469; EU430616); SA3 (10685; EU151482; EU430613); PO1
(10686; EU151487; EU430607); RT1 (10687; EU151490;
EU430606); BU1 (10688; EU151489; EU430611); CE1 (10689;
EU151486; EU430620); NOVb1 (10690; EU151476; EU430603);
MATA-1 (7581; EU151467); MATA-2 (7583; EU151468); MATD-1
(7596; EU151484); MATD-2 (7595; EU151485). The four latter
mating-type tester strains were not sequenced on calmodulin
(available Genbank sequences were used and are reported in
Fig 2 along with their accession numbers). Additional strains
of Fv and Fp from different hosts and/or geographical origin
were also tested; they were obtained from public collections
and are listed in Table S1.

AFLP analysis
Strains were grown in Czapek-Dox mineral medium (Sigma)
in still culture for two weeks. Mycelium was harvested by filtration and frozen in liquid nitrogen, and DNA extracted by
the CTAB method (Murray & Thompson 1980). The AFLP protocol followed Vos et al. (1995) with minor modifications
(Lanteri et al. 2004). Briefly, 5 ml (400500 ng) DNA was double-digested with EcoRI and MseI and ligated to adapters.
Digested and ligated DNA fragments were pre-amplified
with primers carrying one selective base (EcoRI A and
MseI C), and selectively amplified using primers carrying
two or three selective bases. Initially, 12 primer pairs (four
EcoRI and three MseI primers) were tested against four templates, and the outcome of this pilot resulted in the choice of
the four primer combinations E AAT/M CAA, E AAT/
M CAG, E ACT/M CAA, E ACT/M CAG. AFLP amplicons were resolved through 5 % denaturing polyacrylamide
gels, and visualized by silver staining as described (Bassam
et al. 1991).

Data scoring and analysis

AFLP reactions were repeated at least twice in order to assure consistency. Electrophoretic patterns were documented
using a commercial gel documentation system (Quantity

I. Visentin et al.

One Programme, BioRad). Each fragment was assumed to

represent a single locus and only reproducible polymorphic
bands were scored as present (1) or absent (0). The binary
data were used to calculate the polymorphism information
content (PIC) by applying the simplified formula 2f (1f )
(Anderson et al. 1993) for expected heterozygosity, where f
represents the percentage of individuals in which the
marker is present. The binary matrix was imported into
NTSYS-pc v1.80 (Rohlf 1993) to perform a cluster analysis.
Genetic similarity among isolates was calculated according
to Jaccards similarity index (JSI) (Jaccard 1908), using the
SIMQUAL routine. The similarity coefficients were used to
construct a dendrogram by UPGMA with 1000 bootstraps, using PHYLIP package [http://evolution.genetics.washington.
edu/phylip.html (Felsenstein 1993)]. A co-phenetic matrix
was produced using the hierarchical clustering system, by
means of the COPH routine, and correlated with the original
distance matrices for AFLP data, in order to test for the association between the cluster in the dendrogram and the JSI
matrix. Mantel tests (Mantel 1967) were performed to check
the correlation between the similarity matrices generated by
each AFLP primer combination and the global similarity
matrix. A principal coordinate (PCO) analysis was also performed, based on the triangular matrix of genetic similarity
estimates, and the first two axes were plotted graphically.

Fertility test and vegetative compatibility

Before crossing, each strain was PCR genotyped using the
fusALPHAfor/rev and fusHMGfor/rev primer pairs (Table 1)
to check which idiomorphic allele they carried at the MAT locus (Kerenyi et al. 2004; Yun et al. 2000). Strains tested for sexual compatibility (PI1, CM3, SA3, BU1, PO1, RT1 and CE1) were
co-cultivated on carrot agar in combination with each of the
mating-type tester strains MATA-1, MATA-2, MATD-1 and
MATD-2 (obtained from the Institute of Sciences of Food Production, Bari, Italy) to create a set of crosses following Klittich
and Leslie (1988). The female strain was grown on carrot agar
and sprinkled with a conidial suspension obtained from water
agar plates of the male. The cultures were taken as fertile
when cirri were observed as extruding from the perithecia.
The carrot agar plates were incubated for four weeks at 23/
24  C under 12/12 h light/darkness. A vegetative compatibility
group (VCG) assignment for all strains was based on complementation between nitM and nit1 mutants grown on minimal
medium (Correl et al. 1987). Pairs of vegetatively compatible
isolates produced vigorous aerial growth at the contact site
of the mycelia of two nit mutants. Isolates that exhibited
this morphology were assigned the same VCG.

HPLC analysis of fumonisins

20 g samples of cracked maize kernel were combined with

2 ml water each, and autoclaved twice (120 C, 1 h). Each maize
aliquot was inoculated with 3 ml of sterile water containing
106 conidia of each isolate in turn, and maintained at room
temperature under 12/12 h light/darkness with daily manual
shaking. The quantity of FB1 and FB2 in the culture was determined by HPLC 20 d after inoculation, and cultures containing
little or no FB1 and FB2 were re-analysed for FB3 content.

The ITS region as a taxonomic discriminator

1141

Fumonisins were detected by described methods (Adejumo

et al. 2007).

52
74
65

VP2
GE1

77

Morphological identification and toxin production

67

A collection of 100 Fusarium strains producing microconidial

chains was assembled from 25 different fields covering the
Piedmont territory (Fig S1). Monoconidial cultures were
scanned for presence/absence of polyphialides to discriminate
between Fv and Fp. The results of this morphologically based
classification are given in Table S1. A sample of 14 isolates,
each originating from a different field and including some Fv
and Fp based on morphological scoring, was selected for further analyses. All Fv isolates were highly toxigenic in vitro; FB
production was more variable among Fp isolates (Table 2).

SA3
CHI1

79

Results

MATA-1

MATA-2

65

PI1

CH2

85
72

FR3

91

CM3
CA2

83

SAL4
MATD-1

54
52

PO1
RT1

65
88

MATD-2

55

CE1

AFLP analysis and genetic relatedness

BU1

Table 3 AFLP profiling of a sample of Fv and Fp isolates.

TNB: total number of bands, NPB: number of polymorphic
bands, %: percentage of polymorphic bands, PIC:
polymorphic information content, r: correlation between
the similarity matrices generated by each individual
primer combination and the global similarity matrix
PC
E AAT/M CAA
E AAT/M CAG
E ACT/M CAA
E ACT/M CAG
Average
Total

TNB

NPB

PIC

33
32
46
57

30
28
42
51

90.9
87.5
91.3
89.5

0.318
0.324
0.252
0.352

0.879
0.912
0.954
0.964

42
168

38
151

89.9

0.311

0.927

NOVb1
0.25

0.50

0.75

1.00

Jaccard's Similarity Coefficient

Coordinate 2

The four primer combinations amplified 168 fragments, of

which 151 were polymorphic. The number of polymorphic
fragments per primer combination ranged from 28 to 51
(Table 3). All the genotypes were characterized by a unique
banding pattern. The E ACT/M CAG combination produced the greatest number of polymorphic fragments, the
highest PIC and could discriminate, on its own, between
all 14 isolates and the testers. The lowest JSI value was
0.194 (Fusarium subglutinans NOVb1 and CM3), while the
highest (0.922) applied to the contrast MATA-1 and SA3
(JSI values of the other comparisons are available
on
request
from
the
authors).
The
resulting
UPGMA dendrogram is given as Fig 1. The co-phenetic correlation coefficient between the data matrix and the cophenetic matrix for the AFLP data was 0.975, indicating
a close fit between the dendrogram clusters and the JSI
matrix from which they were derived. As expected, isolate
NOVb1 was the most highly differentiated from the other
isolates (mean JSI 0.250). Two major clusters (bootstrap
probability  88 %) were resolved: clusters A and B correspond to isolates identified by their morphology as Fv
and Fp, respectively. The principal coordinate scatter plot
confirmed the clustering of the genotypes. The first two
axes accounted for, respectively, >49 % and >19 % of the

0.25

CM3

RT1
MATD-2
PO1
MATD-1
CE1
BU1
-0.05

MATA-2
VP2
SAL4
GE1 SA3 CHI1
CA2 PI1
MATA-1
CH2
FR3

-0.35

-0.65

NOVb1
-0.95
-0.70

-0.41

-0.13

0.16

0.45

Coordinate 1
Fig 1 UPGMA dendrogram (A) and principal coordinate
analysis based on Jaccards similarity coefficient (B) of 19
Fusarium isolates. Genotypic data generated by AFLP profiling with four primer combinations. Numbers associated
with each dendrogram node represent the proportion of
1000 bootstrap samples in which the particular clade was
found. Only percentages above 50 % are shown.
variation. The former distinguished the isolates belonging
to cluster A from those belonging to cluster B, with
NOVb1 in an intermediate position; while the latter separated NOVb1 from the rest of the collection.

Calmodulin sequence analysis

A phylogeny analysis was carried out based on the partial calmodulin gene sequences derived from the 14 strains (Fig 2A).

1142

I. Visentin et al.

both Fv) belonged to the same VCG, resulting in the identification of 13 distinct VCGs for 14 isolates. Such a high level of inter-strain incompatibility is consistent with previously
observed rates of differentiation between Fv isolates (Desjardins et al. 1994).

CM3
SA3
GE1
CH2
SAL4
100

VP2

A
PCR amplification using VERT1 and VERT2, Fp3F and Fp4R,
VER1 and VER2, PRO1 and PRO2, FUS1 and FUS2 primer
pairs

AF158315
CA2
PI1
FR3
CHI1
NOVb1
53 CE1
BU1
100
AF291057
57
0.030

0.025

0.020

0.015

0.010

0.005

RT1
PO1

0.000

MATA-2
SAL4
SA3
GE1
CH2
NOVb1
66 VP2
PI1
CM3
MATA-1
FR3
CHI1
CA2
MATD-2
100 BU1
CE1
67 PO1
RT1
MATD-1

0.002

Fig 2 Phylogeny based on a multiple alignment of the

CL1CL2A amplified fragment of the calmodulin gene (A)
and the ITS1ITS4 amplified fragment of the ITS (B). Both
Neighbour-joining trees were constructed with Kimuras
two-parameter model with 1000 bootstrap replicates.
Edge length is indicated in terms of substitution rates per
nucleotide, together with bootstrap % values above 50 %.

This indicated the existence of two major groups. One group

was centred on AF291057, a known type-Fv sequence, and
the other on AF158315, a type-Fp sequence. The cluster pattern derived from this analysis perfectly matched the one generated on the basis of AFLP genotypes.

The results of the PCR genotyping are summarized in Table 2,

where expected and obtained results for each isolate and species are easily visualized. The VERT1VERT2 (Patino et al. 2004)
and VER1VER2 (Mule et al. 2004) primer pairs generated
amplicons of the expected size from most of the strains. The
exceptions were, for VERT1VERT2: CM3 (Fv, but negative),
PO1, BU1 and CE1 (Fp, but positive). VER1VER2 amplified inappropriately, though only weakly, from Fp strains PO1, RT1, CE1
and the MP D testers (Table 2). The sample of 14 isolates plus
testers was tested with the Fp-specific PRO1PRO2 (Mule et al.
2004) and Fp3FFp4R (Jurado et al. 2006) primer pairs. PRO1
PRO2 amplified the expected w600 bp product from five Fp
strains out of six (PO1, RT1, BU1 and CE1, along with the
MATD-1 tester). Fp3FFp4R did not amplify from three (RT1,
MATD-1, MATD-2) out of the six Fp strains expected to be positive (Table 2). The FUS1FUS2 primer pair (Murillo et al. 1998)
amplified from all isolates, so all Fp strains were false positive
(Table 2). Thus to summarize, VERT1VERT2, which was
designed to specifically amplify only Fv DNA, amplified some
25 % of the Fp strains and NOVb1 (Fusarium subglutinans), while
VER1VER2 (Fv-specific) amplified weakly from the DNA of all
but one of the Fp strains. The FUS1FUS2 primers (specific for
Fusarium moniliforme, i.e. Fusarium verticillioides sensu Nirenberg 1976) amplified from the DNA of all Fp isolates as well.
PRO1PRO2 primers, which target the calmodulin gene, effectively identified all the Fp strains except for one tester isolate,
whereas Fp3FFp4R (IGS region) gave a positive result for just
50 % of the Fp isolates.

ITS-RFLP and ITS sequence analysis

The digestion of the w600 bp amplicon generated by primers
ITS1 and ITS4 (White et al. 1990) with AluI, MboI, TaqI, HaeIII
or HinfI produced two profiles for each endonuclease
(see Table 4). The MP testers A and D were associated with, respectively, profiles A and B, so the inference was that the Fv
isolates would have ITS-RFLP profile A and Fp isolates would
have profile B. A comparison of ITS amplicon sequences
among the sample of 14 isolates plus testers also showed
two clear groups (Fig 2B). Fp isolates differed from Fv types
by a 6 bp insertion, together with a few single base substitutions (Fig S3). Group A and B correspond to Type I and Type
II ITS2 (ODonnell & Cigelnik 1997).

Fertility and vegetative compatibility

As far as sexual fertility, PO1, RT1 and CE1 (among the isolates
tested) did not mate with the tester strains under the conditions imposed. Isolate BU1 was attributed to MP D (Fp) and isolates PI1, CM3 and SA3 to MP A (Fv). As far as vegetative
compatibility, only 2 isolates out of the 14 (SA3 and VP2,

Discrimination between Fv and Fp by primers targeting the

ITS region
Two primers, proITS-R and verITS-F, were designed to anneal
to the polymorphic region of the ITS amplicon (Fig S3), and
were intended to be combined with the universal primers

The ITS region as a taxonomic discriminator

1143

Table 4 Sizes of restriction fragments obtained from digestion of the ITS region from restriction profile A (associated to Fv
tester strains) and B (associated to Fp tester strains). Values in brackets refer to bands too small to be detected on agarose
gel but inferred from sequence analysis
Restriction
profile
A
B

Enzyme
AluI

MboI

HinfI

TaqI

HaeIII

332, 114 (61)

404, 114

305, 110 (60, 29, 3)

174, 143, 110 (60, 28, 4)

245, 156, 91 (8, 7)

266, 245 (8)

192, 118, 85 (59, 53)

215, 192 (59, 53)

347, 93 (67)
281, 93 (77, 68)

ITS1 and ITS4 in simplex or multiplex reactions. Fig 3A shows

the primer positions and orientations, along with expected
amplicon sizes. All 14 isolates and the tester strains for MP A
and MP D were tested (Fig 3B). A w600 bp fragment corresponding to the ITS1/ITS4 product was generated from all templates, providing a positive amplification control. Fp and Fv
isolates produced species-specific amplicons of about 390
and 170 bp, respectively. Both this PCR assay and the ITSRFLP test were extended to all 100 isolates (Table S1), and the
classifications based on PCR all matched those based on morphological analysis. Similarly, proITS-R and verITS-F were
assayed with corresponding results on the MP A and D tester
strains (Table 2) and on 9 isolates of Fv and 7 Fp from different
countries of origin (USA, Italy, Spain, China) and/or hosts
(maize, ornamental palm tree, asparagus, wheat) (Table S1).

Discussion
One prerequisite for a detailed understanding of the ecological
behaviour of the fungi belonging to the Gibberella fujikuroi
complex is to have a robust and reliable means of

A
172bp
ITS1
verITS-F
proITS-R

ITS4

390bp

600 bp
400 bp
200 bp

Fig 3 A. Placement of the PCR primers ITS1ITS4, verITS-F

and proITS-R in the ITS region. B. Representative multiplex
PCRs using primers ITS1ITS4, verITS-F and proITS-R.
Lanes 12: Fv tester strains MATA-1 and MATA-2. Lanes
34: Fp tester strains MATD-1 and MATD-2.

discriminating between the various species. Although host

range and morphology are normally rather species-specific,
Fp and Fv are quite similar to one another at the morphological
level, and are often both present in infected maize. Distinguishing between them has been based mainly on their genetic diversity and on their sexual incompatibility with one
another (ODonnell et al. 1998). Here, we have characterized
monoconidial strains of fumonisin-producing Fusarium isolated from pink rotted maize kernels. The majority of these
(83) were Fv and the rest (16) mainly Fp. One Fusarium subglutinans was identified from the same sampling and retained in
the collection as a supplementary member of the G. fujikuroj
complex. The Fv and Fp strains could be distinguished from
one another by their AFLP and ITS-RFLP profiles, and by the
DNA sequence present in the ITS and the calmodulin gene.
Species classification based on any of the molecular analyses
was in agreement with conclusions based on sexual crosses
and vegetative morphology.
Because Fv and Fp are so similar to one another at the morphological level, and because they are often simultaneously
present in diseased maize kernels, the availability of a simple,
cheap and reliable diagnostic method is clearly desirable, and
a variety of PCR-based analyses to be used in combination
might be the best compromise between reliability and cost effectiveness. A number of PCR-based diagnostic assays have
been published as able to discriminate between these two species with a high level of accuracy (Jurado et al. 2006; Patino et al.
2004). The effectiveness of some of them was, however, limited when tested against the present collection of Italian Fv
and Fp isolates (with VER1VER2 and PRO1PRO2 performing
best). Similarly, conflicting results were obtained by others
with primer pairs VERT1VERT2 and PRO1PRO2 on Fp isolates
from Allium fistulosum in Japan (Dissanayake et al. 2009). A
probable explanation for the observed discrepancies is that
the primer sequences were designed on the basis of too limited a sample of fungal strains. Since the extant Fusarium
species-specific primers proved not all sufficiently robust
in our conditions, we searched for additional amplification
targets, and the ITS region was identified as a likely candidate.
The ITS sequence of the sample of 14 isolates and four tester
strains showed that species-specific polymorphisms were indeed present. Most likely, they reflect the relative intragenomic abundance of the nonorthologous type I and type II
ITS2 (ODonnell & Cigelnik 1997). The resulting PCR assays
proved to be an effective, species-specific diagnostic for Fv
and Fp, regardless of the geographical and host origin.
Sequence variation within the ITS region on its own can
discriminate between Fv and Fp, but ITSs cannot be considered phylogenetically informative more widely within the genus Gibberella and particularly are not sufficient for species

1144

discrimination between Fv and F. subglutinans, among maize

pathogens (this work and Mule et al. 2004; ODonnell & Cigelnik 1997). However these two species can be distinguished relatively easily from one another at the morphological level.
Overall, we conclude that sequence variation within the ITS
region should be exploited as a taxonomic diagnostic, particularly in the context of discriminating between Fv and Fp. The
ITS-RFLP and PCR assays described here provide a simple
and reliable means of discriminating between Fp and Fv isolates, and should be added to the toolbox of mycologists
researching the fumonisin-producing pathogens of maize.

Acknowledgements
The authors are thankful to Dr. Mariangela Girlanda for discussing the results, to Marzia di Maio and Federica Mattio
for technical help, and to Dr. Ursula Hettwer for fumonisin
analysis. Work supported by Regione Piemonte Progetti
CIPE Tecniche di controllo delle micotossine del mais per
impieghi alimentari e zootecnici and Progetto pilota per la
produzione di cereali ad uso alimentare a basso contenuto
in micotossine.

Supplementary information
Supplementary information associated with this article
can be found in the online version at doi:10.1016/
j.mycres.2009.07.011.

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