Postharvest Biology and Technology 34 (2004) 179–186
Use of fluorescent amplified fragment length polymorphism
(fAFLP) to identify specific molecular markers for the
biocontrol agent Aureobasidium pullulans strain LS30
F. De Curtis a,∗ , L. Caputo b , R. Castoria a , G. Lima a , G. Stea b , V. De Cicco a
a
Department of Animal, Plant and Environmental Sciences, University of Molise, Via De Sanctis, 86100 Campobasso, Italy
b Institute of Sciences of Food Productions (ISPA), CNR Via Amendola, 122/O Bari, Italy
Received 6 October 2003; accepted 18 May 2004
Abstract
Molecular fingerprinting of biocontrol agents is pivotal both for environmental monitoring and registration purposes. Fluorescent amplified fragment length polymorphism (fAFLP) analysis was utilised for the first time to investigate the intraspecific
variability of the yeast-like fungus Aureobasidium pullulans, in order to identify specific molecular markers for its strain LS30,
an effective biocontrol agent against major postharvest pathogens on different crops, and to pave the way to the development
of molecular-based tools for unequivocal tracking of this agent after its release in the environment. Forty-eight isolates of A.
pullulans from phyllosphere and carposphere of several crops from different sites of Southern Italy and Greece were analyzed
by using four couples of primers. A pairwise comparison of fAFLP patterns was performed, for each primer pair, by using Dice
similarity coefficient (SD ). Four matrices were generated and, subsequently, averaged and combined for constructing a single
dendrogram, in which clustering of fingerprints was performed with the unweighted pair groups (UPGMA). In the combined
dendrogram, most of the isolates grouped into three main fAFLP clusters with levels of similarity ranging from 0.18 to 0.35.
Only two isolates (AU73 and AU91) were very similar in all fAFLP patterns. Only primers AC/CA yielded three DNA sized
fragments that appeared to be specific for LS30.
© 2004 Elsevier B.V. All rights reserved.
Keywords: fAFLP; Aureobasidium pullulans; Biological control; Fingerprints
1. Introduction
Application of biocontrol agents (BCAs) such
as bacteria, yeasts and yeast-like fungi proved to
be effective in reducing postharvest fungal diseases
on different crops (Janisiewicz and Korsten, 2002).
∗
Corresponding author. Fax: +39 0874 404678.
E-mail address: [email protected] (F. De Curtis).
Modes of action of these BCAs have been studied in
order to enhance the ones playing a crucial role in the
efficacy and reliability of these microbes (Droby and
Chalutz, 1994; Castoria et al., 2001, 2003; Grevesse
et al., 2003). Efficacy of BCAs is affected by environmental factors, microbial competition, sexual
recombination as well as their conservation conditions (Hofstein et al., 1994; Guetsky et al., 2002).
In this regard, molecular characterization of BCAs
0925-5214/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.postharvbio.2004.05.008
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F. De Curtis et al. / Postharvest Biology and Technology 34 (2004) 179–186
can provide tools for assessing their genetic stability,
for tracking these agents in practical conditions and,
also, for meeting registration requirements (Hofstein
et al., 1994; Bidochka, 2001; Whipps and Lumsden,
2001). Among the several methods based on phenotype and DNA polymorphism analyses (allozymes,
RAPD-PCR, UP-PCR, REP-PCR, RFLP, PFGE, SSR,
DGGE, etc.) which allow characterisation of microorganisms more efficiently than classical morphological,
physiological and biochemical assessments, amplified
fragment length polymorphism (AFLP) has proven to
be a very promising tool (Vos et al., 1995; Olive and
Bean, 1999; Lima et al., 2003). Key features of AFLP
are its ability to analyze polymorphisms of the entire
genome and to combine the reliability of restriction
fragment length polymorphism (RFLP) analysis with
the flexibility and power of polymerase chain reaction
(PCR) (Vos et al., 1995; Mueller and Wolfenbarger,
1999). Polymorphisms detected by AFLP fingerprinting are multilocus markers, which allow the individuals to be genotyped or differentiated on the basis of
respective alleles (Mueller and Wolfenbarger, 1999;
Olive and Bean, 1999). The AFLP band patterns can
be used for many purposes, and have been shown to
be able to type microorganisms at an isolate level (Vos
et al., 1995; Olive and Bean, 1999). Recently, the use
of fluorescein and digoxigenine-labeled primers has
been proposed for visualizing DNA fragments produced in AFLP analyses, as is the case of fluorescent
AFLP (fAFLP) (Vrieling et al., 1997; Huang and Sun,
1999). In this technique, fluorescent PCR fragments
of each sample derived from AFLP reactions (restriction, ligation with adapters, preselective and selective
amplifications) are loaded automatically in a capillary
electrophoresis (CE) system that allows discrimination and quantification of DNA products differing by
a single base up to 250 bp. Safety of the fluorescent
dyes, automated sample loading, faster separation,
higher resolution and uniform sample electrophoresis are major advantages of CE-based fAFLP, which
significantly increases precision, reproducibility, ease
and speed of analysis (Huang and Sun, 1999).
Aureobasidium pullulans (de Bary) Arnaud is an
epiphytic yeast-like fungus. Some strains, such as
LS30 and L47 display a high biocontrol activity
against the major postharvest pathogens on different
stored crops (Lima et al., 1999; Ippolito et al., 2000).
It has been proposed that efficacy of these BCAs
could significantly be enhanced by their application in
preharvest conditions (Ippolito and Nigro, 2000). The
widespread distribution of A. pullulans in the phyllosphere and carposphere of different plant species
(Blakeman and Fokkema, 1982; de Hoog et al., 2000)
is a relevant limitation for re-isolation and study of
specific biocontrol strains by using classical morphological techniques.
The aim of our study was the comparison of several
isolates of A. pullulans by using automated fAFLP,
in order to obtain both the fingerprint of strain LS30
and to identify strain-specific DNA fragments to be
used in the development of molecular-based tools for
monitoring this biocontrol strain.
2. Materials and methods
2.1. Aureobasidium pullulans strains
Forty-eight strains of the yeast-like fungus A. pullulans were selectively isolated from the surface of
fruits and vegetables, as described by Wilson et al.
(1993), from various locations in Southern Italy and
in Greece (Table 1). The reference strain DSM2404 of
A. pullulans was purchased from Deutsche Samelung
Microbiologike Zeitung, Berlin (DSMZ). Each pure
isolate was stored at −80 ◦ C in 15% glycerol, or kept
on Nutrient Yeast Dextrose Agar (NYDA) at 4 ◦ C for
long-term storage before use.
2.2. Extraction of genomic DNA
Extraction of total genomic DNA of A. pullulans strains was performed according to Hofman
and Winston (1987) with some modifications. Each
strain was grown overnight in 3 ml of yeast extract
Bacto-Peptone Dextrose Broth at 23 ◦ C and 175 rpm.
Cells were collected by centrifugation at 5000 rpm,
washed twice with sterile distilled water, resuspended
in 100 ␮l of lysis buffer [10 mM Tris–HCl pH 8, containing 2% Triton X-100, 1% sodium dodecyl sulfate
(SDS), 100 mM NaCl, 1 mM EDTA] and extracted
with one volume of phenol/chloroform/isoamyl alcohol (25:24:1) in the presence of 0.3 g of acid-washed
glass beads (425–600 ␮m diameter). Tubes were vortexed for 3 min and centrifuged at 13,000 rpm for
3 min at room temperature. The clear upper phase of
F. De Curtis et al. / Postharvest Biology and Technology 34 (2004) 179–186
181
Table 1
Epiphytic isolates of Aureobasidium pullulans used in this study and original source
Strainsa
Host
AU92
AU29, AU66, AU96, AU99, LS30
AU15/2, AU18-2A, AU58
AU100
AU23, AU25, AU98
DSM2404b
AU76
AU20/1, AU28, AU34-2, AU62, AU72, AU74, AU82, AU104, AU111, LS200
AU33, AU69
LS3, LS6
AU31-1, AU42/2
AU61, AU73, AU80, AU91, AU94, AU95, AU112, AU121
AU32/1
AU53, AU57, AU63
AU17/2, AU45/1, AU68, AU24
AU117
Almond tree
Apple tree
Apricot tree
Barley
Cherry tree
Deteriorated army supplies
Fig tree
Grapevine
Lemon tree
Mushroom decay
Oak
Olive tree
Orange tree
Pear tree
Plum tree
Sugar beet
a The prefix “AU” and “LS” before the item number refers to the yeast and yeast-like fungi collection of the Plant Pathology Laboratory,
Campobasso, Italy. The strains were isolated from different sites of Southern Italy and Greece.
b The strain DSM2404 was purchased from Deutsche Samelung Microbiologike Zeitung, Berlin (DSMZ), Germany.
each sample was transferred to a fresh tube. The DNA
was precipitated at −20 ◦ C for 15 min by adding 2 vol.
of isopropanol and 0.1 vol. of 3 M Na Acetate buffer
pH 8 and by centrifugation at 13,000 rpm and 4 ◦ C for
5 min. The DNA pellets were washed with cold 70%
ethanol, dried for a few minutes and resuspended in
an appropriate volume of MilliQ nuclease free water.
The extracted DNA in the samples was quantified by
electrophoresis on 0.7% agarose gel in the presence
of an appropriate marker (Lambda DNA/HindIII,
PROMEGA, Madison, Wisconsin, USA).
2.3. Fluorescent AFLP (fAFLP) analysis
The fAFLP analysis was performed according to
the manufacturer’s protocol (AFLP Microbial Fingerprinting Kit, Applied Biosystem, PE Corporation,
Foster City, California, USA). Briefly, 10 ng genomic
DNA from each isolate was digested with 5 U of
EcoRI and 1 u of MseI (New England Biolabs, Hitchin,
Hertfordshire, United Kingdom) and, in the same
reaction mixture, 1 ␮l each of solutions of 20 ␮M
MseI-adaptor and 2 ␮M EcoRI-adaptor were added for
ligation in 11 ␮l (final volume) of restriction-ligation
buffer and incubated at room temperature overnight.
Four microlitres of 20-fold dilutions of each
restriction-ligation reaction were processed in a pre-
selective PCR in 20 ␮l (final volume) mixture containing 0.5 ␮l of 1 ␮M preselective primer for EcoRI
(5 -GACTGCGTACCAATTC-3 ), 0.5 ␮l of 5 ␮M preselective primer for MseI (5 -GATGAGTCCTGAGTAA-3 ) and 15 ␮l of AFLP Amplification Core Mix
(Applied Biosystem, PE Corporation). The amplification was performed with a 9700 GeneAmp PCR
system (Applied Biosystem, PE Corporation) using
the following cycle parameters: 20 s at 94 ◦ C, 30 s
at 56 ◦ C and 2 min at 72 ◦ C for 20 cycles. After
amplification, samples underwent a 20-fold dilution
with nuclease free TE0.1 (20 mM Tris–HCl, 0.1 mM
EDTA, pH 8.0) and EcoRI-MseI-ended fragments
from each sample were further amplified in 10 ␮l (final volume) reaction mixture containing fluorescent
dye-labeled selective primers for EcoRI [labeled at 5
ends with either blue (FAM), green (JOE) or yellow
(NED) fluorescein-based fluorophores] and unlabeled
selective primers for MseI. Each selective amplification was performed by using four different selective
primer pairs for EcoRI and MseI sites, bearing at
3 ends the following selective nucleotides, respectively: AC/CC (with EcoRI selective primer labeled
with FAM); AT/CG (with selective primer for EcoRI
labeled with NED); AC/CA (with selective primer
for EcoRI labeled with FAM); G/CT (with selective
primer for EcoRI labeled with JOE) (AFLP Microbial
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F. De Curtis et al. / Postharvest Biology and Technology 34 (2004) 179–186
Fingerprinting Kit, Applied Biosystem, PE Corporation). The selective reactions were carried out according to the AFLP microbial fingerprinting protocol by
using the following parameters: denaturation at 94 ◦ C
for 2 min and extension at 74 ◦ C for 2 min; annealing
temperature at 74 ◦ C for 30 s and subsequent decrease
by 1 ◦ C per cycle until 56 ◦ C; 20 cycles at 56 ◦ C and
a final extension of 30 min at 60 ◦ C.
where nXY is the number of common peaks in two fingerprint profiles and nX + nY is the total amounts of
peaks. A single similarity matrix was constructed by
averaging the Dice values derived from four similarity
matrices. The clustering of all fingerprints (dendrogram) was performed with the unweighted pair group
method by using average (UPGMA) linkages (Sneath
and Sokal, 1973) with NTSYSpc software (Applied
Biostatistics).
2.4. Analysis of fragments
Each sample was prepared for analysis by adding
1 ␮l of 10-fold diluted selective PCR products to loading buffer mix [25 ␮l of deionised formammide and
0.5 ␮l of GeneScan-500 (ROX 500 size standards,
Applied Biosystems)]. The samples were denatured
for 2 min at 95 ◦ C and quickly cooled on ice prior
to loading the capillary electrophoresis system filled
with denaturing Performance Optimized Polymer 4
(POP-4) (Applied Biosystems). The set-up of the ABI
PRISM 310 Genetic Analyzer (Applied Biosystems)
was done according to the manufacturer’s instructions.
Fluorescent dyes attached to the DNA fragments were
excited by laser and detected using filter C of ABI
PRISM 310. Afterwards, the data (displayed as peaks
in electropherogram files) were analyzed by using the
ABI GeneScan analysis software (Applied Biosystems, PE Corporation). The fragment sizes were determined by comparison with internal size standards
(GeneScan-500), limiting analysis to fragments between 50 and 500 bp in size and allowing a resolution
of ±1 bp. Reproducibility of peaks in electropherograms was checked by repeating fAFLP reactions on
the isolates examined in this study.
2.5. Data processing and construction of
dendrograms
For the analysis of fragments, output files were exported to Genotyper (Applied Biosystem) and elaborated. A binary matrix was obtained and analyzed
with NTSYSpc software (release 2.0; Applied Biostatistics Inc., Setauket, New York, USA) by using the
band-based Dice similarity coefficient (SD ), according
the following formula (Nei and Li, 1979):
SD =
2nXY
nX + n Y
3. Results
Peaks of fragments generated by primers AC/CA
and specific to strain LS30 are shown in Fig. 1, in
which the electropherogram region with the above
mentioned peaks (398, 431 and 457 bp) is compared
to the same region of electropherograms pertaining
to strains with the highest similarity to the biocontrol strain (see also dendrogram in Fig. 2), i.e. LS200,
AU104, AU62, AU63 and AU76.
Each primer combination yielded a minimum and
a maximum number of highly distinguishable fAFLP
peaks each corresponding to a different DNA sized
fragment, depending on the isolate of A. pullulans,
and a total amount of isolate-specific or common
fragments (data not shown). The combination G/CT
yielded the widest range and the highest number of
different fragments, 5 (isolate AU24) to 86 (isolate
DSM2404), and also a 51 bp fragment that was shared
by most of the isolates (42 out of 48), whereas no
fragment was common to all isolates, whatever the
primer pair used in the analyses (data not shown).
The highest number of isolate-specific fragments (45)
was produced, in the context of this study, by primers
AC/CA (data not shown).
Calculation of the four similarity matrices and averaging of inherent SD coefficients gave rise to a single dendrogram, grouping the isolates according to
the mean similarity level (Fig. 2). The dendrogram
obtained contained three main clusters with similarity levels ranging from 0.18 to 0.35. The largest main
cluster contained about 77% of the isolates and the
total pairwise comparison of the different fingerprints
scored similarity levels ranging between 0.35 and 0.78.
The highest similarity level (0.93) was recorded, in
one of the other two clusters, for isolates AU73 and
AU91.
F. De Curtis et al. / Postharvest Biology and Technology 34 (2004) 179–186
183
Fig. 1. Comparison of peak patterns, in the range 290–460 bp of electropherograms, corresponding to DNA sized fragments generated by
the selective primer pair AC/CA in the fAFLP reaction of strain LS30 and of strains with the highest degree of similarity to LS30 (see
also dendrogram in Fig. 2), i.e. LS200, AU104, AU62, AU63, AU76. Peaks of DNA sized fragments that appear to be specific for LS30
are filled in black.
4. Discussion
Biocontrol strains of yeasts and yeast-like fungi are
becoming a strong alternative to chemicals to control postharvest diseases, or for integration with such
chemicals (Janisiewicz and Korsten, 2002). Some biocontrol yeast-based formulations have been registered
and are being commercialized throughout the world
(Whipps and Lumsden, 2001; Wraight et al., 2001;
Janisiewicz and Korsten, 2002). Aureobasidium pullulans strains have been shown to be highly effective in
controlling postharvest fungal decay of several crops
(Leibinger et al., 1997; Schena et al., 1999; Ippolito
and Nigro, 2000). Modes of action of A. pullulans biocontrol strains involve competition for nutrients and
space, induced resistance and production of lytic enzymes, while no synthesis of antibiotics by strain LS30
was detected and, for this reason, this BCA is a good
candidate for registration (Droby and Chalutz, 1994;
Ippolito et al., 2000; Janisiewicz et al., 2000; Castoria
et al., 2001).
Application of postharvest BCAs before harvest
could enhance their efficacy since their activity is essentially preventive (Ippolito and Nigro, 2000; Droby,
2001), and this is true also for A. pullulans biocontrol
strains when used in preharvest applications in combination with reduced rates of fungicides (Lima et al.,
2003). In this regard, utilization of these strains in the
field needs accurate monitoring strategies for assessments of population dynamics, viability and, genetic
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F. De Curtis et al. / Postharvest Biology and Technology 34 (2004) 179–186
Fig. 2. Dendrogram obtained from fAFLP analyses with four different selective primer sets on 48 isolates of Aureobasidium pullulans
representing the similarity degree. Clusters were constructed with the unweighted pair groups method analysis (UPGMA) by combining
and averaging four similarity matrices, each derived from a single selective primer pair.
stability, which are necessary also for registration procedures (Schena et al., 2000; Bidochka, 2001; Wraight
et al., 2001). Since A. pullulans is a widespread
microorganism in the environment, molecular tools
able to differentiate closely related strains are needed
(Yurlova et al., 1995; Schena et al., 1999; Sabate
et al., 2002). Random amplified polymorphic DNA
(RAPD) and arbitrary primed polymerase chain reaction (ap-PCR) have been successfully used to obtain
information on the genetic complexity of a natural
epiphytic population (Welsh and McClelland, 1990;
Schena et al., 1999), and as the basis for developing a
specific monitoring tool for a biocontrol strain (L47)
of A. pullulans (Schena et al., 2002). However, the
use of relatively low stringency in PCR-reactions in
these techniques increases the chance of non-specific
priming (primer mismatches) and, consequently, the
risk of artificial polymorphisms, which could make
time-consuming the development of monitoring tools
(Mueller and Wolfenbarger, 1999). The relatively low
level of reproducibility in different laboratories has
limited the use of these techniques, whereas AFLP
scores from duplicate test samples revealed average
errors of 0–2% (Mueller and Wolfenbarger, 1999).
In this paper, fAFLP analysis was used for the first
time to assess the intraspecific variability of A. pullulans isolates from different plant sources (Table 1).
The use of four sets of primers for MseI and of fluo-
F. De Curtis et al. / Postharvest Biology and Technology 34 (2004) 179–186
rescent dye-labeled primers for EcoRI yielded highly
reproducible patterns of accurately sized fragments.
We obtained more than five thousand-sized fragments
from all tested isolates. Among these, the primers
G/CT generated the largest number of fAFLP fragments but the lowest number of strain-specific fragments (data not shown). This is probably due to the
presence of only one selective nucleotide on one of
the labeled primers for EcoRI.
The data sets from each fAFLP analysis were used
to calculate levels of similarity among patterns of sized
fragment with the band-based Dice similarity coefficient (SD ). In the pairwise comparison of such fingerprints of different isolates, this “stringent” coefficient attributes double or single weight to the matched
and mismatched fragments with the same size position, respectively, and no relevance to the shared absence of a given fragment. The greater the number
of common fragments the higher was the SD coefficient between two given isolates. The dendrogram
deriving from the average matrix of similarity coefficients (Fig. 1) shows that a high degree of variability
was detected within all clusters. This high intraspecific variability is in agreement with other investigations performed on other strains of A. pullulans by
using Ap-PCR (Welsh and McClelland, 1990; Schena
et al., 1999), or universal primers (Up-PCR) (Bulat and
Mironenko, 1992; Yurlova et al., 1995). A high level
of similarity was recorded only in the case of isolates AU73 and AU91. These same isolates were very
similar to each other with all primer combinations
used and, even considering matrices of SD coefficients
from single primer pairs separately, clustered together
in all inherent dendrograms (data not shown). The
case of AU73 and AU91 was the only one suggesting a possible relationship between high similarity
and source crop plant (olive tree) and/or geographical origin (data not shown). Groupings of isolates,
in fact, do not appear to be related to source crop
plant (or geographical origin). However, further studies are necessary to assess the existence of any possible
correlation.
Finally, as in the main objective of this investigation, the application of the fAFLP technique allowed
us to identify possible specific molecular markers for
the biocontrol strain LS30. In particular, three fragments that were specific for this strain were detected,
at least in the frame of the isolates examined in this
185
study. These fragments should be sufficiently different
in their size so as to be purified for developing specific
probes or primers for sequence-characterized amplified regions (SCAR). These primers can be properly
adapted in order to obtain TaqMan, Beacons or Scorpion primers for identification and real-time quantification of the biocontrol strain LS30, through the emission of a hybridization-specific fluorescent signal, as
already described for A. pullulans L47 (Whitcombe
et al., 1999; Schena et al., 2000).
Acknowledgements
This work was funded by MIUR (Italian Ministry of
Education, University and Scientific Research, Project
PRIN 2002073389 001: Necrotroph pathogens of
pome fruits and antagonist yeasts during storage: role
of oxidative stress in pathogenesis, in the mechanisms
of action of antagonists and interactions involving
antagonists, pathogens and fungicides).
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