Mode of action of milk and whey in the control of grapevine powdery mildew

Abstract

Grapevine powdery mildew, caused by the fungus Erysiphe (Uncinula) necator, is a major disease affecting grape yield and quality worldwide. In conventional vineyards, the disease is controlled mainly
by regular applications of sulfur and synthetic fungicides and, in organic agriculture, by sulfur and botanical and mineral
oils. Research has identified milk and whey as potential replacements for synthetic fungicides and sulfur in the control of
powdery mildew. Electron spin resonance and scanning electron microscopy were used to investigate the possible mode or modes
of action of milk and whey in the control of powdery mildew. Electron spin resonance experiments showed that various components
of milk produced oxygen radicals in natural light, which may have contributed to the reduction of severity of powdery mildew
on treated leaves. Milk and whey caused the hyphae of E. necator to collapse and damaged conidia within 24 h of treatment. Hydrogen peroxide, applied as a source of free radicals, also caused
collapse of the hyphae of E. necator but did not damage conidia, and appeared to stimulate germination. Lactoferrin (an antimicrobial component of milk) ruptured
conidia, but damage to hyphae was not evident until 48 h after treatment. The results support the hypothesis that free radical
production and the action of lactoferrin are associated with the control of powdery mildew by milk.

Full text

CSIRO PUBLISHING
www.publish.csiro.au/journals/app Australasian Plant Pathology, 2006, 35, 487–493
Mode of action of milk and whey in the control of grapevine powdery mildew
P. CrispA,D,T.J.Wicks
B,G.Troup
Cand E. S. ScottA
ASchool of Agriculture, Food and Wine, University of Adelaide, Waite Campus, PMB 1,
Glen Osmond, SA 5064, Australia.
BSouth Australian Research and Development Institute, GPO Box 397, Adelaide, SA 5001, Australia.
CFaculty of Engineering, School of Physics and Materials Engineering, Monash University, Vic. 3800, Australia.
DCorresponding author. Email: peter.crisp@adelaide.edu.au
Abstract. Grapevine powdery mildew, caused by the fungus Erysiphe (Uncinula) necator, is a major disease
affecting grape yield and quality worldwide. In conventional vineyards, the disease is controlled mainly by regular
applications of sulfur and synthetic fungicides and, in organic agriculture, by sulfur and botanical and mineral oils.
Research has identified milk and whey as potential replacements for synthetic fungicides and sulfur in the control of
powdery mildew. Electron spin resonance and scanning electron microscopy were used to investigate the possible
mode or modes of action of milk and whey in the control of powdery mildew. Electron spin resonance experiments
showed that various components of milk produced oxygen radicals in natural light, which may have contributed to
the reduction of severity of powdery mildew on treated leaves. Milk and whey caused the hyphae of E. necator to
collapse and damaged conidia within 24 h of treatment. Hydrogen peroxide, applied as a source of free radicals,
also caused collapse of the hyphae of E. necator but did not damage conidia, and appeared to stimulate germination.
Lactoferrin (an antimicrobial component of milk) ruptured conidia, but damage to hyphae was not evident until
48 h after treatment. The results support the hypothesis that free radical production and the action of lactoferrin are
associated with the control of powdery mildew by milk.
Additional keyword: organic viticulture.
Introduction
Vitis vinifera (European grape) cultivars are grown in many
countries for wine, dried fruit and table grape production, but
are most commonly used for wine production in Australia
and Europe. Powdery mildew, caused by the fungus Erysiphe
(Uncinula) necator, is a major disease of grapevine in many
countries (Magarey et al. 1997) and causes losses due to
damage to berries, reduced bunch weight (Chellemi and
Marois 1992) and a decrease in wine quality (Ough and
Berg 1979).
E. necator is controlled by regular applications of a
number of materials, most commonly sulfur, as a wettable
powder, dust or flowable formulation, oils, inorganic salts
and/or synthetic fungicides. In organic viticulture, sulfur and
vegetable oil formulations are mainly used to control powdery
mildew but other materials such as foliar coatings, e.g. clay
and silicates, and inorganic salts are used by some growers.
Research has identified potential replacements for
synthetic fungicides and sulfur in the control of powdery
mildew, including milk and whey (Crisp et al. 2002). Weekly
applications of milk at concentrations of up to 50% proved
more effective than weekly applications of fenarimol and
benomyl in the control of powdery mildew of zucchini
(caused by Sphaerotheca fuliginea) (Bettiol 1999). Milk,
applied at rates between 1 : 5 and 1 : 10 (v/v), and whey
powder at 25 g/L, provided effective control of grapevine
powdery mildew in greenhouse and field trials (Crisp et al.
2002). Milk and whey appear to act as contact fungicides
against the superficial powdery mildew fungus.
There have been a number of explanations for the action
of milk, including the anti-fungal action of the fatty acids,
the production of free radicals when exposed to UV light
or the creation of osmotic imbalance due to salts and
other components. Bettiol (1999) suggested that milk may
have a direct effect on the fungus or may induce systemic
resistance to powdery mildew in zucchini. There is also
evidence that exposure of milk to the ultraviolet radiation in
sunlight results in the photogeneration of superoxide anions
(Korycha-Dahl and Richardson 1978) and oxygen radicals
that interfere with the cell membranes of Phytophthora
infestans (Jordan et al. 1992). The production of free radicals
when methionine and riboflavin have been exposed to UV
© Australasian Plant Pathology Society 2006 10.1071/AP06052 0815-3191/06/050487

488 Australasian Plant Pathology P. Crisp et al.
light has been shown to control powdery mildew (Tzeng and
DeVay 1989). Free radical production can be measured using
electron spin resonance spectrometry (ESR) (Tzeng and
De Vay 1984, 1989).
Components of milk and whey, particularly lactoferrin
and lactoperoxidase, have been extensively researched as
antimicrobial agents for use in human medicine and food
preservation (Batish et al. 1988; Modler et al. 1998; Kuipers
et al. 1999; Sallmann et al. 1999; Kanyshkova et al. 2000;
Samaranayake et al. 2001). Lactoferrin, an 80 kDa iron-
binding glycoprotein, binds to the membranes of various
bacteria and fungi, causing damage to membranes and loss
of cytoplasmic fluids (Batish et al. 1988; Kuipers et al. 1999;
Sallmann et al. 1999; Samaranayake et al. 2001). However,
research has focused largely on Gram-negative bacteria and
other species primarily involved in food spoilage, and on
fungi related to human health, especially Candida spp.
(Kuipers et al. 1999; Kanyshkova et al. 2000; Samaranayake
et al. 2001). The optimal concentration of lactoferrin for
control of Escherichia coli and Salmonella typhi is 0.2 g/L
(Batish et al. 1988). The concentration of lactoferrin ranges
from 20 to 200 mg/L in bovine milk and from 56 to 164mg/L
in whey (Riechel et al. 1998).
Lactoperoxidase, a known antimicrobial protein, is present
in milk at ∼30 mg/L (Modler et al. 1998). The ability of
lactoperoxidase to control E. coli, Lactococcus lactis and
some other bacteria varied with the concentration of hydrogen
peroxide and thiocyanate, and was reduced in the presence
of low or excessive concentrations of hydrogen peroxide.
Whey contains 5.6–28.6 mg/L hydrogen peroxide (Modler
et al. 1998). Research into the ability of lactoperoxidase to
control fungi appears limited.
Establishing the mode of action of milk and whey will
provide information that will assist the optimisation of spray
timing and intervals in the vineyard. The identification of
the active fractions of milk and whey may also enable the
development of additional commercial controls of powdery
mildew that are suitable for use in organic and conventional
vineyards. Thus, ESR and scanning electron microscopy
(SEM) were used to investigate the mode of action of milk
on grapevine powdery mildew.
Methods
Detection of free radical activity
Milk, whey and selected milk components, including lactoferrin and
apo-lactoferrin (iron depleted), were assessed for the presence and
production of active oxygen radicals using ESR. A Varian E-12 EPR
spectrometer, operating at X-band (∼9.1 GHz) at the Department of
Physics at Monash University, Melbourne, Victoria, was used. All
measurements were performed at room temperature (∼22◦C). The
specimens were placed in standard, special quartz EPR tubes, with
an internal diameter of ∼2 mm. The sensitivity of the apparatus was
confirmed using the standard ‘weak pitch’ sample supplied by Varian,
which gives a stable signal for carbon radicals in pitch that is easily
calibrated against other known standards. All dry samples were tested
undiluted for free radical signals. The apparatus was not sufficiently
sensitive to observe free radical signals at the dilutions used in the field.
Scanning electron microscopy
Infected leaf tissue treated with various test materials was examined
in a Philips XL30 field emission SEM at Adelaide Microscopy. The
microscope was equipped with an Oxford Instruments CT1500 HF Cryo.
The cryogenic technique, involving minimal specimen preparation, was
selected to reduce the likelihood of damage to hyphae and conidia during
preparation.
The following treatments were examined as aqueous solutions: milk
(1 : 5 and 1:10 dilutions of full cream pasteurised bovine milk); whey
(30 g/L whey powder); whey protein (30 g/L); lactoferrin (from bovine
colostrum, 20 mg/L, Sigma Chemicals); lactoperoxidase (from bovine
colostrum, 10 g/L, Sigma Chemicals); hydrogen peroxide (1mL/L);
Synertrol Horti-Oil (2 mL/L, Organic Crop Protectants Pty Ltd) and
Synertrol Horti-Oil (2 mL/L) mixed with potassium bicarbonate (4g/L
Ecocarb, Organic Crop Protectants Pty Ltd). These application rates
were equivalent to rates that were used in previous greenhouse or field
experiments (Crisp et al. 2002).
Young, near-fully expanded leaves with no noticeable powdery
mildew were excised from V. vinifera cv. Viognier or cv. Cabernet
Sauvignon that had been grown in the greenhouse. The leaves were
surface sterilised by soaking in a solution of 0.5 g/L sodium hypochlorite
and Tween 80 for 3min. The leaves were then rinsed in sterile distilled
water three times and allowed to dry on sterile paper towels before
being placed in tissue culture dishes (Falcon), 100mm in diameter
and 20 mm deep, containing 1.5% agar (Bitek, Difco Laboratories,
Michigan, USA). Leaves were placed adaxial side uppermost on four
sterile toothpicks with the petiole embedded in the agar (Evans et al.
1996). Spores of E. necator, collected from vines maintained in tissue
culture using a modified cyclone separator, were brushed onto the leaves
using a sterile artist’s brush (Evans et al. 1996). The leaves were then
incubated in a growth cabinet for 2 weeks at 25◦C with a 12h light/dark
cycle to allow the fungus to grow on the leaf surface and form conidia.
Leaves on which no powdery mildew developed were discarded.
Test materials were applied using an Atomizer reagent sprayer
(Alltech Associates Inc., Belgium) to optimise leaf coverage. In some
experiments, half of the leaf surface was protected by a plastic shield to
examine treatment effects on the same leaf and to explore the possibility
of systemic activity. The dishes were left open in a laminar flow cabinet
to allow the sprayed leaves to dry; the lids were then replaced and the
dishes were stored at ∼15/25◦C night/day, in natural light. Additional
plates containing leaves treated with milk, whey, lactoperoxidase or
lactoferrin were incubated in darkness by wrapping the plates in foil.
Leaf segments, ∼10 ×5 mm, were cut from the treated leaves and frozen
in semi-solid nitrogen, then transferred under vacuum to the preparation
chamber. In the case of leaves that were partly shielded, the leaf segment
was cut to represent both treated and untreated leaf surfaces. The sample
was coated with platinum, then placed on the microscope stage (held at
<–150◦C) and examined. Leaves were examined 24 h after treatment,
unless otherwise stated.
Leaves treated with milk 30 min, 6, 12, 18 or 48 h prior to
examination were also observed to assess response over time. Additional
leaves treated with whey, whey protein and lactoferrin were incubated
in natural light for 48 h prior to examination. Infected leaves, untreated
or sprayed with sterile, reverse osmosis water 24 h prior to examination,
were used as controls.
Observations of the hyphae and conidia of E. necator on untreated
leaves and on leaves sprayed with sterile, reverse osmosis water only
were used as a reference when observing fungal structures on leaves
sprayed with the test materials. The percentage of damaged hyphae
was estimated based on the relative proportion of hyphae that appeared
undamaged and similar in appearance to hyphae on untreated and water-

Control of grapevine powdery mildew Australasian Plant Pathology 489
sprayed leaves. Estimates of damage to the conidia were based on counts
of conidia that appeared damaged and those that appeared similar to
conidia on untreated and water-treated leaves.
A second series of experiments was conducted using leaves with
older colonies of E. necator bearing cleistothecia, which were collected
from the greenhouse, placed directly into tissue culture dishes, as above,
and treated 24 h prior to examination. Untreated leaves were used as
a reference for the appearance of natural deterioration of hyphae and
conidia, to allow comparison with those structures following treatment
with test materials.
Results
Detection of free radical activity
EPR spectrometer signals and outputs for mixtures are
qualitative rather than quantitative and, while showing the
presence or absence of free radical species, do not provide
accurate information on their relative abundance, as they
do for a single substance. All samples tested in the EPR
spectrometer showed a symmetrical free radical signal with
no structure and a linewidth of ∼4 gauss. The control,
sterile, reverse osmosis water had no free radical signal. Both
lactoferrin and apo-lactoferrin produced a signal with the
same shape and structure and of a similar signal to noise
ratio; with the saturation behaviour of the signal, this suggests
that the signal is at least partially independent of the iron
interaction of the molecule.
Scanning electron microscopy
Approximately 90% of E. necator hyphae from 2-week-old
colonies on untreated and water-treated leaf segments
appeared turgid and undamaged, with the remaining 10%
either collapsed or damaged (Fig. 1). Likewise, more than
90% of conidia appeared undamaged 24 h after spraying with
water. There was no obvious difference in the appearance
of conidia and hyphae on samples collected from control
leaves incubated in natural light or where light was excluded.
Conidia produced germ tubes at one end and close to the
leaf surface. Colonies that were more than 6 weeks old
displayed large areas of collapsed hyphae, and conidia
were often collapsed and cracked (Fig. 1b). Cleistothecia
on untreated leaves were roughly spherical in shape and
appeared turgid.
Milk (1 : 10 dilution) or whey (25 g/L), applied to leaves
incubated in natural light 24 h prior to examination, resulted
in extensive damage to both hyphae and conidia but not to
cleistothecia. Most (80–90%) of the hyphae were collapsed
and, in some cases, appeared to have ruptured (Fig. 2a).
A similar proportion of conidia were collapsed, cracked
or ruptured with extrusion of cell contents along the
cracks (Fig. 2b). However, undamaged conidia were able to
germinate. No symptoms were noted on fungal structures on
untreated areas of the same leaves. For treated leaves kept in
the dark for the 24 h prior to examination, appearance of the
conidia was similar to that on leaf segments incubated in light,
but only 20% of the hyphae were damaged. Cleistothecia
appeared undamaged and similar in appearance to those on
untreated leaves.
The extent of damage to fungal structures on leaf samples
observed 30 min after spraying with milk was difficult to
assess accurately as the samples were not completely dry
and milk droplets obscured much of the leaf surface. The
cytoplasmic extrusions were present on ∼20% of conidia
observed 6 h after treatment. Up to 80% of the hyphae
observed were damaged at this time; however, many were
only partially, rather than completely, collapsed as was the
case with samples treated 12, 24 or 48 h previously. There
were no obvious differences in the extent of damage to conidia
and hyphae of E. necator on samples from leaves sprayed with
milk and observed 12, 24 or 48 h after treatment.
Application of whey protein to the leaves exposed to
natural light and maintained at 20–25◦C for 24 h prior to
examination resulted in damage to fungal structures similar
to that observed on leaves that had been sprayed with
milk and incubated in darkness. The majority of hyphae
(>80%) were turgid and similar in appearance to those on
untreated leaves. Conidia displayed cracking and rupturing.
Cleistothecia appeared slightly collapsed and dehydrated
(Fig. 3). Examination of samples 48 h after application of
whey protein showed complete collapse of conidia and
collapse of up to 50% of the hyphae.
When leaves were sprayed with lactoferrin 24 h prior
to assessment and incubated in natural light at 20–25◦C,
∼70% of the hyphae of E. necator appeared undamaged.
The remaining 30% had lost turgidity and, in some places,
appeared to have ruptured. Approximately 90% of the conidia
were cracked and ruptured, with leakage of cell contents.
Leaves sprayed with lactoferrin 48 h prior to assessment and
incubated similarly, showed collapse of ∼50% of hyphae and
all conidia (Fig. 4).
Up to 20% of conidia had split on leaves 24 h after
treatment with lactoperoxidase and incubation in natural light
at 20–25◦C, but the remaining 80% appeared undamaged and,
in some cases, to have germinated normally. Approximately
70% of hyphae appeared turgid and undamaged, whereas the
remaining 30% had lost turgidity and collapsed.
Hyphae were collapsed and ∼50% of conidia appeared
disfigured where mixtures of Synertrol Horti-Oil and Ecocarb
were applied to infected leaves. The integrity of hyphae was
maintained only in areas that were not treated. The conidia
did not show cytoplasmic extrusions, but germ tubes had
collapsed. Residues were more noticeable on leaves sprayed
with Synertrol Horti-Oil plus Ecocarb than on leaves sprayed
with other materials in the experiments. The damage to
hyphae and conidia seen on leaves sprayed with Synertrol
Horti-Oil plus Ecocarb was not evident on leaves sprayed
with Synertrol Horti-Oil alone.
Approximately 80% of the conidia treated with
hydrogen peroxide 24h prior to SEM examination appeared
undamaged, but the hyphae had collapsed and ruptured

490 Australasian Plant Pathology P. Crisp et al.
(a)
(b)
Appressoria
Hyphae
Deteriorated
hyphae
Deteriorated
conidia
Fig. 1. Vine leaf surface with Erysiphe necator sprayed with water.
E. necator on the upper surface of a detached leaf of Vitis vinifera cv.
Viognier, sprayed with sterile reverse osmosis water 24 h prior to
examination and incubated in natural light at 20–25◦C. (a) 15-day colony
and (b) 42-day colony.
Cleistothecium
Intact appendages
Fig. 3. Vine leaf surface with cleistothecium of Erysiphe necator,24h
after whey treatment. Cleistothecium on the upper surface of detached
leaf of Vitis vinifera cv. Viognier collected from the greenhouse, sprayed
with 30 g/L whey protein. After treatment, leaf was incubated in natural
light at 20–25◦C.
(a)
(b)
Crack in cell wall
Collapsed hyphae
Collapsed conidium
Ruptured conidia
Fig. 2. Vine leaf surface with 15-day colony of Erysiphe necator,24h
after milk treatment. E. necator on the upper surface of detached leaf
of Vitis vinifera cv. Viognier, sprayed with a 1 : 10 dilution of milk
and exposed to natural light at 20–25◦C after treatment. (a) Collapsed
hyphae and conidia and ruptured conidia are indicated by arrows.
(b) Close up of ruptured conidium.
Appressorium
Collapsed
conidia
Collapsed
hyphae
Fig. 4. Vine leaf surface with 15-day colony of Erysiphe necator,48h
after lactoferrin treatment. Conidia of E. necator on the upper surface
of detached leaf of Vitis vinifera cv. Viognier, sprayed with 20 mg/L
lactoferrin. After treatment, leaf was incubated in natural light at
20–25◦C.

Control of grapevine powdery mildew Australasian Plant Pathology 491
Germ tubes
Collapsed
hyphae
Collapsed
conidium
Collapsed appressorium
Collapsed
conidium and
germ tube
Collapsed
germ tubes
Germinating
conidium
Collapsed
hyphae
(a)
(b)
Fig. 5. Vine leaf surface with 15-day colony of Erysiphe necator,24
and 48 h after hydrogen peroxide treatment. Hyphae and conidia of
E. necator on the upper surface of detached leaf of Vitis vinifera cv.
Viognier, sprayed with 0.1% hydrogen peroxide solution, showing
collapsed hyphae and germinating conidia. After treatment, leaf was
incubated in natural light at 20–25◦C. (a) 24 h after treatment and (b)48h
after treatment.
(Fig. 5a). The remaining conidia had collapsed completely.
Over 50% of the intact conidia on the hydrogen peroxide
treated leaf segments appeared to have germinated (Fig. 5a),
compared with ∼10% of conidia on untreated leaf segments.
Cytoplasmic extrusions were visible on some partially
collapsed hyphae on leaf segments treated with hydrogen
peroxide. Approximately 50% of the germ tubes that had
emerged from conidia appeared to have collapsed when
examined 48 h after the application of the hydrogen peroxide
solution to leaves that were incubated in natural light
(Fig. 5b).
Discussion
Several of the materials tested caused conidia and hyphae
of E. necator to collapse after incubation for 24 h in
natural light. Milk, whey and whey protein damaged both
hyphae and conidia, whereas damage 24 h after treatment
appeared to be restricted to conidia or hyphae in the case
of lactoferrin and hydrogen peroxide, respectively. However,
both hyphae and conidia were affected 48 h after treatment
with these materials. When infected leaves were incubated
in the dark after treatment with a 1 : 10 dilution of milk,
the damage to conidia was unchanged but the damage to
hyphae was greatly reduced compared with those incubated in
natural light.
Free radicals were produced by all samples when exposed
to natural light. Free radicals have been shown to reduce
the severity of powdery mildew and may have contributed
to the reduction in powdery mildew severity observed in
greenhouse and field experiments. Tzeng and DeVay (1989)
found that exposure of methionine, riboflavin and sulfur-rich
amino acids to light resulted in the production of free radicals
that was associated with reduced severity of powdery mildew
on grapevines. In the field, fluctuations in the intensity of solar
UV radiation may influence the production of free radicals
from the milk or whey solids. Should it occur, this variation
in free radical production might have implications for the
timing of spray applications in the field. Spraying on bright,
warm days could result in greater free radical production
than application on cooler, cloudy days. This may also have
implications for the efficacy of the test materials in cool
climates where the intensity of natural light is lower, leading
to a need to increase concentrations of materials or reduce
spray intervals.
Hydrogen peroxide, which is known to cause DNA strand
breaks and membrane disruption (Hoffman and Meneghini
1979; Imlay and Lin 1988) at a concentration similar to
that in milk, damaged hyphae and appeared to stimulate
germination of conidia that were not ruptured. However,
examination of powdery mildew-affected leaf segments
48 h after application revealed that ∼50% of the germ tubes
emerging from such conidia had collapsed. Whether this
was a direct effect of the hydrogen peroxide, failure of the
germ tubes to form penetration pegs or appressoria, the
induction of a plant defence response or some other effect is
not known.
SEM observations of powdery mildew on leaves treated
with a 1 :5 or 1 : 10 dilution of milk and incubated in natural
light for 6–48 h revealed damage to both hyphae and conidia
that was not evident on leaves sprayed with water or left
untreated. The damage to hyphae of E. necator sprayed with
a 1 : 10 dilution of milk or 0.1% hydrogen peroxide was
similar, whereas only the conidia treated with milk appeared
damaged, suggesting that milk had fungicidal properties. This
supports the results from previous greenhouse trials, in which
milk and whey appeared to provide eradicative control of
grapevine powdery mildew (Crisp et al. 2000).
When light was excluded after treating powdery mildew-
affected leaves with a 1: 10 dilution of milk, the damage
to hyphae was considerably less extensive than when leaves
were incubated in natural light; conidia were ruptured

492 Australasian Plant Pathology P. Crisp et al.
regardless of the presence of light. The reduced damage
to hyphae in the absence of light suggests that the
fractions of milk active against hyphae and conidia in the
24 h after application are different or that they act in a
different manner. Riboflavin–methionine mixtures reduced
the severity of disease due to P. infestans only when
exposed to light, which generated hydroxyl radicals or
superoxide anions depending on pH (Jordan et al. 1992).
If the damage to hyphae was caused by induction of
resistance in the plant due to the application of milk and
whey, exclusion of light should not reduce the efficacy of
the treatments.
The rupturing of conidia of E. necator was observed
on leaves sprayed with milk, whether incubated in natural
light or in darkness, and on leaves treated with whey, whey
protein and lactoferrin. If lactoferrin bound to membranes of
E. necator in these experiments and altered their permeability,
possibly disrupting the osmotic balance of the cells
(O. Schmidt, pers. comm.), increased internal pressure might
cause such rupture. Further research is required to establish
if lactoferrin binds to the cell membranes of E. necator and is
responsible for this damage. However, the visually identical
nature of the damage caused to conidia by lactoferrin, milk
and whey suggests that lactoferrin is an active component
of milk and whey in these reactions. Although lactoferrin
caused more collapse of E. necator hyphae at 48 h than at
24 h after exposure, the majority of the damage caused to
hyphae after treatment with milk, whey or hydrogen peroxide
occurred within 24 h of application. Possibly, components
of milk other than lactoferrin also caused damage to
the hyphae, or the interaction with other components of
milk increased the activity of lactoferrin against hyphae
(Crisp et al. 2003).
Application of lactoperoxidase, while resulting in damage
to ∼20% of the conidia and 30% of the hyphae of E. necator
on sprayed leaf segments, caused less damage than did
milk or whey. However, the ability of lactoperoxidase to
control E. coli, L. lactis and other bacteria varied with the
concentration of hydrogen peroxide and thiocyanate added
(Modler et al. 1998). Damage may have increased if suitable
amounts of thiocyanate or hydrogen peroxide had been
included with lactoperoxidase. The extent of damage to
E. necator observed in the experiments reported here suggests
that lactoperoxidase is a component of milk and whey which
is active in the control of powdery mildew and, as thiocyanate
and hydrogen peroxide are present in milk and whey, the
effects of milk and whey on powdery mildew may be greater
than when lactoperoxidase is applied alone. Further in vitro
and greenhouse experiments are needed to determine the
effect of the additives if lactoperoxidase is to be used in the
control of powdery mildew.
Although some active components of milk and whey, such
as lactoferrin, have been identified, further evaluation of their
mode of action is required. A thorough understanding of
the mechanisms by which components such as lactoferrin
interact with E. necator to cause physical damage will assist
in calculating spray timing and concentrations to achieve
maximum cost-effective disease control.
Investigation of other components of milk is required to
assess their ability to control powdery mildew alone and in
combination. A thorough understanding of the mechanisms
may assist in the development of new products for the control
of powdery mildew and other plant diseases. For example, if
a second protein or molecule is involved in the interaction
of E. necator and lactoferrin, mixtures of the two may be
more effective than either compound used in isolation. As
other components of milk that contribute to the control of
powdery mildew are identified, their modes of action should
be investigated.
Milk, whey, lactoferrin, and mixtures of botanical oil
plus bicarbonate have potential as alternatives to sulfur and
synthetic fungicides for the control of powdery mildew in
grapevines. Spray programs involving milk or whey and oil
plus bicarbonate mixtures would reduce concerns about the
perceived negative impacts of relying on applications of oil
plus bicarbonate or sulfur all season.
Acknowledgements
The research was conducted with support from the
Australian Research Council and industry partners, Temple
Bruer Wines, Glenara Wines and Mountadam Vineyard.
Special thanks to David Bruer from Temple Bruer Wines
for his trust, knowledge and commitment to the project
and Leigh Verrall from Glenara Wines for advice and
experience in organic viticulture. Also, thanks to the staff at
Adelaide Microscopy for assistance with Scanning Electron
Microscopy.
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Received 25 January 2006, accepted 6 June 2006
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