2
.
5
. Field experiments The efficacy of the antagonists A42 and B11
against bunch rots of wine and table grapes was evaluated during 1996, 1997 and 1998 on ‘Thomp-
son Seedless’ and ‘Superior Seedless’ table grapes and ‘Sauvignon blanc’ wine grapes in
vineyards located in the southern coastal plains Lachish and Golan heights Yonatan. Un-
treated vines and chemically treated vines served as controls. Details of the field experiments con-
ducted on wine and table grapes in 1996 – 1998 are shown in Table 1. Antagonists were grown for 48
h in 1 l bottles with NYDB, on a rotary shaker as described above. Cells were pelleted, resuspended
in tap water at the initial concentration and di- luted ten fold except in the 1996 experiment with
table grapes, where the cultures were used at the original concentration. Experimental plots con-
sisted of one to seven vines per treatment in the different experiments, arranged as randomized
blocks with four replicates. The antagonists and chemical controls were applied two to five times
until run-off, with a motor-driven back-sprayer. The incidence of decay in the wine grape experi-
ments was determined on the day of harvest. Forty clusters were sampled from each plot and
scored according to the causal agent of the decay and the percentage of rot. In the table grape
experiments, no decay developed in the vineyard and rot was evaluated only after storage. Approx-
imately 3 kg of grapes were harvested from each plot and packed in plastic boxes which were
wrapped in polyethylene bags to create high rela- tive humidity. In 1996, the last spray was applied
on the day of harvest and the grapes were picked after the clusters had dried. In the ‘Thompson
Seedless’ experiments in 1997 and 1998 there were two harvests: the first one week after the penulti-
mate spray, and the second after the clusters had dried following the last spray. Rot development
was evaluated after 3 – 4 weeks storage at 0°C followed by 3 – 4 days at 20°C. After arc-sin trans-
formation of the data, analysis of variance was carried out by Duncan’s multiple range test using
the SAS GLM SAS Ins. Cary, NC procedure.
3. Results
3
.
1
. Screening for antagonists among epiphytic microflora
The antagonistic activity, against B. cinerea of
Table 1 Details of field experiments conducted on wine and table grapes in 1996–1998
1998 1997
1996 Wine
Table Table
Wine Wine
Table A42, B11
A42, B11 Isolates
A42, B11 A42, B11
A42 A42
1:1 1:10
1:10 Dilution
a
1:10 1:10
1:10 4 vines
3 vines Replicate size
b
7 vines 1 vine
7 vines 3 vines
S.B. T.S. S.S
Cultivar S.B.
c
T.S. S.B.
T.S. Weekly
14 days Superior 10–14 days 10-days
Weekly Spray frequency
Weekly Weekly
Thompson 3
2 Superior 4 Thompson No. of sprays
5 4
5 4
− +−
− +
− Augmentation
spray Iprodione table grapes Fluazinam
Iprodione 0.075
d
Pyrimethanil 0.08 Chemical control
Benzamidazole 0.025 0.05 wine grapes
a
Dilution factor from the concentration at the stationary phase.
b
Four replicates in each experiment.
c
‘Sauvignon blanc’, S.B.; ‘Thompson Seedless’, T.S.; ‘Superior Seedless’, S.S.
d
Active ingredient.
Table 2 Efficiency of vineyard yeast isolates in suppressing rot caused
by B. cinerea on detached ‘Thompson Seedless’ berries
a
Percent inhibition No. of isolates
No. of isolates 1995
1993 of rot
b
25 0–20
27 20–40
24 7
15 6
40–60 18
60–80 1
5 –
80–90 –
90–100 1
39 90
Number of tested isolates
a
Detached ‘Thompson Seedless’ berries were wounded with a pin, treated with 10 ml antagonist cell suspension and inocu-
lated with 10 ml of 5×10
4
conidiaml suspension of B. cinerea.
b
Percent of control.
3
.
3
. Biocontrol on small bunches The infection levels in the small bunches, inocu-
lated by spraying the pathogen cell suspension and without wounding the berries, were lower
than those in the detached wounded berries. Final infection levels in the control treatments were 70,
72 and 76, respectively, for Aspergillus, Botrytis and Rhizopus Fig. 2. The isolates were equally
effective in reducing the decay caused by all three fungi. B11 significantly P = 0.05 reduced decay
development by Rhizopus, Aspergillus and Botrytis to 50, 54 and 63, respectively, in comparison
Fig. 1. Effect of antagonists on rot development in wounded detached berries. Berries were punctured once with a pin 2
mm deep. Ten microliters of antagonist cell suspension was pipetted onto each wound and when dried, berries were inocu-
lated with 10 ml of fungal spore suspension 5 × 10
4
CFUml. Decay development was assessed 4 days after inoculation. Bars
indicate means and standard errors.
isolates collected over 2, years was tested on detached berries. Biocontrol activity of the vari-
ous isolates in reducing the number of decayed berries ranged between 0 and 93 compared to
decay development on wound-inoculated berries to which no isolates were applied Table 2.
Two isolates, A42 and B11, were selected for further studies. These were identified by Centraal-
bureau voor
Schimmeelcultures Baarn,
The Netherlands as Candida guilliermondii A42 and
Acremonium cephalosporium B11.
3
.
2
. Biocontrol acti6ity on detached berries The isolates A42, B11 showing the highest
antagonistic activity in the screening tests were tested for their efficacy to inhibit the three grape
pathogens, B. cinerea, A.niger and R. stolonifer on detached single berries. Results presented in Fig. 1
show that both isolates were significantly effective P = 0.05 in reducing the development of B.
cinerea, A. niger and R. stolonifer when applied at 10
8
CFUml. At lower concentrations both demonstrated reduced efficacy against the three
pathogens. It is worth noting that, of the three pathogens, A. niger was the least affected by both
isolates when applied at 10
7
and 10
6
CFUml.
Fig. 2. Effect of antagonists on rot development on small grape bunches. Small bunches 6 – 10 berries were dipped in
an antagonist cell suspension ca. 5 × 10
7
CFUml. When dry, the clusters were sprayed to run-off with a 5 × 10
4
CFUml fungal spore suspension. Rot percent of decayed berries was
evaluated twice, 4 – 6 days after inoculation. Bars indicate means and standard errors.
number of CFUberry found 3 weeks after a single spray did not differ from that found on
clusters sprayed weekly Fig. 3A. Both isolates survived well on fruit stored for one month at 0
or 20°C Fig. 3B.
3
.
5
. Field experiments Figs. 4 and 5 summarize the effects of the
different treatments on decay development in wine and table grapes, respectively. The predomi-
nant rot pathogen in the experiments on wine grapes, conducted in the Golan heights, was As-
pergillus niger affecting 32, 7 and 37 of the clusters in the untreated control in 1996, 1997 and
Fig. 3. Antagonist survival. Grape clusters were sprayed with a suspension of yeast cells 10
8
CFUml. When the clusters had dried and every following week, five berries were sampled
from each plot into sterile cups with 20 ml of sterile water. The berries were shaken for 1 h on a rotary shaker and serial
dilutions of the wash were plated on BYA in petri dishes. Colonies were counted after 3 – 4 days at room temperature. In
A, lines represent survival on clusters that were sprayed weekly and bars represent survival 3 weeks after a single application.
Survival during storage after a weekly field application B was followed in grapes held at 0° solid line or at 20°C dashed
lines.
with the control bunches, while A42 gave signifi- cant P = 0.05 reductions of 36, 58 and 43,
respectively. The extent of rot increased as the holding time at 20°C increased.
3
.
4
. Sur6i6al of antagonists in the field During the experimental periods in 1996 and
1997, both isolates survived well under the condi- tions existing in the field and storage Fig. 3A and
B are the results from the 1996 experiment. The
Fig. 4. Effect of antagonist sprays on decay development in wine grapes. Vines were sprayed from veraison to harvest at
7 – 10 day intervals five sprays in 1996 – 1997 and four in 1998. Incidence of decay, caused by Botrytis cinerea and
Aspergillus niger, was monitored 1 – 2 days before harvest. Bars indicate means and standard errors see Table 1 for details of
chemical treatment in each year.
the 1996 experiment, had no effect on the percent- age of decayed bunches. In 1997, Fluazinam
0.05 a.i. prevented the development of rot caused by Botrytis cinerea but had no effect on
the incidence of Aspergillus niger. Only in 1998, when Pyramithanil 0.08 a.i. was used as the
chemical control, was there an adequate reduction in total decay incidence compared to the un-
treated control. Neither antagonists reduced de- cay
incidence in
1996 compared
with the
untreated control. B11 had no effect in the 2 years it was applied and therefore was not included in
the experiment in 1998. A42 reduced decay inci- dence significantly in both 1997 and 1998, and
was as effective as the application of the chemicals.
On stored table grapes, Botrytis cinerea was the predominant rot pathogen. Decay severity per-
centage of decayed berries was reduced by both isolates examined in 1996, compared with the
non-treated control Fig. 5. Only A42 reduced decay in 1997 and an augmentation spray of the
yeast, applied on the day of harvest marked in the graph A42 + , did not increase the effect. B11
reduced decay severity in the 1996 experiment, although less than A42; in 1997 it had no effect
and was, therefore, not included in the 1998 ex- periments. Two experiments were conducted in
1998, one with ‘Thompson Seedless’ and the other with ‘Superior Seedless’. Only A42 was included
in these experiments, but no reduction of decay was achieved.
4. Discussion
