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
3
.
1
. Biocontrol acti6ity of A. pullulans isolates In the assays on wounded apples, A. pullulans
isolates varied in their biocontrol activity against B.
cinerea and some of them provided a
significant reduction of decay P = 0.05. In particular, a high level of control was achieved by
the isolates L47 and 198 93 less decay in comparison with the control whereas complete
inhibition of decay was obtained with the isolates 235, 240, LS236 and LS250 Fig. 1. Among the
most effective antagonists on apple, the isolates LS236 and LS250 were selected for further studies
because they also showed a different ap-PCR pattern than L47; isolates 235 and 240 were
discarded because they had the same ap-PCR patterns as L47. Isolate 198 was not differentiated
from isolate LS236 Fig. 6.
At high concentrations 10
8
and 10
7
cellsml, isolates LS236, LS250 and L47 provided a
significant P = 0.01 reduction of decay of grapefruit, cherry tomato and table grape Fig. 2.
In particular, on table grape and cherry tomato, complete inhibition of decay caused by B. cinerea
and A. niger was obtained with isolate L47, while complete inhibition of decay due to A. niger on
table grape was achieved with isolates LS236 and LS250. At a lower concentration 10
6
cellsml, all the
antagonists showed
less decay
control. However, a significant reduction P = 0.05 of B.
cinerea decay on table grape and cherry tomato was obtained with isolates L47 and LS250, while
decay due to A. niger on table grape and P. digitatum on grapefruit was reduced only by
isolate L47.
3
.
2
. In 6i6o assays with small table grape bunches Isolates L47, LS236 and LS250 also were very
effective in reducing the incidence of decay on small grape bunches. For instance, isolate L47
reduced the incidence of decay caused by R. stolonifer, A. niger and B. cinerea by 54.8, 70.7
and 61.3, respectively, as compared to the con- trol Fig. 3. No significant differences were found
between isolates LS236, LS250 and L47 in con- trolling decay caused by the three pathogens
tested.
3
.
3
. Biocontrol acti6ity under field conditions L47 was further tested to control grey mold on
table grape under field conditions. The results shown in Fig. 4 indicate that the chemical treat-
ment iprodione was the most effective treatment in reducing grey mold after 30 days of storage at
0°C followed by 3 or 9 days of shelf-life. Prehar- vest application of L47 four and five sprayings
resulted in a significant reduction of both inci- dence and severity of the decay, as compared to
the control. When evaluated at 3 or 9 days, grey
Fig. 1. Biocontrol activity of different isolates of A. pullulans on injured fruits of apple against B. cinerea. Columns with different letters are statistically different according to Duncan’s multiple range test at P = 0.05.
Fig. 2. Biocontrol activity of isolates L47, LS236, and LS250 on injured fruits against A. niger on table grape, P. digitatum on grapefruit, B. cinerea and R. stolonifer on table grape and cherry tomato. Antagonists were applied at three different concentrations
10
8
, 10
7
and 10
6
cellsml. For each concentration, columns with different letters are statistically different according to Duncan’s multiple range test at P = 0.05, small letters, and P = 0.01, capital letters.
mold decay on grape sprayed four times, was reduced by 33.5 and 27.1, respectively; when the
antagonist was applied five times before harvest, a greater reduction P = 0.01 in disease severity
was achieved 49.5 and 30.1, respectively.
Populations of isolate L47 rapidly increased on grape berries under field condition. During the
period starting from the first application until harvest, the number of cfuberry increased from
1 × 10
3
to 1.6 × 10
4
. During cold storage, the population of A. pullulans further increased from
1.6 × 10
4
to 5.9 × 10
4
cfuberry Fig. 5.
3
.
4
. Characterisation of epiphytic antagonists The genetic characterisation of the antagonists
by ap-PCR showed an extremely high variability in the natural epiphytic population of A. pullulans
isolated in Southern Italy Figs. 6 – 8. Only a few isolates showed the same band patterns with the
three primers tested. Particularly, isolates 235 and 240 showed the same patterns as L47, isolate 198
showed the same patterns as LS236 and isolate 379 showed the same patterns as 380. Isolates
showing the same ap-PCR patterns, showed a similar biocontrol activity Fig. 1. All the other
isolates showed different ap-PCR patterns with at least one of the primers used. The isolates ob-
tained from grapes treated with L47 in the field
Fig. 4. Incidence of gray mold on table grape treated in the field with isolate L47, Rovral iprodione or untreated con-
trol. The grape was sprayed four A or five B times at weekly intervals and the rots were evaluated after 4 weeks of
storage at 0°C followed by 3 and 9 days of shelf-life. For each assessment columns with different letters are statistically dif-
ferent according to Duncan’s multiple range test at P = 0.05, small letters, and P = 0.01, capital letters.
Fig. 3. Biocontrol activity of isolates L47, LS236 and LS250 on small table grape bunches against B. cinerea, A. niger and
R. stolonifer. Columns with different letters are statistically different according to Duncan’s multiple range test at P =
0.01.
had RAPD-PCR patterns characteristic of the L47 isolate Fig. 9.
4. Discussion
