prior to harvest Biggs, 1995, it may be advanta- geous to apply antagonists before harvest. For
this approach to be successful, putative biocontrol strains must be able to tolerate low nutrient
availability, UV-B radiation, low temperatures, and climatic changes. The yeast-like fungus Au-
reobasidium pullulans de Bary Arnaud is one of the most widespread and well-adapted sapro-
phytes, both in the phyllosphere Blakeman and Fokkema, 1982 and in the carposphere. A. pullu-
lans has a high tolerance to desiccation and irradi- ation Elad et al., 1994; Kohl et al., 1995, and
has been considered as a possible biocontrol agent of postharvest diseases Bhatt and Vaughan, 1962;
Leibinger et al., 1997.
A. pullulans is characterised by an extreme vari- ability and genetic instability Cooke, 1959; Bulat
and Mironenko, 1992; Yurlova et al., 1995. Mor- phological and cultural characteristics alone are
not sufficient to assess interspecific variability and to differentiate closely related strains. Random
amplified polymorphic DNA RAPD and arbi- trary primed polymerase chain reaction ap-PCR
techniques can be valid means for characterising naturally occurring micro-organism populations
and to obtain preliminary information about the genetic complexity of a natural epiphytic popula-
tion Welsh and McClelland, 1990; Williams et al., 1990.
Biological control agents differ fundamentally from chemical fungicides in that, in order to be
effective, they must grow and proliferate on the plant surface. Therefore, effective antagonists
must become established in crop ecosystems and remain active against target pathogens during pe-
riods favourable to plant infection Lo et al., 1998. The survival ability of biocontrol agents, in
terms of population size, survival period, and distribution in crops, needs to be surveyed and
compared with biocontrol effects. The most fre- quently used methods for the detection and quan-
tification of microbes in ecosystems is the viable-cell count, typically reported as colony
forming units cfu per unit area or sample weight Li et al., 1996. However, the introduced strains
are
difficult to
distinguish from
indigenous strains. RAPD markers allow the unequivocal
recognition of the selected antagonistic strain af- ter its introduction into the phyllosphere Gullino
et al., 1995. The aims of this research were: i to obtain
preliminary information about the genetic diver- sity of a natural epiphytic population of A. pullu-
lans, isolated in Southern Italy, by using ap-PCR; ii to evaluate the potential of several isolates of
A. pullulans as biocontrol agents; iii to determine the effectiveness of an isolate of A. pullulans L47
previously tested in Southern Italy Lima et al., 1996, 1997; Nigro et al., 1997 in a new environ-
ment Israel and on a different table grape vari- ety; iv to identify and to monitor the population
of the antagonist L47 in the field using RAPD- PCR.
2. Materials and methods
2
.
1
. Isolates Isolates of A. pullulans were obtained from the
surface of several untreated fruits and vegetables from Southern Italy as described by Wilson et al.
1993. For long-term storage, A. pullulans iso- lates were suspended in 15 glycerol and frozen
at − 80°C, or kept on PDA at 4°C for short-term storage. Pathogen isolates Penicillium digitatum
Pers., Botrytis cinerea Pers., Aspergillus niger Tiegh., Rhizopus stolonifer Ehrenb. were obtained
from decayed grape berries and kept on potato dextrose agar PDA at 5°C.
2
.
2
. Preliminary screening of antagonists All the isolated antagonists were tested on ap-
ples cv. Golden delicious wounded with a pin at four sites around the stem-end to a uniform depth
of 2 mm. Isolates were grown in 25 ml of Nutrient Yeast-extract Dextrose Broth NYDB in 100 ml
Erlenmeyer flasks on a rotary shaker at 150 rpm for 48 h. Cells were harvested by centrifugation
1100 × g for 15 min, rinsed twice in sterile deionized water, and resuspended in distilled wa-
ter to the initial concentration of about 10
8
cells ml. A water suspension 20 ml of antagonist cells
10
7
cellsml was pipetted into each wound, and two hours later the wounds were inoculated with
20 ml of B. cinerea spore suspension 5 × 10
4
sporesml. The proven isolate L47 and two additional ones
LS236 and LS250 were selected for further stud- ies and tested for their biocontrol activity against
decay of grapefruit, caused by P. digitatum, table grape, caused by B. cinerea, A. niger and R.
stolonifer, and cherry tomato, caused by B. cinerea
and R.
stolonifer. Grapefruits
were wounded to a uniform depth of 3 mm at three
sites around the stem-end using a dissecting needle, while table grape berries and cherry toma-
toes were wounded to a uniform depth of 2 mm at one site in the equatorial zone. Twenty microlitres
of cell suspension of the antagonist at three differ- ent concentrations 10
8
, 10
7
and 10
6
cellsml were pipetted into each wound, and 2 h later the
wounds were inoculated with 20 ml of pathogens spore suspension 5 × 10
4
sporesml. The wounds treated with water were used as controls.
In all the experiments, fruits were maintained at 20°C at high RH and the percentage of infected
fruits was evaluated after 5 days. In table grape and cherry tomato tests, each treatment was repli-
cated three times and each replicate consisted of ten fruits; in grapefruit and apples, five replica-
tions of five fruits were used. Each test was re- peated at least twice.
2
.
3
. In 6i6o assays with small table grape bunches Isolates L47, SL236 and SL250 were also tested
for their biocontrol efficacy on small table grape bunches with approximately ten berries each,
against B. cinerea, A. niger and R. stolonifer. Small bunches were dipped in an antagonist sus-
pension 10
7
cellsml obtained as described be- fore, or in water control. Two hours later,
bunches were sprayed with a spore suspension of the pathogens 10
5
sporesml of B. cinerea, 5 × 10
4
sporesml of A. niger and R. stolonifer. Each treatment was replicated four times and each
replicate consisted of eight small bunches. The treated bunches were stored at 20°C at high RH
and the percentage of rotted berries was evaluated after 5 days.
2
.
4
. Field tests The trials were conducted on seedless table
grape cv. Thompson in a commercial vineyard located in Lachish Israel using L47. A cell
suspension of the antagonist 10
7
cellsml was sprayed four or five times before harvest at
weekly intervals starting on July 15th. In the first trial four sprayings, the last spraying was
carried out one week before harvest; in the second trial five sprayings the last spraying
was done 2 h before harvest. In addition to the untreated grape control, four treatments with
Rovral iprodione 25 a.i. at 0.15, similar to the schedule used for the antagonist, were also
included. For each treatment, four replications of three grapevines, arranged in a randomised
block
design, were
considered. From
each replication, 8 – 10 bunches were harvested, placed
in commercial plastic boxes and covered with plastic sheeting.
The incidence of natural infections caused by B. cinerea was evaluated after 30 days of
storage at 0°C followed by 3 – 9 days of shelf-life at 20°C using the following empirical scale: 0,
bunch without rots; 1, 0 – 10 of rotted berries; 2, 10 – 25 of rotted berries; 3, 25 – 50 of
rotted berries; 4, 50 – 75 of rotted berries; 5, more than 75 of rotted berries. This empirical
scale
made it
possible to
calculate the
McKinney index McKinney, 1923, expressing the weighted average of the disease severity
as actual percentage in terms of the max- imum disease severity. The index was cal-
culated
by the
formula: M
i
= [
d×fT
n
× D]100, where d is the degree of disease
severity assessed
on the
bunch and
f its
frequency, T
n
is the total number of the bunches examined healthy and diseased and D the
highest degree of disease intensity occurring on the empirical scale.
The data were submitted to variance analysis and the mean values were compared using
Duncan’s multiple range test. Percentages were converted into Bliss angular values arcsin
before analysis.
2
.
5
. Population studies on the fruit surface in the field and during storage
To assess the survival of isolate L47 on the fruit surface, the antagonist was sprayed just once at a
concentration of 10
7
cellsml and epiphytic popu- lation was evaluated on grape berries five times, at
weekly intervals. Grape berries were picked the first time 2 h after the application of the antago-
nist July, 14th and then after 7, 14 and 21 days. The epiphytic population was also evaluated at
the beginning August, 11th and at the end of the storage period September, 10th. Each replication
consisted of five berries which were shaken in 100 ml of sterile distilled water on a rotatory shaker at
150 rpm for 30 min. The rinse water was diluted and plated on PDA plates 0.1 mlplate. The
plates were incubated at 25°C, and after 3 – 4 days the colonies showing the typical morphological
characteristics of A. pullulans were recorded. Some of these colonies were randomly selected
and genetically analysed, as described later, to assess if they belonged to the introduced antago-
nist or to other natural occurring A. pullulans strains.
2
.
6
. Characterisation of epiphytic antagonists To characterise the natural epiphytic popula-
tion of A. pullulans the following primers derived from minisatellite or repeat sequences ap-PCR,
were used: GACAGGACAGGACAG GACA- G
3
, GACAGACAGACAGACA GACA
4
, and CAGCAGCAGCAGCAG
CAG
5
, Freeman
and Shabi, 1996. RAPD-PCR reaction, with the primer 5-ACC-
CGGTCAC-3 OpD-20, from Operon Technolo- gies, Inc. USA, was used to identify the isolate
L47 after field treatment and re-isolation. Single colonies showing the characteristic morphology of
A. pullulans were randomly selected from Petri dishes and analysed. A pure culture of isolate L47
was used as a control.
The primers used for ap-PCR and RAPD-PCR were preliminarily selected to be the best ones
providing a greater number of polymorphisms. Genomic DNA was extracted by the method of
Hofman and Winston 1987 with some modifica- tions. One loop of antagonists taken from a
colony actively growing on a PDA plate, was suspended in Eppendorf tubes containing 100 ml
of breaking buffer 2 Triton X-100, 1 SDS, 100 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA.
Acid-washed glass beads 0.3 g, phenol 50 ml, chloroform 48 ml and isoamyl alcohol 2 ml were
added to the solution to make a final concentra- tion of 10
7
– 10
8
cellsml. Eppendorf tubes were vortexed at high speed for 5 min, and then spun
in a microfuge at room temperature for 5 min. For ap-PCR, amplification reactions were per-
formed in a total volume of 20 ml, containing 10 – 100 ng of genomic DNA, 50 mM KCl, 10
mM Tris – HCl, 0.2 mM each of dATP, dCTP, dGTP and dTTP, 1.5 mM MgCl
2
, 1 unit of Taq polymerase Promega, Madison, WI, USA and 1
m M primer. The reactions were incubated in a
programmable Thermal Controller PTC-100
tm
, Peltier-Effect Cycling, MJ Research, Inc, USA,
starting with 5 min of denaturation at 94°C, followed by 30 cycles consisting of 30 s at 94°C,
30 s at either 60°C [for CAG
5
] or 48°C [for GACA
4
, and GACAC
3
] and 1.5 min at 72°C Freeman and Shabi, 1996.
For RAPD-PCR, reactions were performed in a total volume of 25 ml, containing about 25 ng of
genomic DNA, 50 mM KCl, 10 mM Tris – HCl, 100 mM each of dATP, dCTP, dGTP and dTTP,
2 mM MgCl
2
, 0.5 unit of Taq polymerase and 5 pmols of a single ten-base primer. The reactions
were incubated in a programmable Thermal Con- troller starting with 5 min of denaturation at
94°C, followed by 45 cycles of 1 min at 94°C, 1 min at 36°C, and 2 min at 72°C. Negative con-
trols no template DNA were used in every ex- periment to test for the presence of contamination
in the reagents. The amplification products were analysed by electrophoresis in 2 agarose gels in
TAE buffer at 70 V for 1.5 h, and detected by staining with ethidium bromide.
3. Results
