ABSTRACT
EFFICACY OF PHEROMONE MATING DISRUPTION FOR
VINE MEALYBUG CONTROL
The vine mealybug (VMB), Planococcus ficus (Signoret), is an established
vineyard pest in California along with almost all other wine grape growing
regions. The influence of the pheromone, [S] lavandulyl senecioate, is expected to
form a cost effective mating disruption system across commercial scale vineyard
plantings. Proprietary pheromone puffer systems, standard pheromone dispensing
cards and delta traps were all used to gather data on infestation level and VMB
movement. The experiments show that whenever pheromone is present in the
vineyard with any level of VMB infestation there will be a mating disruption
effect. Pheromone puffer systems produced significant improvements over
insecticide treatments alone with up to 50% reduction in total damage to the
harvested crop. Standard pheromone dispenser cards supported highly effective
mating disruption with only 2% of the total crop affected in any way when cards
were deployed at 175 per acre. Flight monitoring through pheromone traps
indicates male VMB populations are unaffected by high summertime temperatures
and are unable orient to pheromone point source plumes against average daily
wind conditions. Visual damage assessments also indicate that the correct density
and loading of pheromone dispenser can influence extremely strong mating
disruption and lead to effective economic VMB control in conjunction with low
impact insecticide treatments.
David John Langone
May 2013
EFFICACY OF PHEROMONE MATING DISRUPTION FOR
VINE MEALYBUG CONTROL
by
David John Langone
A thesis
submitted in partial
fulfillment of the requirements for the degree of
Master of Science in Viticulture and Enology
in the Jordan College of Agricultural Sciences and Technology
California State University, Fresno
May 2013
APPROVED
For the Department of Viticulture and Enology:
We, the undersigned, certify that the thesis of the following student
meets the required standards of scholarship, format, and style of the
university and the student's graduate degree program for the
awarding of the master's degree.
David John Langone
Thesis Author
Kaan Kurtural
Kent Daane
Sonet VanZyl
Viticulture and Enology
Entomology
University of California, Berkeley
Viticulture and Enology
For the University Graduate Committee:
Dean, Division of Graduate Studies
AUTHORIZATION FOR REPRODUCTION
OF MASTER’S THESIS
I grant permission for the reproduction of this thesis in part or in
its entirety without further authorization from me, on the
condition that the person or agency requesting reproduction
absorbs the cost and provides proper acknowledgment of
authorship.
X
Permission to reproduce this thesis in part or in its entirety must
be obtained from me.
Signature of thesis author:
ACKNOWLEDGMENTS
I would like to acknowledge West Coast Grape Farming, Bronco Wine
Company, California Department of Food and Agriculture, and the Kearney
Agricultural Research Center for the funding and support of this project. I would
also like to thank the VERC lab team including Geoffrey Dervishian, Kerry, Buse,
and Tiffany. A special thanks goes out to the professors that helped with this
research including Kaan Kurutral, Kent Daane, and Sonet vanZyl. I also
acknowledge the continued support of my family and friends throughout my
education.
TABLE OF CONTENTS
Page
LIST OF TABLES ................................................................................................. vii
LIST OF FIGURES ............................................................................................... viii
INTRODUCTION .................................................................................................... 1
LITERATURE REVIEW ......................................................................................... 5
Vine Mealybug Biology .................................................................................... 5
Refuge Effects ................................................................................................... 7
Grapevine Leafroll Virus Vectors ..................................................................... 8
Ant Interactions ............................................................................................... 11
Flight Ability ................................................................................................... 17
Central Nervous System Habituation .............................................................. 18
Parasitoid Activity ........................................................................................... 20
Pheromone Responses/Puffer Systems ........................................................... 22
MATERIALS AND METHODS ........................................................................... 24
Pheromone Deployment Device ..................................................................... 24
Field Sites ........................................................................................................ 25
Treatment Applications ................................................................................... 25
Male Vine Mealybug Flight Activity .............................................................. 26
Cluster Damage Assessments ......................................................................... 27
Dormant Season Crawler Activity .................................................................. 28
Pheromone Device Placement and Spatial Analysis ...................................... 29
Movento/Insecticide Standard Grower Applications ...................................... 30
Pheromone Influence on Male Vine Mealybugs............................................. 30
RESULTS ............................................................................................................... 33
vi
Page
Cluster Damage Assessment (Site 1) .............................................................. 33
Cluster Damage Assessment (Site 2) .............................................................. 33
Male Vine Mealybug Trapping (Site 1) .......................................................... 34
Male Vine Mealybug Trapping (Site 2) .......................................................... 35
Pheromone Dispenser Field Trial ................................................................... 35
Male Vine Mealybug Response ...................................................................... 36
DISCUSSION......................................................................................................... 39
2011 Experiment Proceedings ........................................................................ 39
2012 Experiment Proceedings ........................................................................ 43
CONCLUSION ...................................................................................................... 48
REFERENCES ....................................................................................................... 49
APPENDICES ........................................................................................................ 54
APPENDIX A: TABLES ....................................................................................... 55
APPENDIX B: FIGURES ...................................................................................... 58
LIST OF TABLES
Page
Table 1. Chi-square analysis of percentage of cluster damage in site 1, as
affected by puffer pheromone application, 26 August 2011. .................. 56
Table 2. Chi- square analysis of percentage of cluster damage in site 2, as
affected by puffer pheromone application, 2 September 2011. ............... 56
Table 3. Percent Cluster Damage by Treatment 16 September 2012 ................... 57
LIST OF FIGURES
Page
Figure 1. Cumulative Male Vine Mealybug Trap Counts 2011 ............................ 59
Figure 2. Percent Cluster Damage 2011 ................................................................ 60
Figure 3. Denair 2012 pheromone dispenser loading trial map represented by
block. Values represent mg/card, letters represent total area loading.
A = 25 g/acre. B = 37.5 g/acre. .............................................................. 61
Figure 4. Male vine mealybug pheromone response pattern repetition 1. ............ 62
Figure 5. Male vine mealybug pheromone response repetition 2. ........................ 63
Figure 6. Male vine mealybug pheromone response repetition 3 ......................... 64
INTRODUCTION
Vine mealybug infestations in California vineyards are a threat to fruit
quality and lead to severe economic loss. Vine mealybug infestations threaten
yield and quality in wine vineyards as well as raisin, table grape and juice
vineyards. After VMB introduction to California in 1994, the pest spread north to
several other grape growing regions including Napa and Sonoma counties (Millar
et al. 2003). California holds 891,000 acres of vineyard and 60,000 are estimated
to have VMB (Daane et al. 2009). This represents a distinct threat to economic
profitability in the $5 billion grape industry. Almost 6.9 million tons of grapes are
produced between wine, table and raisin grapes in California and every grape
production area in the state has some level of VMB presence (de Borbon, Gracia
et al. 2004). Growers have little tolerance for the insect, as even small infestations
can lead to significant economic losses due to the unique biology and behavior of
the insect. The latest methods to provide control of the invasive VMB are through
pheromone mating disruption. As an alternative approach to VMB control, mating
disruption retains certain advantages over classical insecticide approaches and
biological control efforts. Growers also sacrifice sustainable growing practices as
expensive and polluting insecticides are applied in efforts to control or eradicate
VMB infestations. Grower testimony and survey indicates that VMB is of critical
concern when considering vineyard management, especially in commercial scale
wine grape production. New methods are required for the grape industry in
California to continue production of high quality fruit by sustainable means.
Mating disruption methods along with improved chemical applications represent
the most promising form of VMB control yet.
2
Current control methods for VMB are based on synthetic chemical
applications, especially in large commercial scale vineyard operations. Contact
insecticides such as Clorpyrifos and Methomyl contribute to air pollution and
disrupt vineyard processes due to extended reentry intervals and other exposure
precautions. Organophosphate class chemicals, like clorpyrifos are highly
restricted and targeted for phase out by the Environmental Protection Agency due
to high ecological impact. Organophosphate contact chemicals are expensive and
not entirely effective due to the refuge effects of VMB. Because of unique VMB
reproduction, multiple insecticide applications may be required each growing
season to prevent crop loss surpassing economic thresholds. Developments of
third generation imidacloprid systemic insecticides are effective in controlling
VMB and have less detrimental environmental impacts (Chang 2002). Grower
testimony will indicate that over multiple growing seasons even the most efficient
and effective insecticides do not provide economically sustainable control of the
VMB.
Mating disruption of VMB using pheromone has been an effective
treatment in preliminary trials and initial research in California vineyards. The sex
pheromone of the VMB has been identified as lavandulyl senecioate the racemic
ester of lavandulol. The pheromone is highly attractive to the male VMB, which
take flight when exposed to the compound (Hinkens et al. 2001). When applied in
the vineyard the pheromone compound induces the mealybugs to search for
female crawlers on reproduce. Soon after taking flight the male mealybugs tire
and perish. This process is aided by the fact that male mealybugs have no mouth
parts and cannot feed for sustenance (Millar et al. 2003). Other aspects of VMB
biology make mating disruption a favorable approach for infestation control and
reproductive disruption. Mating disruption, through reproductive disruption and
3
lower mealybug density, can have a significant influence on crop damage and
economic loss in commercial scale vineyard operations.
Emergency permission to use the sex pheromone is granted through the
State of California Department of Pesticide Regulation, though there is no
previous use of lavandulyl senecioate as a pest control agent. Previous
commercial use of the sex pheromone compound entailed pheromone application
in lures within sticky traps to monitor male VMB presence and or flight
distribution patterns. This manner of use is common in other locations including
Argentina, South Africa, Egypt and Israel (Daane et al. 2009). This study will
deploy the VMB sex pheromone in aerosol puffer devices to evaluate the
effectiveness of mating disruption in commercial scale vineyard situations.
Evaluation of mating disruption on large-scale operations has yet to be conducted
for VMB. Like other mating disruption efforts, aerosol puffer application of
lavandulyl senecioate provides species selective, non-toxic and cost effective way
to control VMB. However, this is the first study to utilize this method in vineyard
treatment areas totaling more than 100 acres.
Along with pheromone emitter devices, experiments evaluating the effect
of the pheromone plume were conducted using basic lure and trap methods.
Evaluating the pheromone plume and male mealybug movement is a key
component to understanding spatial organization and efficient deployment of
control programs. Information on summer mealybug activity is limited, especially
with regard to upper temperature limits and influence of the wind on male flight.
Our research will evaluate spatial influence of the pheromone in field settings
under ambient temperature and average prevailing wind conditions. Sensitivity
and selectivity of individual pheromone components is a crucial step in
understanding VMB biology and supports advances in mating disruption
4
technology. Monitoring the male mealybug activity with relation to a female
source and point source pheromone emitters will shed much light on the biology
and behavior of VMB. Sensitivity, selectivity and responses to lavandulyl
senecioate were evaluated in out experiments to deepen understanding of insect
biology and supports integrated control measures in large scale vineyards.
Vine mealybug sex pheromone mating disruption through timed aerosol
spray application has certain complications inherent to the device system. Spatial
distribution and effective coverage by the puffer devices is unknown in this
particular application. Zone of influence and device density within treatment
blocks is related to cluster damage reduction. In general little is known about
mating disruption of VMB. Less information is available regarding VMB
infestations and control in commercial scale vineyard applications. There is also a
significant gap in knowledge regarding the physical and economic damage that
VMB can cause in untreated vineyard blocks. This experiment goes in depth with
regard to pheromone activity, biology, and behavior of VMB under field
conditions in the San Joaquin Valley.
LITERATURE REVIEW
Vine Mealybug Biology
Plannococcus ficus (Signoret), commonly known as VMB, is native to the
Mediterranean basin where it was first seen feeding on young grapevine canes in
southern Israel. In addition to defoliating and weakening vines, the mealybug
excretes honeydew, which becomes a substrate for sooty mold (Berlinger 1977).
The mealybug moved through more than ten synonyms until gaining its current
classification from Signoret in 1875. The VMB is described as an ovate soft scale
insect about 4 mm long with white waxy secretions covering a flesh colored body.
The outer edge of the VMB is adorned with short spines, also covered in white
mealy wax. Unlike the other mealybug species, the VMB lacks any posterior
filaments and thus is distinguishable from P. citri only by counting the number of
glandular ducts. Due to various biological characteristics, the VMB is particularly
invasive and destructive in California vineyards. Plannococcus ficus has been
observed in 17 California counties as of 2005 and is capable of occupying various
hosts including common weeds like Malva neglecta (Daane et al. 2006). Vine
mealybug also has very specific spatial distribution patterns which almost always
result in a clumped distribution within vineyard sites. Insects are usually highly
concentrated on groups of two to three vines in random spatially distinct locations
throughout the vineyard. This makes sampling field sites extremely difficult due
to large sample sizes that are required to overcome the large margin of error.
Vineyard contour maps show that mealybug infesations are typically clustered on
2-9 vine sections of the vineyard (Geiger and Daane 2001). Statistically
significant sampling patterns are possible but difficult to achieve in commercial
production vineyards.
6
After comprehensive evalutation of the P. ficus life cycle it was determined
that male characteristics appear after the third nymphal instar. Female mealybugs
are flightless while the males are capable of flight and have distinct morphology.
Several nymphal life stages lead to adulthood when females release sex
pheromone to entice flying males for copulation. Each female egg sac, often
containing more than 300 eggs, is also covered in filamentous waxy strands,
characteristic of the female mealybug crawlers (Walton and Pringle 2004). Up to
seven generations in one vineyard growing season have been recorded, especially
in favorable climates with warm spring temperatures. Fast generation time of P.
ficus is the main reason the insect receives priority treatment in California
vineyards. VMB reaches peak reproductive levels at 21°C and is capable of
producing more than 300 eggs at 25°C. VMB is capable of producing many eggs
between 21°C and 25°C making its presence in California vineyards particularly
damaging (Walton and Pringle 2005). In classical studies VMB populations
reached one or two peaks during the growing season and remained at low
population level under bark layers during winter. In spring, the crawlers move
upward and onto green tissue where the life stages and population reach a peak
around May or June. The population of hidden mealybugs under bark layers
reaches a peak at the same time as mealybugs on exposed parts of the vine
(Berlinger 1977). The timing of peak population density of VMB in California
has yet to be evaluated thoroughly but it has been observed that there are multiple
peaks in population level over the growing season.
The many life stages of the VMB occur over various time frames and vary
based on average ambient temperatures. After 1 week in the egg stage the VMB
hatches into a first nymphal instar. Three instar stages last around 1 week each
after which differentiation into male or female occurs. Adult males live for as
7
little as one day under hot conditions after hatching from third nymphal instar
crawlers (Walton and Pringle 2005). It was previously understood that the life
cycle of male VMB was short lived as temperatures increase but we find in current
experiments that the males take flight and reach lures at temperatures exceeding
37°C. We observed these activity patterns in pheromone lure experiments while
also observing that large numbers of males are released at an interval of about 2
weeks. In our insectary populations of VMB the population is flooded with first
nymphal instar insects hatched from the ovisacs left by adult female crawlers.
Refuge Effects
Early in the history of VMB research it was observed that the insects are
hidden well beneath layers of plant tissue or below the soil surface. The VMB
clearly seeks out the deepest layers of bark to take refuge under. Investigators
realized as early as 1977 that standard insecticide sprays would be ineffective
against the VMB. The insects remain protected underneath the bark layers for the
lifecycle of the vine. At ideal temperatures the majority of the population will
move onto the green tissues of the plant while some crawlers remain protected
under bark layers (Berlinger 1977). In California where the valley reaches high
temperatures quickly, mealybugs tend to stay under the bark layers of the
grapevines. The exact temperature thresholds for VMB populations in California
are yet to be determined and could provide crucial information regarding proper
treatment timing or necessity. Refuge patterns and movement of VMB to different
locations on the grapevine has yet to be completely understood. Overall
distribution of VMB in vineyards is another subject yet to be investigated
regarding the biology of the insect.
8
After moving to the upper portions of the vine canopy during the first warm
temperatures the VMB remains protected in bark crevices or under new shoot
growth close to the cordon split. Remaining under bark layers or in protected
crevices on the vine renders chemical insecticide sprays ineffective. Other studies
have observed this trend and note that the insects often hide under bark at the soilair interface (Godfrey et al.). Many research articles and authors site the ability of
the VMB to reside continually under bark layers above or below the soil level
(Daane et al. 2006). Observations and consistent monitoring during by laboratory
personnel also confirmed that VMB crawlers are primarily found under the bark
layers on the trunk or under the cordon arm. The entire insect population is almost
never in completely exposed locations and in the San Joaquin Valley the
temperatures appear to have less of an effect on reproduction than in Coachella
Valley (Daane et al. 2002). The refuge activity of VMB populations is most likely
influenced strongly by ant activity and parasitoid presence so attributing refuge to
temperature alone is difficult.
Grapevine Leafroll Virus Vectors
Grapevine Leafroll viruses are common to all grape growing regions but
until was understood about virus transmission. Grapevine Associated Leafroll
Virus 3 (GLRaV-3) is an Ampelovirus of serious concern globally and in the San
Joaquin Valley. The virus does not spread naturally between vines but recent
expansion of the virus in northern coastal areas is attributed to VMB infestations.
The virus leads to dramatic reductions in yield, delayed maturity and strong dark
leaf color (Golino et al. 2002). Recent research indicates that VMB and other soft
scale insects vector the virus by feeding on different vines. One VMB crawler in
the first or second larval instar stage can transmit the GLRaV-3 virus between
9
grapevines. Up to 70% transmission has been observed within the Planococcus
ficus and Pseudococcus longispinus, the long tailed mealybug (Douglas and
Kruger 2008). Rapid advancement of the virus in certain grape growing regions is
attributed to VMB presence, specifically the VMB due to its unique refuge
behavior and fast reproduction. Until the 1980s it was believed that the GLRaV-3
was spread only through nursery stock but it is understood now that many species
of soft scale insects and mealybugs transmit the infection. Vine mealybug, in
addition to eight other mealybug species, is able to efficiently transmit GLRaV-3
and Grapevine virus A. Research indicates that VMB transmits leafroll virus
semi-frequently but virus strains and varietal resistance should be evaluated when
considering virulence. Virus transmission was elevated in first instar VMB stages
and almost non-existent in adult crawlers. Virus inoculation by VMB larvae can
vary with differing acquisition times on infected material but reach a maximum at
around 48 of feeding on infected wood. In this time frame virus inoculation into
clean plant material can proceed in as little as 1 hour. The next step would be to
examine the feeding methods of early instar mealybugs and study the condition of
the stylet mouthpart. Though the condition of the stylet is not examined research
indicates that the virus does not circulate within the insect body before being
transmitted. The virus is lost from the VMB crawlers in four days and they do not
transmit the virus to egg sacs and young (Tsai et al. 2008). GLRaV-3 can spread
through large vineyards rapidly due to mealybug activity. Plant longevity and
fruit quality are compromised as a result of GLRaV-3. Reports from Argentina
show that in leafroll incidence increased from 26 to 84% in 1 year, with the VMB
as the only apparent vector (de Borbon et al. 2004). Leafroll damage is one of
many convincing arguments to explore alternative controls for the VMB. P. ficus
10
efficiently transfers GLRaV-3 to grapevine hosts in most studies (Engelbrecht and
Kasdorf 1990).
Leafroll virus research and awareness is growing fueled mostly by grower
concerns. Substantial research efforts are aimed at preventing, eradicating and
hindering VMB infestations, as it is the main culprit in fast spreading epidemics of
leafroll disease. Leafroll virus acquisition rates by P. ficus vary between the
different ampeloviruses and closterovirus families. Transmission of type I virus is
absent in P. ficus and results are convoluted within attempts to explain
transmission of type II. No transmission of Closterovirus I occurred with P. ficus
and only sporadic transmission occurred with closterovirus II (Engelbrecht and
Kasdorf 1990). The methods of this experiment are sound but the vine clone
could be contributing to variance in the data. Future investigation into VMB
transmission of GLRaV-3 or any other type should incorporate multiple source
and recipient vines to account for varietal transmission factors. Currently,
research suggests that GLRaV-1 is not transmitted by P. ficus, which follows
closely with the results from Englebrecht and Kasdorf (1990). Multiple studies
show us that virus nor vector explain differential results of virus transmission.
The mechanisms of GLRaV-3 transmission go beyond mealybug biology or virus
properties. Virus transmission as it relates to the host species or grape varietal is
only partially understood and should be examined in future studies. Various
suggestions support the idea that the stylet mouthpart is not simply an inoculation
needle, confirmed by the negative transmission of particular viruses. Inoculation
success rates with different stage mealybugs are another intrinsic problem with
controlled transmission experiments. First instar insects may be more resilient and
have mouthparts that are so small that physical movement by the experimenter
will not damage them, where the opposite may be true for larger second and third
11
instar VMB crawlers. Careful timing must also be considered where larger VMB
specimens are used, as they do not feed from vine material before molting and
moving to the next instar stage. Despite problematic experimental procedures
related to these issues there is a significant effect of virus transmission with the
VMB species at any virus titer, confirming the notion that crawlers can transmit
virus infections at any point during the year (Petersen and Charles 1997). Careful
consideration must be made in making conclusions on virus transmission when so
many test give contrasting results and other aspects of the insect biology are
involved. These findings do elevate the importance of identifying VMB
infestations and controlling spread of the insect to uninfected areas.
Ant Interactions
The argentine ant, Linepithema humile [Mayr], has a crucial and symbiotic
relationship with P. ficus. Most often finding mealybug-infested vines starts with
observing unusual amounts of ant activity on the vine cordon, catch wires and drip
tubes. Ants continue to feed on the sweet discharge from P. ficus and are believed
to tend the insects, moving them from vine to vine and removing debris that could
inhibit efficient feeding. Statistics also indicate that infestations tended by ants
have a significantly lower incidence of parasitism by resident populations of
parasitic wasps or predation events. These are a few of many proposed ideas to
explain the strong statistical interaction of ants and VMB. In practice observations
continue to show that increased levels of ants exacerbate infestation levels and in
situations where ants are excluded infestation levels decreases dramatically.
Studies have confirmed the strong ant interaction with mealybugs but few
have suggested a solution to controlling ants sustainably or proposed a model to
understand how far or fast ants can transport the insect. Recent research has
12
targeted the L. humile with selective methods to deliver strong insecticides that
capitalize on the colony social structure. Imidacloprid and spinosad insecticide
treatments are registered for use in vineyard settings and have provided effective
control of ant populations and subsequently mealybug infestations. Using sugar
based insecticide treatments prevents loss to other beneficial insects that would be
lost in a spray-based program. These new treatments are key to integrated pest
control approaches as well as preserving natural biological control for growers that
are sustainably oriented. Treating Argentine ants according to their social
structure should contain them more effectively and limit the incidence of large
conglomerate ant populations. Large ant “supercolonies” are one reason for the
elevated pest status of L. humile. The conglomerate colonies are unique in that ant
“families” lack aggression to other members allowing what appears as a single
colony to inhabit vast areas such as vineyards (Cooper et al. 2008). These
attributes allow ants to colonize vast areas but their unique colony structure also
promotes exploitation through slow acting insecticides that infiltrate nests.
Limiting ants that thrive in irrigated disturbed habitats is critical to
controlling the populations of scales, aphids and most importantly VMB
infestations. Because ants feed mainly on carbohydrates, systemic control of the
species is possible relatively simple. Liquid bait containing the insecticide nearly
eliminates exposure to non-target species such as predatory insects or pollinators.
Spray treatments for ants are also dysfunctional in that they cannot target insects
within the underground nest, just as spray insecticides will not influence VMB
under bark layers or on roots. Trophallaxis, the feeding of one ant by another, is
common in ant colonies rendering insecticide baits useful for the entire growing
season. Laced liquid carbohydrate baits allow insecticide activity deep within the
ant colony and provide superior control to granular protein baits. Testing shows
13
that any concentration of ant control influences infestation levels. Limitations in
this course of action include the severity of the ant colony and area utilization or
foraging distance. Movement across and down rows was uniform indicating that
trellising does not facilitate ant activity and therefore should not explain VMB
infestation spread along rows at any distance. Applying ant bait stations in April
and May provide the best season long control of colony development and larvae
reproduction. Ant baits include thiamethoxam, imidacloprid and boric acid type
solutions deployed in the manner stated above (Nelson and Daane 2007).
Deployment of species selective, low impact treatments can be instrumental in
accomplishing long term economically successful control of VMB infestations in
California vineyards.
Using carbohydrate based liquid insecticide baits for invasive ant species
should be utilized as a multiyear strategy and alongside other integrated controls
for hemipteran pests. Due to the efficient and invasive nature of argentine ants
control can be costly but does yield effective control of other insects and even
plant or vertebrate populations. Overall liquid baits provide season long control
and species selective impact with relatively low application costs. In Daane’s
experimentation with liquid bait stations all applications were applied with
simulated commercial methods. Ant activity is evaluated by measuring the level
of sucrose bait solution remaining. VMB infestation levels are conducted in a
similar manner as this study with cluster damage assessment ratings. Damage to
vine fruit was reduced in 12 of 14 treatment blocks but reduction in mealybug
presence was only effected by ant control in seven plots (Daane et al. 2008). This
type of results speaks to the idea that ants facilitate movement and spread of VMB
damage in vineyard settings. Daane also indicates that all of the tested insecticides
work with ants when applied through sucrose liquid bait stations. One potential
14
problem with season long control with one application is having the bait station
emptied by large populations of ants or losing efficacy of the applied insecticide.
The second scenario was confirmed in this trail where the imidacloprid bait station
lost potency after exposure to sunlight in a clear polyurethane container. Exact
parameters for long term hemipteran suppression and bait station density within
vineyards have yet to be evaluated.
Similar trials in California’s central coast found matching results as VMB
was suppressed by most insecticide bait station treatments. In cases where VMB
populations were disrupted by insecticide sprays for other pests the pattern did not
remain. In 2006, Daane suggests that boric acid baits stations have significant
effects compared to imidacloprid stations that had lesser effects on the ant
population density. An apparent shift in ant counts in August is likely attributed to
ant populations remaining in the vine canopy to tend active VMB insects that are
feeding and producing honeydew. With ants in the canopy and on clusters the
ground and lower vine counts yielded less ant population but only during August
trials. There is some decline in populations as the third instar stages reach
adulthood and hatch into males or remain as females, and a resurgence of VMB
populations into September as the new generation becomes active.
Creating exclusion treatments for ant activity is difficult due to the highly
variable vine structure. Peeling bark, crevices and rotted vines create passages
where ant can access the cordon despite sticky barriers or insecticide treatments on
the lower portion of the vine. Exclusion of ants is also dependent on sources of
forage at different points during the growing season. Ants may naturally avoid the
canopy when VMB density is low during a time of high temperature or during
developmental stages where feeding adults are absent. Observations indicate that
preference over liquid sucrose bait is common and ants may avoid bait stations
15
completely when grape exudate and honeydew is available (Daane et al. 2006).
Ant forage preference causes the timing of sucrose bait application to become a
critical component of season long ant control. Reducing ant populations early
when food sources are low is critical to prevent season long infestations as well as
reduce the amount of secondary forage sources from feeding VMB infestations.
Having low populations of ants starting in February will prevent fast increases in
VMB reproduction and spread. Tending of mealybugs by ants does become a
critical component to large area vineyard management, as well as having the
advantage of low impact product that fits well with other integrated pest
management strategies.
Other factors such as bait station size can impact the effectiveness of ant
control trials. Together, with higher bait station density and early season
deployment, effective control of VMB damage can be achieved with ant control
alone. Applications range from 85 to 620 bait stations which are unrealistic for
commercial scale production but could serve to protect smaller vineyards that
institute sustainable or low impact farming methods. Targeting reproducing ant
colonies early in the growing season could allow for fewer products to be used
while maintaining significant effects on ant population. Overall, experiments
carried out in 2000 and 2001 show that barrier and exclusion treatments have little
or no effect while sucrose bait stations have significant effects if well timed and
maintained over the season (Daane et al. 2007). This type of experiment shows
that even unlikely treatments can have substantial effects if adjusted and calibrated
to fit the system where they are in place.
Ants tending VMB in California vineyards are expected to create better
mealybug habitat or improve the fitness in the environment. Ant interactions vary
from site to site but generally favor VMB activity and correspond to higher
16
population density when tended consistently. VMB can be tended but have no
different distribution than insects in ant excluded situations (Daane et al. 2007).
This creates a situation where ant presence corresponds to high population density
but movement of first instar VMB by ants does not change the distribution. Also,
it is proposed that insects are protected from parasitoids and predators by ant
activity through spatial refuge. Despite this likely activity, distributions remain
unchanged with regard to presence or absence of ants, refuting the previous
hypothesis. It is more likely that ants simply remove extra honeydew excretions,
which allows access to feeding sites and therefore higher fitness. This process of
habitat improvement has been observed in field trails but does not allude to overall
fecundity. It may be more likely that parasitoid and insecticide interactions
explain the distribution and high density of VMB in commercial vineyards.
Increased population density is a result of ant interactions but cannot be explained
by movement or spatial refuge from parasitoids. Increased fecundity due to
sanitation of honeydew excretions is the likely explanation of high populations
where ants are abundant. These results have been found in the southern
hemisphere also. Shiraz field trials in South Africa show a strong linear
correlation between ant population and VMB populations. Parasitoid effects from
ant presence were also obtained and revealed highly significant values for the
negative effect of ants on biological control by parasitoid (Mgocheki and Addison
2010).
Although positive statistical analysis was gained from ant and VMB
interactions no exclusion study was performed to confirm the validity of the
increased population level. Populations of VMB were aggregated in locations
lacking ant activity and random when associated with ants. This is consistent with
other studies where L. humile is the dominant ant species. L. humile was also
17
observed to be more aggressive when VMB populations were low, probably due to
a lack of honeydew forage. Previous studies have confirmed the relationship of
mealybugs and ants but actual infestation percentage is a rare number to obtain.
Mgochecki proposes that as little as 2% infestation of VMB will warrant
insecticide control (Mgocheki and Addison 2010). Insecticide treatments can also
have a strong influence on parasitoid impacts with relation to ant interactions.
VMB interactions vary among ant species but will always have lower parasitism
when ants are excluded from the infestation area. Presence of L. humile strongly
influences parasitism rates by common resident parasitoids of California. In cases
with both aggressive ant species and mediocre parasitoid types parasitism can be
reduced by as much as 70% (Mgocheki and Addison 2009). The most aggressive
ant activity always results in higher protection from parasitoids. Warm, dry
regions exacerbate ant activity and are a likely explanation of frequent ant activity
in wine grape growing regions of California and South Africa. Parasitoid
fecundity is strongly influenced by ant activity; especially in cases where sensitive
parasitoids avoid any type of movement from the host or ants (Mgocheki and
Addison 2009). The interactions with ants should be evaluated more closely in
California vineyards where ants achieve high populations. Natural parasitoid
interactions could dramatically reduce costs if efficient ways of combating ant
colonies are developed. Key issues like these demand further research attention
by integrated pest management specialists.
Flight Ability
One of the basic stipulations of successful pheromone mating disruption is
that the male must follow a false pheromone plume, even against wind movement
(Stelinski et al. 2003). Insects influenced by wind patterns will not respond to
18
pheromone plumes, especially when those sources are infrequent, highly
concentrated deployment devices. Male VMB insects are less than 1 mm and
could easily be moved with wind direction. Other trials indicate that the male is
able to move more than 50 m against prevailing wind directions but could also be
carried past one pheromone plume to another trap causing more randomized
distribution. Flight patterns also take place in cohorts where most males release in
a matter of weeks (Millar et al. 2002). Like Planacoccus citri it is observed that
the male VMB takes flight during morning exposure to light when air conditions
are calm (Zada et al. 2008). The male VMB may remain in the vicinity of the
insect infestation because it has low flight ability. These experiments will
evaluate the ability of the VMB to fly against the least intense wind patterns and
conclude if this aspect of VMB biology is appropriate for mating disruption.
Flight ability of the VMB is in question now. Other insect studies reveal
that pockets of air with varying pheromone concentrations cause proper mating
flight patterns. Air pockets with high and low levels of pheromone induce codling
moth males to follow the pheromone plumes against air currents. This type of
flight ability and behavior has yet to be observed in natural VMB reproduction or
pheromone treatments (Welter et al. 2005). Pheromones applied through puffer
systems and also standard pheromone cards will allow the experiments to evaluate
how density affects the flight patterns of the male VMB. Although pheromone
treatments are successful in lepidopteran pest, the question remains with respect to
VMB.
Central Nervous System Habituation
Pheromone mating disruption studies focused on Choristoneura rosaceana
(obliquebanded leafroller) indicate that habituation by the insect to the pheromone
19
occurs at relatively low concentrations. The olfactory sensory centers become
overwhelmed by high concentrations of pheromone in field settings causing long
lasting adaptation. When the pheromone is presented to the C. rosaceana
olfactory neurons become habituated to pheromone exposure and show 40 to 60%
response reduction. Moreover, a distinct threshold concentration exists, above
which habituation results in 40 to 60% loss of response lasting up to 12.5 minutes.
Picogram level concentrations of pheromone initiate the long lasting negative
adaptation, which is much higher in concentration than that used for normal sexual
communication (Stelinski et al. 2003). The concept of olfactory neuron
habituation could likely influence the efficacy of pheromone products used in
VMB mating disruption. Habituation in the field could inhibit the effectiveness of
deploying pheromone products. In field observations in leafroller insects indicate
that habituation occurs when insects are held within a few centimeters of a
pheromone source. VMB males could be subject to some habituation if olfactory
responses are similar. This situation could present problems for mating disruption
efforts as male mealybugs would be left in a dormant state in the field, effectively
increasing their lifespan. Puffer applications increase the likelihood that males are
exposed to high levels of pheromone and therefore habituation conditions. High
levels of pheromone may also suppress the sexual activity of VMBs, as suggested
in leafroller studies, but the flight strength of the VMB male remains to be seen
(Stelinski et al. 2003).
Long lasting adaptations to pheromone exposure indicate time frames for
returning to normal response are as short as 1 minute or up to 96 hours (Stelinski
et al. 2003). Immobility responses to high levels of pheromone would cause the
VMB to respond after pheromone exposure stops. In a multiple day recovery
response scenario the male could easily overcome pheromone exposure over time
20
and subsequently respond to natural pheromone levels from mating female VMB.
Experiments of this type have not occurred and would require use of specialized
equipment like electroantennograms and gas chromatography mass spectrometers
(Stelinski et al. 2003).
Parasitoid Activity
Natural enemies of the VMB can provide significant control of the insect
and should be included in pest control strategies. Parasitoid wasps utilize the
VMB crawlers for their own reproductive cycle by laying eggs inside the larger
adults. The parasitoid wasps have resident populations in California due to
previous releases to control other pests in citrus and nut crops. The resident
populations exist naturally and add a level of control without any additional cost to
the grower. Additionally, many insecticides, which are ineffective in treating
VMB, are harmful to the wasp populations. Losing this level of control by
applying agrochemicals is unnecessary, especially after considering other
management strategies such as pheromone mating disruption. Several species of
parasitoid wasps that control VMB exist in California. Anagyrus Pseudococci (A.
pseudococci) is the most common and effective natural enemy but other species
are recovered from parasitized VMB crawlers. Leptomastidea abnormis and
Pseudophycus species are also present in California (Walton and Pringle 2005).
Unique characteristics of their biology make them suitable for integrated pest
management programs and beneficial interactions exist between the wasp and
VMB pheromone deployed for mating disruption.
Parasitism rates are highly variable due to the phenology of the VMB and
other abiotic and biotic factors. VMB under bark layers or in crevices are
protected from parasitoids but are still partially accessible. Research suggests that
21
less than 1% of mealybugs are parasitized when hidden under bark layers or vine
crevices. Generally parasitism rates relative to the entire population in a vineyard
are not high. Rates may be as high as 25% (Daane et al. 2006) but are usually
lower in the range of 13 to 20% (Walton and Pringle 2005). Much of the success
of a parasitoid species depends on threshold temperatures which are commonly
between 12°C and 36°C. Studies indicate that parasitoids are well adapted to
survive higher and lower temperatures given daily variation in temperature
change. The San Joaquin Valley is the perfect environment for parasitoids with
high daytime temperatures and moderate nighttime cooling (Daane et al. 2004).
Walton also found that no parasitized VMB crawlers were recovered from the
trunk below the soil surface. These observations support the idea that VMB
patterns and refuge habits strongly inhibit parasitism and possibly predation. As
demonstrated in our research and observations Walton also demonstrates that
resident populations of VMB remain under bark layers on the trunk at all times
during the year (Walton and Pringle 2004).
Parasitism and predation of the VMB is a secondary approach that falls
under the category of biological control. Biological control methods are
recognized as sustainable within the industry as most locations have resident
parasitoid species as a result of past control efforts of other agricultural pests. The
San Joaquin Valley contains resident parasitoids of VMB from past efforts to
control citrus crop pests. Though parasitism can be effective in certain situations,
generally it is inconsistent due to the tendency of the VMB to take refuge under
the soil surface or bark layers of grape vines. Though these aggressive parasitoids
and predators exist in California at no additional cost to growers, the biology and
population dynamics of the VMB prevent these insects from providing adequate
control.
22
Pheromone Responses/Puffer Systems
Pheromone mixtures of lavandulyl senecioate are the identified compounds
for pheromone mating disruption of VMB. The compound is a racemic mixture
but no preference is observed for either the (S) or (R) enantiomer of the
compound. The parent compound of lavandulyl senecioate is lavandulol which is
also produced naturally by VMB in extremely small amounts. Lavandulol is
inhibitory to attraction of the male VMB in doses comparable to normal
lavandulyl senecioate pheromone. The inhibitory nature of lavandulol is not
understood (Millar et al. 2002). Some studies indicate that the (R) enantiomer of
lavandulyl senecioate is inactive completely and has no effect in a field trial (Zada
et al. 2008).
Pheromone mating disruption studies where puffer devices were used to
implement and deploy pheromones have been effective. Codling moth is one
example of a pheromone trial that was successful when the insect population was
low to moderate. These experiments also suggest the proximity of other potential
insect sources plays a key role in the effectiveness of the treatment. Pheromone
lure traps suffer suppression when tested areas are exposed to higher dosage
pheromone mating disruption regimes. This observation suggests an immediate
impact of the puffer-dispensed pheromone on codling moth (Welter et al. 2002).
Though downwind locations experienced higher trap shutdown, there was no
overall damage difference in puffer trials. Over all the experiments carried out by
the Welter lab, damage is relatively uniform despite trap shutdown effects from
pheromone applications. Although clues can be drawn for comparison to VMB
activity, the distinct biological differences suggest that the flight patterns and
movement potential is much different than codling moths. Our experiments will
23
evaluate the flight strength of VMB considering the results of these pheromone
trials.
MATERIALS AND METHODS
Pheromone Deployment Device
The pheromone emission system is modeled after a simple aerosol spray
dispenser. The device is a simple rectangular polyethylene case about 30 cm tall.
The case is orange brown and contains the battery pack, electronic timer and
housing for spray can aerosol formulation of the mating disruption compound.
The electronic timing system is battery powered, batteries being contained within
the back of the apparatus. The electronic timing system activates a simple
mechanical aperture to depress the active ingredient aerosol spray per the setting
and timing of the programmer. The system is placed 3.0 m above the vineyard
floor. The puffer system is supported by 7.62 cm Polyvinyl chloride (PVC) piping
and secured with metal wire.
Pheromone card dispensers were used in the second season due to legal
constraints on the timed pheromone emission device. Checkmate VMB-XL
dispensers were applied at six different loadings with two rates equating to field
density of 25 g/acre and 37.5 g/acre. The two rates were replicated nine times
with three full replications of each dispense loading. Dispensers contained 200mg
and 300 mg for the 125 cards per acre loading. Vineyard blocks with 143 mg and
214 mg of pheromone produced the 175 dispensers per acre placement. Vineyard
blocks with 100 mg and 150 mg dispensers per acre produced the densest plots
with 250 cards per acre. This trial should have an immediate impact on the mating
pattern of the insects and reduce exponential increases in population in the first
year.
25
Field Sites
Two sites were selected for pheromone mating disruption, both in the San
Joaquin Valley. All research plots are located within commercial vineyards east
of Denair, CA (lat. 37.4°N, long. 120.4°W, 79m absolute elevation) and Herald,
CA (lat. 38.4°N, long. 121.6°W, 47m absolute elevation). Vines within treatment
plots are trained to a bilateral cordon at 1.4 m with 2.1 m x 3.4 m (vine x row)
spacing. Trellis support consisted of two foliage support wires at 1.7 m with a 0.2
m T-top. Vines were pruned mechanically to a 100 mm bearing surface during the
dormant season after any possible risk of frost. Over the growing season shoots
were vertically positioned downward to reduce intravine shading. Fertilizer
application consisted of 16 kg/ha nitrogen applied 1 week post-anthesis. The
vineyards were drip irrigated with pressure compensating emitters spaced at 1.1 m,
or two emitters per vine, delivering 2.3 L/h. Root zone irrigation was based on
crop coefficient (Kc) of 0.2 and 80% of reference crop (grass) evapotranspiration
information obtained from local CIMIS weather station data. Root zone irrigation
began in late April and was interrupted before bloom, allowing water potential to
decrease below -1.0 MPa to control shoot growth. Regulated deficit irrigation
(RDI) was imposed at both sites after fruit set and consisted of 70% of daily ETo
when leaf water potential was below -1.2 MPa.
Treatment Applications
Experimental design was organized in a randomized complete block design
with two mating disruption treatments and three replications of each treatment.
Treatment replicates consisted of 4.05-hectare plots in uniform vineyards 96 vines
long by 56 vines wide. Five pairs of rows in each treatment block consist of every
other vine moving into the row and two of three sample vines moving out of the
row to equate to 112 vines in each row pair. Treatment replicates were spaced by
26
at least 1600 m. Treatment replicates were assigned randomly consistent with the
RCBD experimental design and consisted of exactly 5376 vines each. Six
replicate plots in each site were selected from areas previously infested with high
levels of VMB. Three treatment blocks at each site were pheromone treated while
the other three were left as control plots. Lavandulyl senecioate was applied to
treatment plots starting on 27 May 2011 in Herald, CA (site 2) and 3 June 2011 in
Denair, CA (site 1). Distribution of the pheromone was achieved through
Checkmate Puffer dispensers positioned at 1.8 m above the vine canopy using 50
mm PVC stakes. Puffer dispensers were applied at two per acre at site 1 and three
per acre at site 2, arranged in a grid pattern in all treatment plots. Puffer
dispensers were timed to emit 1.3 g of active pheromone on a 24-hour cycle over
the entire growing season. Checkmate VMB XL pheromone dispensers were hung
on the perimeter rows on every other vine and also on every other head row vine
emitting 3.1 g of active ingredient as a protective boundary plume.
Male Vine Mealybug Flight Activity
Male VMB trapping is initiated by placing two traps in each 4.05 hectare
treatment replicate plot. Delta shaped sticky traps are loaded with Scenturion
VMB lures by Suterra and impregnated with 100 ug of active lavandulyl
senecioate. Traps are replaced every 4 weeks to maintain pheromone
concentration and weekly counts of each trap monitor VMB captures. The
Scenturion lure is placed within a standard three fold sticky card trap that
preserves the insects in clear adhesive. The adhesive traps are folded into a three
sided cylinder, orange on the outside and printed with a grid pattern on the
adhesive inner side. The edge of each side is also shaped and folded down to
prevent unwanted dust or debris from entering the trap. These same male VMB
27
flight traps are used in the second phase of the experiment where plume studies are
evaluated in relation to male flight ability. The density of pheromone released by
the lure is not expected to influence the density and distribution of the puffer
applied pheromone trials. Results are compared to growing degree data supplied
by CIMIS and analyzed on SAS version 9.2.
Cluster Damage Assessments
Cluster damage assessments will be made in 10% of each replicate plot,
equating to 560 vines in each treatment. For each of five pairs of vine rows in
treatment blocks, samples were taken on every other vine moving up the row and
two of three vines coming down the adjacent row. This sampling method equates
to 112 measured vines in each of five pairs of rows. Measurements are taken with
clusters at 16 brix and from clusters in contact with the bark at the head of the vine
where the cordons meet. One cluster per vine will be removed from each vine and
rated on a 0-3 scale to assess VMB damage. 0 = no damage, 1 = very little
damage, 2 = some damage with honeydew and VMB wax residue, and 3 =
complete destruction and economic loss of cluster. Dormant season and pre
harvest damage assessments will also be used to estimate the total infestation level
and potential crop damage based on the same categorical system described above.
Data collected will be analyzed by Pearson chi-square test or the “goodness of fit”
in SAS version 9.2.
Pheromone devices within the 12 vine blocks were arranged in a grid
pattern across the block with 10 vine rows between rows lined with applicators.
Devices will be placed at a rate of two per acre in site one and three per acre in site
2. Cluster samples will be assessed along the same vine row as dispensers and
total 112 samples in adjacent rows. A single cluster from 48 vines will be sampled
28
in each row every other vine. Additional samples (64) are taken from the adjacent
row, two of three along the 96-vine row; each sampling ending six vines (12.8 m)
from the vineyard edge. The pattern of 48 samples moving north and 64 samples
moving south is consistent throughout the sampling. This totals 560 samples per
treatment replicate.
Damage assessments in 2012 consisted of 60 observations in each treatment
replicate and 20 control observations in each control treatment. Observations were
taken from two rows from treatment plots that consisted of eight rows each, 13
rows including five buffer rows between adjacent treatments. Each block
consisted of six different pheromone loadings supplied by Suterra LLC. The entire
experiment totaled three blocks and 21 different treatment plots including controls.
Controls were sampled on random rows in the same vineyard with the same
technique but in areas north of the pheromone trials to avoid wind interference and
drift.
Dormant Season Crawler Activity
Delayed dormant season VMB crawler activity will be evaluated on a
categorical basis on each vine sample described in the vineyard treatments above.
The same sample vines from pre-harvest cluster damage assessments were used to
evaluate delayed dormant crawler survival, activity and parasitism. Each vine is
sampled by removing two bark sections to examine the phellem and phellogen
layers underneath. Small 5 X 7 cm sections of bark were removed at the split of
the cordon on the under side of the vine and also at the base of a spur half way to
the end of the cordon. If vines had been cane pruned in the dormant season, one 5
X 14 cm area of bark was removed from the lower portion of the trunk where bark
layers are thickest. These locations are believed to be highly active areas during
29
the dormant season and likely locations for dormant VMB female crawlers to
overwinter. After phellem peel observations of residue, mummified crawlers,
residual egg sacs, and live crawlers were made and recorded in a categorical
system. The residual VMB activity rating system was based on the cluster damage
assessment model. Ratings labeled 0 equated to no activity, 1 was some, 2
significant activity and 3 equated to residue and activity completely covering the
phellem peel area. Delayed dormant observations occurred on March 14, 2012
and March 27, 2012, completing the evaluation of all treatment blocks within a
short time frame.
Pheromone Device Placement and Spatial Analysis
Distribution and geographic location of pheromone devices will be made
using GPS, global positioning system handheld device (Delorme Earthmate
PN60). Layers within mapping software include sample vine positions,
pheromone device positions and wind vectors. Each treatment replicate will
utilize mapping layers to create geo spatial analysis under a spatial interpolation
model. Vineyard data points are overlaid within ArcGIS software version 8.2 to
create zone of influence patterns and geographical distributions (Kurtural 2006).
Geo-statistical spatial interpolation, also known as Kriging, will be used to analyze
categorical vine damage ratings across spatial components. Semiveriograms will
be used to compare damage ratings of points to spatial dependence based on
distance between values. Universal Kriging methods along with thin-plate
smoothing splines will be used to analyze data from the ArcGIS software system
and make sure that analysis is carried out with minimum curvature between
control points (Chang 2002). Using these spatial distribution techniques will yield
30
the best possible statistical analysis of categorical damage assessment ratings over
large commercial scale vineyard areas.
Movento/Insecticide Standard Grower Applications
Recent insecticide developments have produced a tetramic acid derivative
under the brand name Movento. This spirotetramat compound is extremely
effective in laboratory and greenhouse studies and is currently being implemented
in field settings. The ambimobile compound is capable of xylem and phloem
transport, creating protection for all plant tissues after a simple drip irrigation
application. Phloem transport is a more desired delivery method and foliar
applications of spirotetramat products enable this type of treatment for pests that
take refuge and protect themselves from typical insecticide sprays. The compound
also has a favorable impact environmentally and is safe to handle for applicators
(Bruck et al. 2009). Spirotetramat compounds are currently the preferred
treatment for mealybug infestations in California vineyards. Pheromone mating
disruption is designed to complement standard grower insecticide applications
which include systemic spirotetramat compounds. Movento was applied at the full
label rate of 71.80 to 95.73 ml per hectare. The full label rate was applied in the
spring of 2011 to all treatment blocks and before deployment of mating disruption
pheromone. Movento was applied once during the growing season and not every
30-day interval as suggested by the manufacturer.
Pheromone Influence on Male Vine Mealybugs
Analysis of the pheromone plume with respect to male mealybug behavior
was evaluated in pistachio orchards to facilitate a field study and prevent
inoculation of clean grapevines. The experiment explains the relationship between
standard wind conditions and the distance or direction that male mealybugs are
31
able to fly. Experiments ran in July and August in Fresno County at the Fresno
State campus. The experiments evaluate the effectiveness of the pheromone
plume and the threshold wind value that the male mealybugs are capable of
resisting in a field setting.
The experiment consisted of 50 folding sticky traps with rubber septa lures
loaded with VMB pheromone. The lures were loaded with 100 micrograms of
pheromone and all supplies were provided by Suterra LLC. Fifty total traps were
placed in a grid pattern in the 9.5 hectare pistachio field on the North West side of
the Fresno State campus (lat. 36.8°N, long. 119.8°W, 90 m absolute elevation).
Traps were hung at 2 m above the orchard floor on the northwest quadrant of
individual trees. Minimum distance between traps was 36.6 m and traps were
located half that distance from the male VMB source at the center of the block.
Butternut squash infected with VMB was placed in the orchard to supply male
insects. Insects were at proper third instar stages when males are likely to emerge
and attempt to mate when influenced by the pheromone. Each cohort of VMB
insects is left in the field for 10 days, providing one treatment replicate. The
number of male trap catches was recorded three times during each trial at 2-day
intervals. Lured traps were placed in the pistachio field and remained in the same
locations for all the experiments. Different sets of VMB, sourced from different
insectaries were placed in the center of the block to provide male VMB. Each set
produced one repetition of the experiment and had four individual recordings for
each trap. The entire experiment is repeated four times totaling 16 observations of
male VMB movement.
Prior to the experiment, lured pheromone traps were placed in the field as
controls to ensure no outside mealybug source. Control traps were placed in a grid
pattern with 21 m between traps in a total of 16 locations. This placement
32
occurred on July 12 and was recorded July 13, 14 and 16. Lured traps were placed
in a grid pattern at the center of the pistachio orchard acting as controls between
treatments. These 20 traps steadily decreased in male mealybug catches over three
consecutive days, August 8th, 9th and 10th. Lured traps and the mealybug source
experiment started on July 18. Again, 10 un-lured control traps were placed with
the 50 lured traps to test against random flight patterns. The 10 untreated control
traps were spaced 21 m. Control and lure traps were recorded with the PN-60
GPS device and loaded into Delorme Earthmate software to extract coordinates.
Coordinates were used in ARC Map to generate spatial interpolation maps as
visual representations of the trap counts and male mealybug movement over time.
Wind speed, direction and daily mean temperature were recorded from CIMIS
weather station data available online.
RESULTS
Cluster Damage Assessment (Site 1)
Puffer dispenser experiments run in Denair, CA (Site 1) show that a
significant difference in VMB damage can be found in treated and untreated plots.
After observing the cluster damage during the final days of the growing season
overall ratings indicated very little damage from VMB. Fig. 2 indicates that total
damage ratings other than zero cover almost 94.17% of the test vines in control
plots with no pheromone application. In plots treated with VMB pheromone the
total number of test vines with a zero damage rating is 97.12% (p<0.0002). Third
category ratings in the same trial result in twice the amount of damage rendering
fruit unmarketable. The control plots contained 0.72% unmarketable fruit where
treated plots contained only 0.36% unmarketable fruit from observed vines.
Damage can be reduced by as much as 50% (Table. 1). Reductions in crop
damage by VMB are significant and could have a strong potential impact on
treatments of various mealybug species in the future. The result of standard
grower insecticide treatments along with pheromone application through puffer
devices yields clean vineyards and low overall damage ratings.
Cluster Damage Assessment (Site 2)
Similar patterns were found in Site 2 (Herald, CA), except with much lower
overall damage in the vineyard blocks. Total VMB damage to the site was less
than 3% overall. These extremely low values make quantifying economic
sustainability extremely difficult. Pheromone products are expensive to apply
when considering minor but significant differences between controlled and treated
areas. Site 1 damage totaled around 4.45% for all observed vines, where Site 2
has fewer than 3% total damage. Such a small amount of damage makes statistical
34
analysis difficult due to lack of data points registering values other than zero to
promote the effectiveness of the Pearson Chi Square test. The positive results
show that 98.92% of the treated plots fall into the zero rating category or
otherwise completely clean. Control plots in the same three blocks total 95.26%
zero ratings in observed vines (Table. 2). All blocks, especially the pheromone
treated areas, are essentially free of VMB infestation and lost almost no fruit to
crop destruction by the pest. Only 20 total vines sampled contained a fruit cluster
that was categorized as a three level rating or unmarketable. Most of the 19
observations were in control plots and only a single unmarketable cluster was
found in pheromone treated areas. This translates into 0.03% unmarketable
damage in pheromone treated plots where untreated controls had 0.57% damage
(p<0.0001). This is a significant difference and shows that pheromone treated
plots are highly protected from VMB damage where pheromones are present.
Male Vine Mealybug Trapping (Site 1)
Cumulative trap counts of male VMB in Site 1 display an increasing pattern
in control blocks where numbers of insects caught increases continuously over the
season. Blocks treated with VMB pheromone experienced some trap shut down as
the number of caught males leveled off after some time. Up to the accumulation
of 750 growing degree days in Site 1 the number of males caught in control and
treated blocks increased to the same number. After 750 growing degree days the
number of caught male VMB in pheromone treated blocks increased at only one
other point in the season. Control blocks observed increases in the number of
males caught twice after the 750 growing degree day mark. The total number of
trapped male VMB in the entire site was 34, 12 caught in pheromone treated areas
and 22 caught in control blocks. Cessation of male trapping in pheromone treated
35
blocks indicates that lavandulyl senecioate compounds have a seasonal and long
lasting effect on the biology of VMB. Because Fig. 1 shows a similar pattern of
trap shut down in both treated and control blocks the control effect could be due to
insecticides applied by the grower.
Male Vine Mealybug Trapping (Site 2)
Cumulative trap counts based on growing degree days for Site 2 were
dismissed from the study due to lack of any male VMB catches in any trap over
the entire season of observations. Conclusive results support VMB crawler
activity in the vineyard but 12 traps throughout the blocks yielded only one male
over the entire growing season. This pattern, along with the low damage
assessments support the idea that grower applied insecticides were extremely
effective alongside pheromone applications.
Pheromone Dispenser Field Trial
Trials conducted in Denair, California in 2012 yielded surprising results
from pheromone dispensers simulating puffer emitters. The randomized complete
block design allowed exacting statistical analysis to be carried out to differentiate
between treatments. The control blocks experienced 17.5% damage in each of the
three replicates of the trial. This amount of damage to the clusters is significantly
greater than any other blocks treated with VMB pheromone at any level. This
level of damage contains almost no unmarketable fruit and is considered clean by
most growing standards. Blocks treated with the highest density of pheromone
card dispensers experienced 7.22% damage and 9.44% damage. These plots
contained 250 dispensers per acre loaded with 25 and 37.5 g of active pheromone
in each loading type. This level of damage represents mostly minimal damage and
almost no lost fruit due to honey dew accumulation or sooty mold presence.
36
Plots containing 175 dispensers per acre contained almost no mealybug
damage whatsoever. These plots were 99.44% and 98.33% clean in plots with
loadings of 25 g/acre and 37.5 g/acre respectively. In a vineyard that was highly
infested in previous years this represents inconceivable insect control. Only three
total observations of any mealybug presence were recorded over the 360 vines
sampled in these plots; all of which were category 1 ratings. Along with the
standard grower insecticide applications, this level of pheromone deployment
brings VMB control to near eradication levels.
Pheromone cards at a density of 125 per acre gave the lowest VMB control
levels besides those of the control block where no pheromone was applied. These
cards have 200 mg and 300 mg of active pheromone but only 125 are applied per
acre resulting in the same total loading of 25g/acre and 37.5g/acre respectively.
The total damage for these plots was 10.0% in the 25 g/acre trial plots and 12.78%
in the 37.5 g/acre plots. Each of these plots contained multiple 2 and 3 category
damage ratings from the fruit assessment. This level of damage is approaching the
control plot where no pheromone application took place. This higher dosage of
pheromone per card seems to be negated by the distribution and application
pattern of the dispensers. Results referenced in table 3.
Male Vine Mealybug Response
Trap counts and geostatistical analysis with ArcMap software shows that
over the first repetition of the trial the male VMB are not moving towards a known
pheromone source. Wind speeds in the vicinity of the first repetition never exceed
2.8 m/s (CIMIS) and the average daily temperature is 35.2°C. The directionality
of the wind is WNW based on daily averages from years of climatic wind data
collection (NOAA 2012). Flights of male VMB were substantial and despite
37
warm morning temperatures and low wind velocities the insects did not orient to
upwind pheromone point sources. In each of the four observations males were
consistently caught in the vicinity of VMB source and never upwind from that
source on any observation day. In each trial observation the trap closest to the
VMB source had the highest number of caught males. The closest traps to the
VMB source always constitute the majority of caught insects in any trial day. The
only exception to this was on a test day before the experiment began when eight
total insects were caught in a trap directly downwind from the source.
Repetition 2 of the trial was conducted a week later during the second two
weeks of August; 8/15/2012 to 8/31/2012. High temperatures and low wind
vectors allowed for a successful repeat of the first trial. Average daily high
temperatures were 35.6°C and the average wind speed was 2.0 m/s. The high
temperature was 38°C and the highest recorded wind speed was 2.4 m/s. The
VMB source box was located in the northwest portion of the pistachio orchard to
differentiate from the first trial. Once again the caught VMB males were isolated
around the source or moved slightly downwind. Very strong flights of VMB
males were recorded with all but one day above 100 caught males. The average
flight during all observation days was 157.4 caught males. The flight pattern was
similar to trial one in that the males isolated around the VMB source and also were
influenced by wind direction. The average wind direction in this trial was again
WNW (NOAA 2012).
The last repetition of the flight pattern trial was conducted from October 12
through October 22 in the same pistachio orchard. This trial allowed several
weeks for remaining males to die off with no source of new males present. This 4
week period ensured that the site was clear of any insect activity. Timing of this
trial caused heat unit accumulation to slow dramatically, which affected the trial
38
but did not affect the success of the trial. Heat unit accumulation and average
daily temperature were much lower than other trials and overall VMB insect
activity reflected this. The average maximum air temperature was 24.2°C and the
daily average temperature was only 16.5°C. Wind speed was again not a factor in
this trial with maximum speeds of 2.5 m/s, and an average daily wind speed of
only 1.6 m/s. The VMB source was moved to the northeast portion of the
pistachio orchard in this trial. Maximum trap catches in all traps yielded 51
catches in the second reading. The flight pattern was similar to the first two trials
with the majority of catches isolated around the female VMB source. The flight
pattern is not influenced strongly by wind direction in this trial but lower average
temperatures prevented the VMB population from reaching maximum fecundity.
Results referenced in figures 4-6.
DISCUSSION
2011 Experiment Proceedings
1.1
Site 1 in Denair, CA yielded good overall results for the first year of the
pheromone application trial. The Pearson Chi Square analysis of the treated plots
in Denair shows that when pheromone is deployed in commercial scale vineyards
there is a significant and positive effect on overall VMB damage at the end of the
growing season. The experimental areas are laid out in vineyards with moderate
to severe VMB infestation which yields results that are consistent with common
entomological theories. When considering VMB behavior and biology it is
expected that small populations would be less affected by pheromone mating
disruption than areas with higher population density. In the test plots, which all
contained moderate to high VMB population levels, the pheromone was effective
in limiting damage to the crop. This is a fortunate result considering other
experiments have had no significant differences in damage ratings due to a low
population level of VMB. Damage was reduced in pheromone treated plots and
the worst damage ratings were reduced by 50%. Damage reduction was observed
across all of the treated plots when compared to untreated control plots but the
interaction with standard grower insecticides remains unknown. Control and
treated plots received the same insecticide treatments. These treatments usually
strongly complement pheromone mating disruption trials but that is not a result of
this experiment. Year to year evaluations of insecticide schedules along with
pheromone mating disruption are also yet to be evaluated. Overall, pheromone
mating disruption applied through puffer systems is effective when used alongside
standard grower insecticide applications but the economic feasibility of this
40
control method has yet to be evaluated. The cost of VMB pheromone itself and
the various types of deployment methods make evaluating time and cost
effectiveness very difficult.
1.2
Site 2 in Herald, CA had similar results to Site 1. The mealybug infestation
level prior to starting any pheromone treatments was much lower in Site 2
resulting in less convincing but statistically significant results. Total damage
caused by VMB was reduced by at least 50% when comparing control plots to
treated plots. Although pheromone treated plots had significantly less damage the
plots also had almost no VMB damage in the unmarketable category. No fruit in
any control or treated plots at the site had enough damage to have the buyer reject
the fruit. Thousands of clusters were samples at each site and only 20 from site 2
had a level three category rating or were considered completely unmarketable.
This means that the mealybug population was simply not high enough at the start
of the experiment or the temperature and weather conditions were not favorable in
that year to allow mealybug populations to thrive. This supports our expectation
that low levels of VMB would not allow pheromone mating disruption to be
economically feasible. The pheromone treated plots did have a slightly lower
level of overall fruit damage than control plots but standard grower insecticide
treatments cleaned up most of the infestation. No area had more than 5% fruit
damage to any degree. Once again the next step would be to evaluate when the
VMB population is high enough to justify applying pheromone mating disruption
alongside standard grower insecticide treatments. This experiment has a positive
result but would not be economically sustainable based on the level of achieved
41
control and the cost of the pheromone product. Future research may also consider
standalone pheromone mating disruption on a commercial scale.
1.3
Pheromone assisted trapping of the male VMB in the test plots indicate that
mealybug activity is responsive to the synthetic pheromone compounds found in
the trap lures. As with other studies, the pheromone compounds are effective in
attracting the male VMB to the vicinity of the trap, allowing us to study at least
some proportion of the male VMB population (Walton et al. 2006). The male
VMB are attracted to a lured trap that is 8 inches wide indicating that biological
interaction with the pheromone is occurring to at least that proximity. What is not
understood is when or if a chemical-tactile shift occurs inside the 8 inch proximity.
Simple entomological laboratory experiments would elucidate that information for
the scientific community. The trap counts indicate that in plots where pheromone
is disrupting the mating cycle, the trap counts are significantly lower than in no
mating disruption is deployed. This trend occurred across all three test plots and
was consistent in both years of the study. The population of male VMB in the
pheromone application plots reached a certain level and then stopped increasing;
suggesting that once the population is high enough the pheromone becomes more
effective in disrupting the mating cycle. This is consistent with our expectations
that low population levels would not be affected by pheromone mating disruption
efforts. The monitored control plots are also consistent with the theory that male
VMB population levels will continue to increase without pheromone present.
These results prove that pheromone application is not a stand-alone solution to
VMB control and should be integrated with sustainable chemical application and
other management strategies. Despite no further increase in the male VMB
42
population in treated plots, this does not suggest trap shut down but a possible
long lasting pheromone adaptation. This type of result would be a further increase
in pheromone treated plots after the removal or decrease in pheromone amount.
The dormant male VMB would reemerge or move after “waking” from the long
lasting adaptation (Stelinski et al. 2003). This is another possible scenario that
could be evaluated in laboratory experiments. These results can be referenced in
Figure 1.
These experiments also evaluate some components of VMB biology
pertaining to temperature limitations and upper thresholds of temperature
tolerance. As growing degree days accumulate over the season the VMB
population is not adversely affected. This would be expected but what is not
understood is why the mealybug population does not level off and stops increasing
during periods when the growing degree days are accumulating fastest or during
the peak heat of summer. Figure 1 shows that during the fastest growing degree
day accumulation populations still increase. Traditional knowledge on VMB
suggests that upper temperature thresholds exist around 35° C (Walton and Pringle
2004). This experiment suggests temperatures above 43°C have no effect on or
slow VMB populations. Later experiments also support this conclusion.
1.4
Two lured pheromone traps to monitor male VMB activity were placed in
each block but the lack of overall infestation in Site 2 did not allow for many
catches. The lack of activity in these traps supports the idea that less mealybug
activity will limit the efficacy of pheromone mating disruption applications. The
cluster damage results from the same blocks were significantly improved with
pheromone applications but the margin was so small that the treatment will not be
43
economically feasible. The lack of trap catches as compared to Site 1 is strong
evidence that higher mealybug populations are required to make pheromone
mating disruption feasible. Lower trap catches correspond to a smaller margin for
pheromone efficacy. For this reason the data from these 12 traps, two per block,
have been omitted from the study but the information gathered is still relevant to
the overall research goal.
2012 Experiment Proceedings
2.1
Limitations due to lack of production of the “puffer” devices prevented year
to year replication of the experiment and forced a simulation experiment. The
company that previously produced the “puffer” emitters no longer manufactured
them for legal reasons. Standard pheromone emitter cards were used in place of
the puffer devices and additional wind vector pattern testing was used in another
experiment to simulate the effect of “puffer” application in a commercial vineyard
setting. What we find from this trial is that the hang rate is more important that
the loading of the pheromone itself. The amount of pheromone is not having as
great of an effect as the density of pheromone devices in the vineyard. When 125
or 250 pheromone dispenser cards are hung per acre the effect on the mealybug
population is insignificant when compared to dispensers hung at 175 per acre. The
reasons for this result could be many but most likely relate to the clumped nature
of VMB infestations (Daane et al. 2006). The density of the card placement in this
distribution is likely having an effect on more VMB locations overall than any
other density of hang rate. Another likely scenario is that the hang rate combined
with the loading of 25 or 37.5 g of pheromone per card is the correct amount of
pheromone to cause maximum mating disruption and at the same time prevent
44
over exposure or long lasting adaptation to the pheromone itself (Stelinski et al.
2003). Most likely, the spacing and loading are both correct in 175 card
placements to have maximum effect on VMB mating. The effect of 175
dispensers per acre is extremely high compared to other treatments with levels
exceeding 98% control in both loadings. Whether 25 g or 37.5 g was loaded per
card the treatments had over 98% control with standard insecticide treatments.
Results from the plume study, also conducted in 2012, will also confirm that the
density of the cards is the major factor influencing VMB pheromone mating
disruption. This experiment is also in cooperation with standard grower
insecticide applications and could also be tested over a longer period of time to
ensure pheromone levels are adequate over longer growing seasons.
2.2
The second experiment conducted in 2012 supplemented the pheromone
dispenser study from the same year and results from the previous year. This
experiment is completely unique because it is based on geographical data, which
makes special analysis very powerful. Data in this experiment included wind
direction, daily temperature, pheromone concentrations, and distance. Conducting
the experiment in a pistachio orchard also allowed for a higher level of control
while maintaining the benefits of a field study. The pistachio orchard, which is
not a host environment for VMB, also allowed us to find out how long male VMB
can survive while exposed to the elements. The results from this trial are very
promising and absolutely support the findings of our other trials.
The first conclusion drawn from the pistachio orchard experiment is that
synthetic pheromone compounds designed for VMB are effective in luring and
trapping the male insects. This has been demonstrated in laboratory studies but
45
field trials focused specifically on lure trapping did not previously exist. The
synthetic pheromone compounds used in the lures captured up to 50 male VMB
per delta trap, all from a single VMB source population. The pheromone
compound also lured male VMB from up to 500 feet from the mealybug source
population. The pheromone lures were also effective during and after extended
periods of time exposed to high temperatures up to 42°C. Previous research
indicates that the pheromone probably degrades at high temperatures, especially in
field conditions, but our work shows that even at high temperatures and multiple
days exposed to wind and sunlight, the pheromone is effective. Even after a two
week period with no mealybug source population, the pheromone lures were
successful in trapping dormant males residing in the pistachio orchard. These
results insist that even small 100 ug pheromone lures are effective in disrupting
mealybug mating at high temperatures and long durations under field conditions.
These experiments were replicated in time and consistently produce the same
positive results.
The most critical observation of this experiment was monitoring the
movement of the male VMB over time. This was done by checking each trap
multiple times during the week and removing trapped mealybug bodies to get an
accurate count and movement pattern across the orchard. All 18 trials constituting
three replicates in time show us that male VMB insects stay in the vicinity of the
source VMB infestation on the squash plants. In certain trials the VMB males are
moved with the prevailing wind direction away from the source VMB to the
corners or edges of the pistachio orchard. Under normal prevailing winds the
VMB males are trapped in the vicinity of the VMB source population even though
it is suggested that any wind velocity would move the males downwind.
Prevailing wind vectors in some cases moved part of the trapped male population
46
away from the source location but the majority of trapped males remained near
squash plants. This was observed with wind conditions from the WNW at only 11
km/h. A smaller proportion of the trapped VMB males moved to the North West
corner of the Pistachio block, directly against the prevailing wind force proving
that the males do have the strength to maneuver in moderate wind velocities. The
highest sustained wind velocity (9.5 km/h) did not affect the majority of the male
VMB population. When high numbers of males are released and take flight most
(60%) are trapped within 80m of the VMB source population. These results
indicate that even when exposed to clear pheromone point sources the male VMB
consistently remains in the vicinity of the source population, or the VMB
infestation in a vineyard setting. This supports the concept of blanketing the
vineyard in pheromone to target all infestation locations instead of supplying point
source “puffer” emitters in the field to “attract” the insects away from infestations.
Results from 2012 overwhelmingly support pheromone card dispensers over
puffer systems when considering these new findings of VMB biology. The
experiment does not differentiate attraction to natural or synthetic pheromone but
this could potentially play a role in field mating disruption applications. This
experiment also does not consider a transition to tactile response when proximity
to the pheromone source is reached by the male insect.
Control trials were also conducted before and after a VMB source
population was placed in the orchard to ensure no outside mealybug population
could influence the experiment. These observations between our trials reveal that
there is no male population increase when the female VMB source is absent. This
tells us that there is no long lasting adaptation to the pheromone and no dormancy
activity for the males in the orchard. Dormancy or long lasting adaptation
scenarios would result in decreases in population after initial increases when the
47
female source population was removed. This was not observed. This also tells us
that some of the males may be able to survive for up to 10 days under field
conditions. This is new information in opposition of previous knowledge that the
males could only function for up to 4 days. These findings together are very
strong evidence of distinct biological patterns and will lead to much stronger
control of this pest in the future.
CONCLUSION
Pheromone mating disruption efforts for VMB control have seen many
developments and variations since the concept began in the early 1990s. Our
research focused on the most recent developments with puffer applicators that
utilize aerosol formulations of VMB pheromone. The synthetic pheromone
formulations used in the puffer applicator are effective in luring the male insect
but over large commercial scale sites the puffer point sources do not provide the
proximity necessary. The point source approach cannot cover enough area to
counteract the clustered random infestation pattern of the VMB. Pheromone
dispenser cards are a reasonable option when the correct density and loading is
applied to areas with significant VMB infestation. This is one case where
pheromone mating disruption can be economically feasible. Conducting flight
pattern experiments further confirmed that VMB males are not able to orient
against average prevailing wind conditions, more support for using pheromone
cards instead of puffer applicators. These experiments confirm that targeting
VMB with pheromone must be done comprehensively and is not dependent on
specific ambient temperatures. The pheromone systems deployed in these
experiments are not economically feasible but do provide significantly better
control when applied with standard grower insecticides. Comprehensive
pheromone dispenser application can be economically feasible due to the high
level of control achieved when applied with integrated chemical treatments. This
experiment has been a great step forward in regard to VMB biology, behavior
patterns, and pheromone responses.
REFERENCES
LITERATURE CITED
Berlinger, M. 1977. The Mediterranean vine mealybug and its natural enemies in
southern Israel. Phytoparasitica 5(1): 3-14.
Bruck, E., A. Elbert, et al. 2009. Movento, an innovative ambimobile insecticide
for sucking insect pest control in agriculture: Biological profile and field
performance. Crp. Prot. 28(10): 838-844.
Chang, K. T. 2002. Introdution to Geographic Infromation Systems 1st ed. The
McGraw-Hill Companies, New York.
Cooper, M., K. Daane, et al. 2008. Liquid baits control Argentine ants sustainably
in coastal vineyards. Cal. Ag. 62(4): 177-183.
Daane, K., W. Bentley, et al. 2006. New controls investigated for vine mealybug.
Cal. Ag. 60(1): 31-38.
Daane, K., W. J. Bently, et al. 2009. Application for Emergency Exemption. Dep.
Pestic. Reg., Pesticide Registration Branch (State of California).
Daane, K., R. Malakar-Kuenen, et al. 2002. Abiotic and biotic pest refuges hamper
biological control of mealybugs in California vineyards. Abiotic and biotic
pest refuges in California Vineyards. 1st International Symposium on
Biological Control of Arthropods. 390-397.
Daane, K. M., M. L. Cooper, et al. 2008. Vineyard managers and researchers seek
sustainable solutions for mealybugs, a changing pest complex. Cal. Ag.
62(4).
Daane, K. M., R. D. Malakar-Kuenen, et al. 2004. Temperature-dependent
development of Anagyrus pseudococci (Hymenoptera: Encyrtidae) as a
parasitoid of the vine mealybug, Planococcus ficus (Homoptera:
Pseudococcidae). Bio. Cntrl. 31(2): 123-132.
Daane, K. M., K. R. Sime, et al. 2007. Impacts of Argentine ants on mealybugs
and their natural enemies in Californiaís coastal vineyards. Eco. Ent. 32(6):
583-596.
de Borbon, C. M., O. Gracia, et al. 2004. Mealybugs and grapevine leafrollassociated virus 3 in vineyards of Mendoza, Argentina. Am. J. Enol. Vitic.
55(3): 283-285.
51
Douglas, N. and K. Kruger 2008. Transmission efficiency of Grapevine leafrollassociated virus 3 (GLRaV-3) by the mealybugs Planococcus ficus and
Pseudococcus longispinus (Hemiptera: Pseudococcidae). Eur. J. P. Path.
122(2): 207-212.
Engelbrecht, D. and G. Kasdorf 1990. Transmission of grapevine leafroll disease
and associated closteroviruses by the vine mealybug, Planococcus ficus.
Phytophylactica 22(3): 341-346.
Geiger, C. A. and K. M. Daane 2001. Seasonal movement and distribution of the
grape mealybug (Homoptera: Pseudococcidae): developing a sampling
program for San Joaquin Valley vineyards. J. Econ. Ent. 94(1): 291-301.
Godfrey, K., J. Ball, et al. 2003. Biology of the Vine Mealybug in Vineyards in
the Coachella Valley, California. SW. Ent. 28(3): 183-196.
Golino, D., S. Sim, et al. 2002. California mealybugs can spread grapevine leafroll
disease. Cal. Ag. 56(6): 196-201.
Hinkens, D. M., J. S. McElfresh, et al. 2001. Identification and synthesis of the sex
pheromone of the vine mealybug, Planococcus ficus. Tetrahedron Letters
42(9): 1619-1621.
Kurtural, S. K. 2006. Balanced cropping of'Chambourcin' grapevines and a spatial
decision support system for vineyard site selection in southern Illinois,
Southern Illinois University At Carbondale.
Mgocheki, N. and P. Addison. 2009. Interference of ants (Hymenoptera:
Formicidae) with biological control of the vine mealybug Planococcus ficus
(Signoret) (Hemiptera: Pseudococcidae). Bio. Cntrl. 49(2): 180-185.
Mgocheki, N. and P. Addison 2010. Spatial distribution of ants (Hymenoptera:
Formicidae), vine mealybugs and mealybug parasitoids in vineyards. J. Appl.
Ent. 134(4): 285-295.
Millar, J., W. J. Bentley, et al. 2003. Mating Disruption of Vine Mealybug in
California Vineyards. Vitic. R. Rep. 1-9.
Millar, J. G., K. M. Daane, et al. 2002. Development and optimization of methods
for using sex pheromone for monitoring the mealybug Planococcus ficus
(Homoptera: Pseudococcidae) in California vineyards. J. Econ. Ent. 95(4):
706-714.
52
Nelson, E. H. and K. M. Daane 2007. Improving liquid bait programs for
Argentine ant control: bait station density. Env. Ent. 36(6): 1475-1484.
NOAA. 2012. Climate Wind Data For The United States. National Oceanic and
Atmospheric Administration.
Petersen, C. and J. Charles. 1997. Transmission of grapevine leafroll associated
closteroviruses by Pseudococcus longispinus and P. calceolariae. P. Path.
46(4): 509-515.
Stelinski, L. L., L. J. Gut, et al. 2003. Concentration of airborne pheromone
required for long lasting peripheral adaptation in the obliquebanded
leafroller, Choristoneura rosaceana. Phys. Ent. 28(2): 97-107.
Stelinski, L. L., J. R. Miller, et al. 2003. Presence of long-lasting peripheral
adaptation in oblique-banded leafroller, Choristoneura rosaceana and absence
of such adaptation in redbanded leafroller, Argyrotaenia velutinana. J. Chem.
Eco. 29(2): 405-423.
Tsai, C. W., J. Chau, et al. 2008. Transmission of Grapevine leafroll-associated
virus 3 by the vine mealybug (Planococcus ficus). Phytopath. 98(10): 10931098.
Walton, V. and K. Pringle. 2004. Vine mealybug, Planococcus ficus
(Signoret)(Hemiptera: Pseudococcidae), a key pest in South African
vineyards. A review. S. Afr. J. Enol. Viticult 25.
Walton, V. and K. Pringle 2005. Developmental biology of vine mealybug,
Planococcus ficus (Signoret)(Homoptera: Pseudococcidae), and its parasitoid
Coccidoxenoides perminutus (Timberlake)(Hymenoptera: Encyrtidae). Afr.
Ent. 13(1): 143-147.
Walton, V. M., K. M. Daane, et al. 2006. Pheromone-based mating disruption of
Planococcus ficus (Hemiptera: Pseudococcidae) in California vineyards. J.
Econ. Ent. 99(4): 1280-1290.
Welter, S., C. Pickel, et al. 2005. Pheromone mating disruption offers selective
management options for key pests. Ca. Ag. 59(1): 16-22.
Welter, S. C., F. Cave, et al. 2002. Development of alternative pheromone
dispensing technologies for management of codling moth. Waln. Rsrch. Rep.
2001: 225-227.
53
Zada, A., E. Dunkelblum, et al. 2008. Attraction of Planococcus ficus males to
racemic and chiral pheromone baits: flight activity and bait longevity. J. App.
Ent. 132(6): 480-489.
APPENDICES
APPENDIX A: TABLES
56
Table 1. Chi-square analysis of percentage of cluster damage in site 1, as affected
by puffer pheromone application, 26 August 2011.
Untreated
control
Two puffers·
acre-1
Pearson Chisquare
Rating 0
94.17
Rating 1
3.54
Rating 2
1.56
Rating 3
0.72
Total
100
97.12
2.54
0.42
0.36
100
DF
3
Value
19.8209
Prob
0.0002
Table 2. Chi- square analysis of percentage of cluster damage in site 2, as affected
by puffer pheromone application, 2 September 2011.
Rating 0
Untreated control 95.26
Three puffers·
98.92
acre-1
Pearson Chisquare
DF
3
Rating 1
2.10
0.96
Rating 2
1.50
0.06
Value
46.5832
Prob
0.0001
Rating 3
1.14
0.06
Total
100
100
57
Table 3. Percent Cluster Damage by Treatment 16 September 2012
Control
100 mg/carda
143 mg/carda
150 mg/cardb
a
200 mg/card
214 mg/cardb
300 mg/cardb
Pearson Chisquare
No Damage (0)
Little damage (1) Damaged (2)
Unmarketable (3)
71.67
92.78
99.44
90.56
90.00
98.33
87.22
21.67
5.00
0.56
7.22
5.56
1.11
10.56
6.67
1.67
0.00
2.22
3.33
0.56
1.11
0.00
0.00
0.00
0.00
1.11
0.00
1.11
DF
18
Value
76.2533
P
0.0001
a
25g of total active ingredient deployed over card lifespan
b
37.5g of total active ingredient deployed over card lifespan
APPENDIX B: FIGURES
59
Figure 1. Cumulative Male Vine Mealybug Trap Counts 2011
60
Figure 2. Percent Cluster Damage 2011
61
150b 300b 214b
214b 150b 300b
300b 214b
150b
North
100a 200a 143a
143a 100a 200a
200a 143a 100a
Figure 3. Denair 2012 pheromone dispenser loading trial map represented by
block. Values represent mg/card, letters represent total area loading. A = 25
g/acre. B = 37.5 g/acre.
62
Male Pheromone Response Pattern Repetition 1
WNW Wind
Vector
Insects Trapped
35.5°C
VMB Source
Figure 4. Male vine mealybug pheromone response pattern repetition 1.
63
Male Pheromone Response Pattern Repetition 2
WNW Wind
Vector
36.7°C
Insects Trapped
VMB Source
Figure 5. Male vine mealybug pheromone response repetition 2.
64
Male Pheromone Response Pattern Repetition 3
WNW Wind
Vector
Insects Trapped
29.4°C
VMB Source
Figure 6. Male vine mealybug pheromone response repetition 3
Fresno State
Non-Exclusive Distribution License
(to archive your thesis/dissertation electronically via the library’s eCollections database)
By submitting this license, you (the author or copyright holder) grant to Fresno State Digital Scholar the
non-exclusive right to reproduce, translate (as defined in the next paragraph), and/or distribute your
submission (including the abstract) worldwide in print and electronic format and in any medium, including
but not limited to audio or video.
You agree that Fresno State may, without changing the content, translate the submission to any medium or
format for the purpose of preservation.
You also agree that the submission is your original work, and that you have the right to grant the rights
contained in this license. You also represent that your submission does not, to the best of your knowledge,
infringe upon anyone’s copyright.
If the submission reproduces material for which you do not hold copyright and that would not be
considered fair use outside the copyright law, you represent that you have obtained the unrestricted
permission of the copyright owner to grant Fresno State the rights required by this license, and that such
third-party material is clearly identified and acknowledged within the text or content of the submission.
If the submission is based upon work that has been sponsored or supported by an agency or organization
other than Fresno State, you represent that you have fulfilled any right of review or other obligations
required by such contract or agreement.
Fresno State will clearly identify your name as the author or owner of the submission and will not make
any alteration, other than as allowed by this license, to your submission. By typing your name and date
in the fields below, you indicate your agreement to the terms of this distribution license.
Embargo options (fill box with an X).
x
Make my thesis or dissertation available to eCollections immediately upon
submission.
Embargo my thesis or dissertation for a period of 2 years from date of graduation.
Embargo my thesis or dissertation for a period of 5 years from date of graduation.
David Langone
Type full name as it appears on submission
April 17, 2013
Date