reported to cause decay and are of equal eco- nomic importance to B. cinerea Barkai-Golan,
1981. Rots caused by B. cinerea and R. stolonifer
usually begin to appear at the stage of ‘veraison’ fruit softening when sugar content in the berries
reaches 10 – 12 Hill et al., 1981; Marois et al., 1992. A. niger usually causes decay only at higher
sugar levels 15, although it is one of the fungi found on the surface of healthy grapes at all
stages Zahavi, unpublished data. The relative importance of the several rot-causing fungi, both
in the vineyard and in stored grapes, changes from one season to another, probably because of
differing climatic conditions. Since there is no way of knowing, at the onset of veraison the recom-
mended time to start chemical control programs, which pathogen will be predominant, it is difficult
to recommend a specific chemical control pro- gram in the vineyard. After harvest, SO
2
is gener- ally very effective in preventing the development
of decay Ben Arie et al., 1991, but effectiveness depends on the pathogen and the inoculum load.
Higher amounts of SO
2,
which might be more efficient, cause bleaching of the berries and an
off-flavour. Recently, SO
2
has been removed from the federal government’s ‘generally regarded as
safe’ GRAS list in the USA and its future use on grapes is therefore uncertain.
Public demand to reduce pesticide use, stimu- lated by greater awareness of environmental and
health issues, as well as development of resistance of some of the pathogens to the fungicides, limits
the application of chemicals on agricultural prod- ucts. In recent years much research has focused
on developing alternative control methods against pre- and postharvest decay in grapes as well as
other agricultural commodities Ferreira, 1990; Peng and Sutton, 1991; Wilson et al., 1991, 1993;
Filonow et al., 1996; Fokkema 1996; Harman et al., 1996; Leibinger et al., 1997.
In previous work, we evaluated the activity of the yeast strains, Kloeckera apiculata and Candida
guilliermondii strain U.S.7 in reducing posthar- vest decay of table grapes Ben Arie et al., 1991;
McLaughlin et al., 1992. When applied as a postharvest dip, both antagonists were found to
protect injured and artificially inoculated grape berries and also to reduce the incidence of decay
of naturally infected, non-injured grapes. How- ever, one of the important quality parameters of
table grapes is the presence of a bloom on the surface of the berry. A postharvest dip removes
some of the bloom, thereby adversely affecting the perceived quality of the treated fruit. Therefore,
the possibility of treating the fruit with antago- nists before harvest was evaluated Ben Arie et al.,
1991. The efficacy of the yeast C. guilliermondii in stopping disease development in the vineyard
and in reducing postharvest decay was demon- strated. However, the treatment did not retain its
effectiveness and the incidence of Botrytis decay increased considerably during 4 weeks storage at
0°C. This may be attributed to poor colonization and survival of this particular strain, which had
been isolated from the surface of lemon. We therefore decided to look in the vineyard for
effective antagonists of grape pathogens that are better adapted to the pre- and post-harvest
environment.
In the present paper we describe the isolation of natural epiphytic yeast antagonists from Israeli
vineyards, and the evaluation of their biocontrol activity against bunch rots under laboratory and
field conditions.
2. Materials and methods
2
.
1
. Yeast isolation Epiphytic micro-organisms were isolated by
shaking five grape berries in 10 ml of sterile distilled water for 1 h at 200 rpm on a rotary
shaker Peng and Sutton, 1991. The wash was serially diluted and 30 ml of each dilution were
spread on basal yeast agar BYA containing 20 g glucose, 1 g yeast extract, 10 g protease peptone
and 15 g agar amended with 250 mg Penicillin G to suppress growth of bacteria in 1 l of distilled
water. The Petri dishes were incubated at room temperature for 4 days and yeast-like colonies
were selected randomly according to colour and morphological characteristics, removed with a
sterile plastic loop and transferred to fresh BYA plates to obtain pure cultures. Colonies were held
at 4°C until used. Selected isolates were identified by the Centraalbureau voor Schimmeelcultures
Baarn, The Netherlands.
2
.
2
. Bio-control assay on detached berries Cultures were grown on a rotary shaker at 200
rpm in 50 ml nutrient-yeast dextrose broth NYDB for 48 h at room temperature 20 – 25°C.
Cells were then pelleted by centrifugation and resuspended in an equal volume of sterile distilled
water to give a final concentration of 10
8
– 10
9
cellsml. Conidia of B. cinerea were obtained from 2 – 4
week old PDA cultures incubated at room temper- ature. Spores were suspended in sterile distilled
water, filtered through two layers of cheesecloth and the spore concentration was adjusted to 5 × 10
4
conidiaml. Individual berries of ‘Thompson Seedless’ grapes
were removed from clusters, surface disinfected by dipping for 1 min in 1 vv sodium hypochlorite
pH 11.5 and mounted on masking tape strips glued to PVC pads on the floor of the incubation
box. The berries were punctured with a pin 2 mm deep and 10 ml of an antagonist cell suspension
were pippetted onto the wound site and left to dry for 1 – 2 h, after which the berries were inoculated
with 10 ml of the conidial suspension. Each treat- ment was applied to three replicates of seven or
eight berries. Following treatment, wet filter paper was placed in the boxes which were covered with
polyethylene to maintain high relative humidity. The percent of decayed berries in each replicate was
evaluated after 4 – 5 days at 20°C.
The effect of two isolates A42 and B11, which were the best antagonists in the screening tests, on
the control of decay caused by A. niger and R. stolonifer and the effect of cell concentration on
biocontrol activity were tested on detached berries as described above, with minor changes. The iso-
lates were grown in NYDB and cell concentrations were adjusted to 10
8
, 10
7
and 10
6
cellsml. Conidia of B. cinerea and A. niger and sporangiospores of
R. stolonifer were obtained from 1 – 2 week old PDA cultures incubated at room temperature 20 –
25°C and suspended in sterile distilled water at concentration of 5 × 10
4
CFUml. Analysis of variance, for all laboratory experi-
ments, was carried out using Duncan’s multiple range test.
2
.
3
. Biocontrol acti6ity on grape clusters Biocontrol activity of isolates A42 and B11 was
evaluated against B. cinerea, A. niger and R. stolonifer on small bunches of grapes. Clusters of
‘Thompson Seedless’ grapes were divided to make ‘bunches’ of ten berries. Antagonists were cultured
as described above and diluted 1:10 in sterile distilled water. The bunches were dipped in the
antagonist suspension, allowed to dry for 2 – 3 h and then sprayed with fungal suspensions 5 × 10
4
CFUml of B. cinerea, A. niger or R. stolonifer. Bunches were incubated at 20°C for 4 – 5 days and
percent infection was determined for each bunch. Each treatment consisted of four replicates of five
small bunches.
2
.
4
. Sur6i6al of antagonist yeasts in the 6ineyard Survival of the antagonists under Israeli vineyard
conditions and in storage was determined in the table-grape experiments. Samples were collected on
the first spraying date, after the clusters had dried and thereafter before each spray. Five berries per
plot were sampled aseptically into 150 ml sterile cups containing 20 ml of water and shaken on a
rotary shaker at 200 rpm for 1 h. After serial 1:10 dilutions, 20 ml of each dilution were plated in Petri
dishes containing BYA and the plates were held at room temperature for 3 – 4 days, after which the
number of colonies was counted. Antagonist sur- vival was determined as CFUberry on clusters that
received a spray every week and on clusters that were sprayed only once, at the beginning of the
experiment. Their survival was also followed after harvest on fruit packed as described and held at
either 0 or 20°C. Data analysis mean and standard error was carried out after logarithmic transforma-
tion of the data. On two sampling dates, samples of recovered isolates were compared to pure cul-
tures of the antagonists, using rapid DNA isolation and RAPD PCR with two different sets of primers,
as described by Schena et al. 1999.
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
