Introduction
Various studies revealed that
Botrytis cinerea, the causal pathogen of Botrytis bunch rot, is mostly
associated with rachises, laterals, pedicels and berry bases, and not
with berry skins as previously conceived (Holz et al., 2003). Provided
that sufficient coverage of inner bunch parts was achieved, laboratory
studies have shown that fungicides almost completely reduce the amount
infection and symptom expression of B. cinerea at all growth stages.
The same efficacy was, however, not achieved with the same fungicides
when using conventional spraying methods in vineyards (Van Rooi &
Holz, 2003).
Failure to control Botrytis and other fruit and foliar diseases in
vineyards is often attributed to insufficient coverage of susceptible
tissue. Research regarding spray application to ensure efficient spray
coverage is therefore needed improve disease management of fruit and
foliar diseases in vineyards. Previously, water-sensitive papers were
used in spray application experiments in South Africa. However, to give
a true indication of spray deposits and penetration on certain critical
positions in grape bunches, cards need to be the same size and
orientation as the target, and this method does therefore not give a
good indication of the spray coverage on the 3-dimensional target sites
in bunches (Holownicki et al., 2002). Furthermore, the target to which
fungicides are applied changes constantly, because of the
transformation of grape bunches. Residue recovery techniques were also
used, but did not give a good indication of application quality such as
uniformity or spray distribution (Holownicki et al., 2002). As part of
a research programme aiming at optimising spray application in
vineyards, the Department of Plant Pathology at the University of
Stellenbosch developed a spray cover assessment protocol using
fluorometry, photomicrography and digital image analyses to measure
spray coverage on susceptible grape bunch parts (Brink et al., 2004). Spray Cover Asessment Protocol
Bunches are sprayed with a fungicide and Yellow Fluorescent Pigment®
(400 g/L, EC) (South Australian Research and Development Institute) at
2L/100L mixture (Fig. 1) mixture at the recommended dose. Sprayed parts
are illuminated under six black lights which are installed in a
custom-made hexagonal illumination box (Fig. 2A) that fits closely
around a Nikon SMZ 800 stereoscopic zoom microscope (Fig. 2B). Images
are digitally captured through a stereoscopic microscope at 20 x
magnification using a high-quality photomicrographic Nikon DXM 1200
digital camera (Fig. 2C). Image analysis and enhancements are done with
Image-Pro Discovery version 4.5 for Windows (Media Cybernetics)
software. In order to reduce background noise (Fig. 3A) and enhance
fluorescent pigment, brightness, contrast and gamma settings are
adjusted (Fig. 3B). The total areas of deposited pigment in selected
areas of interest (AOI) are calculated (Fig. 3C) and the percentage
area covered is subsequently calculated for each AOI. 
Figure 1. Mixture of SARDI Yellow Fluorescent Pigment and the fungicide fenhexamid.
Figure 2. Bunch parts were illuminated in a
hexagonal box fitted with 6 black light tubes (A), visualised under a
stereo microscope at 20x magnification (B) and photographed with a
digital camera (Nikon DMX 1200) (C).
Figure 3. Image processing and analysis of a
digital photo taken of a berry skin from a grape bunch that was sprayed
with a mixture of fenhexamid and Yellow Fluorescent Pigment. (A)
Selected objects are UV-illuminated and digitally photographed at 20x
magnification, (B) subjected to several image contrasting and filtering
processes, (C) an AOI selected and the total area of deposited pigment
calculated for each AOI using Image-Pro Discovery image analysis
software.
Figure 4. Gravity feed mist spray gun.
Figure 5. Mean fluorescent pigment coverage (%
area) on berry skin, pedicel and rachis (bunch closure stage only) at
pea size and bunch closure stages and linear regression lines fitted on
spray volume for part x stage combinations.
Validation Of Spray Application Protocol
Dauphine grape bunches (sampled at pea-size and bunch closure) were
sprayed with a mixture of fenhexamid (Teldor® 500 SC, Bayer) at the
recommended dose (75 ml/100 l) and Yellow Fluorescent Pigment® at
2L/100L (Furness, 2000). Spray volumes ranging from 1 to 15 ml were
applied by means of a gravity feed mist spray gun [Fig 4 (ITW DEVILBISS
Spray Equipment Products)]. Fluorescent pigment coverage data for each
spray volume and growth stage were subjected to the appropriate
analysis of variance, linear regression analysis and variance component
analysis using SAS v 8.2 statistical software. Statistical analyses
clearly showed that the described protocol could be used to accurately
determine coverage on the susceptible bunch parts in grape bunches.
Fluorescent pigment coverage had a significant linear fit on spray
volume (Fig. 5). An increase in spray volume generally led to an
increase in coverage. Coverage was significantly influenced by growth
stage and bunch parts. In general, pea size bunches had a higher mean
percentage area coverage on the different bunch parts than bunches
sprayed at bunch closure. This can be explained by higher porosity of
bunches at pea size compared with more compact bunches at bunch
closure. Structural bunch parts were furthermore up to three times more
difficult to cover than berry skins at both stages. Variance component
analysis revealed that variation can be reduced by increasing the
number of bunches, rather than the samples per bunch or measurements
per image.
ConclusionCollectively, these results
clearly showed that spray applications earlier in the season will
result in higher and more effective spray deposition on the susceptible
bunch parts. Disease management would thus be most effective since
structural bunch parts are most susceptible and pathogen inoculum most
abundant during pre-flower to pea-size stages in vineyards (Holz et
al., 2003). The described protocol provides an essential tool that can
be used to study the optimisation of spray application of
agro-chemicals and/or biological control agents in vineyards. Hence,
adequate deposition of active ingredient on the susceptible vegetative
and reproductive parts of grapevines for effective pathogen or pest
control can be facilitated. In future studies, minimum coverage levels
for effective pathogen control will be determined and subsequently be
used as benchmarks to evaluate spray application in vineyards. The
technology developed in the Botrytis-grapevine model will directly
benefit the management of other foliar and fruit diseases of grapevine,
such as powdery and downy mildew as well as diseases or pests in other
cropping systems.
Authors
JAN-COR BRINK 1, GUSTAV HOLZ 1, FRIKKIE CALITZ 2 & PAUL H.
FOURIE * 1 1 Department of Plant Pathology, University of Stellenbosch,
Private Bag X1, Matieland 7602, South Africa 2 ARC Biometry Unit,
Private Bag X5026, Stellenbosch 7599, South Africa *email:
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Literature Cited
- BRINK, J.C., HOLZ G., CALITZ, F.J., & FOURIE, P.H. 2004
Development of a protocol to quantify spray deposits on grape bunches.
7th International Symposium on Adjuvants for Agrochemicals ISAA 2004,
ISAA 2004 Foundation, South Africa 230-236.
- FURNESS, G.O. 2000. SARDI Fluorescent Pigment suspension
concentrate. Fact Sheet 1-2000. South Australian Research and
Development Institute, SARDI. SARDI/ Primary Industries and Resources.
- HOLZ, G., GÜTSCHOW, M., COERTZE, S., & CALITZ, F.J. 2003.
Occurrence of Botrytis cinerea and subsequent disease expression at
different positions on leaves and bunches of grape. Plant Disease 87:
351-358.
- HOLOWNICKI, R., DORUCHOWSKI, G., SWIECHOWSKI, W., & JAEKEN, P.
2002. Methods of evaluation of spray deposit and coverage on artificial
targets. Electronic Journal of Polish Agricultural Universities,
Agricultural Engineering 5 Issue 1: 1-9.
- VAN ROOI, C., & HOLZ, G. 2003. Fungicide efficacy against
Botrytis cinerea at different positions on grape shoots. South African
Journal of Enology and Viticulture 24: 11-15.
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