Foliar Application of Steinernema yirgalemense to Control Planococcus ficus: Assessing Adjuvants to Improve Efficacy

Abstract

The vine mealybug (Planococcusficus) is regarded as a key mealybug pest of grapevines in South Africa, with entomopathogenic nematodes (EPNs) being touted as a potential alternative to chemical control, although their vulnerability to above-ground environmental conditions has limited their use. In this study, tests were conducted to assess the ability of adjuvants to increase the deposition of S. yirgalemense on grapevine leaves. The combination of Nu-Film-P® and Zeba® resulted in significantly more infective juveniles (30) being deposited per 4 cm² leaf disc than with either the control (14.8), or with Nu-Film-P® (23.3), although not significantly more than with Zeba® alone (29.2). The ability of S. yirgalemense, in conjunction with the two adjuvants, to control P. ficus on grapevine foliage was then assessed under controlled conditions. The application of S. yirgalemense with both Zeba® and Nu-Film-P® to P. ficus on leaf discs in a growth chamber resulted in 84% mortality, significantly greater than that attained by the application of S. yirgalemense with either Zeba® (47%), or water alone (26%). Similar results were observed in a glasshouse trial, in which the combination of S. yirgalemense, Zeba® and Nu-Film-P® offered 88% control of P. ficus on leaf discs hung on potted vines, compared with the control that was achieved with S. yirgalemense with either Zeba® (56%) or water alone (30%). This study demonstrates the potential of a combination of S. yirgalemense with adjuvants to give significant control of P. ficus on grapevine foliage, compared with using EPNs alone.

Full text

*Corresponding author: E-mail address: apm@sun.ac.za
Acknowledgements: The authors would like to thank Winetech, the South African Table Grape Industry (SATGI), the Agricultural Research Council (ARC) and
the Technology and Human Resources for Industry Programme (THRIP grant number: TP14062571871), for funding the project. We would also like to thank
D.G. Nel, for assistance with the statistical analysis
Foliar Application of Steinernema yirgalemense to Control Plano-
coccus cus: Assessing Adjuvants to Improve Efcacy
T. Platt2, N.F. Stokwe1,2, A.P. Malan2,*
(1) ARC Infruitec-Nietvoorbij, Private Bag X5026, Stellenbosch 7599, South Africa
(2) Department of Conservation Ecology and Entomology, Stellenbosch University, Private Bag X1, Matieland 7602, South
Africa
Submitted for publication: April 2018
Accepted for publication: July 2018
Keywords: Adjuvants, entomopathogenic nematodes, Steinernema yirgalemense, Planococcus cus, vine mealybug
The vine mealybug (Planococcus cus) is regarded as a key mealybug pest of grapevines in South Africa,
with entomopathogenic nematodes (EPNs) being touted as a potential alternative to chemical control,
although their vulnerability to above-ground environmental conditions has limited their use. In this
study, tests were conducted to assess the ability of adjuvants to increase the deposition of S. yirgalemense
on grapevine leaves. The combination of Nu-Film-P® and Zeba® resulted in signicantly more infective
juveniles (30) being deposited per 4 cm2 leaf disc than with either the control (14.8), or with Nu-Film-P®
(23.3), although not signicantly more than with Zeba® alone (29.2). The ability of S. yirgalemense, in
conjunction with the two adjuvants, to control P. cus on grapevine foliage was then assessed under
controlled conditions. The application of S. yirgalemense with both Zeba® and Nu-Film-P® to P. cus
on leaf discs in a growth chamber resulted in 84% mortality, signicantly greater than that attained by
the application of S. yirgalemense with either Zeba® (47%), or water alone (26%). Similar results were
observed in a glasshouse trial, in which the combination of S. yirgalemense, Zeba® and Nu-Film-P® offered
88% control of P. cus on leaf discs hung on potted vines, compared with the control that was achieved
with S. yirgalemense with either Zeba® (56%) or water alone (30%). This study demonstrates the potential
of a combination of S. yirgalemense with adjuvants to give signicant control of P. cus on grapevine
foliage, compared with using EPNs alone.
INTRODUCTION
South Africa is the twelfth largest producer of wine and table
grapes in the world, producing 2.61% of the world’s grapes
in 2014 (FAO, 2016). Wine and table grape production
therefore is of signicant economic importance to South
Africa, and especially to the Western Cape province, where
the majority of wine and table grape production occurs
(South African Wine Industry Information and Systems
[SAWIS], 2015; South African Table Grape Industry [SATI],
2016).
The vine mealybug, Planococcus cus (Signoret)
(Hemiptera: Pseudococcidae), is a pest of grapevine found
in most grape-producing regions worldwide (Ben-Dov,
1994; Walton & Pringle, 2004). It is the predominant pest
of grapevine in South Africa (Walton, 2003; Walton et al.,
2004), causing damage chiey by transmitting the grapevine
leafroll-associated virus type III (GRLaV-3), which causes
the rolling and discolouration of vine leaves (Bovey et al.,
1980). Mealybugs also damage vines by phloem feeding,
which reduces the ow of plant sap to the fruit, thereby
reducing yield (Millar, 2002); and by depositing waxy
residues and sooty mould-encouraging honeydew, thereby
disguring the grapes (Geiger & Daane, 2001).
Existing measures to control the vine mealybug on
grapevines have tended to focus on chemical control, with
anti-mealybug products using active ingredients such as
imidacloprid, dichlorvos and methidathion (Agri-Intel,
2018). However, due to the potential for harm to non-
target organisms via direct contact, or the contamination
of groundwater, as well as the potential for target insects to
develop resistance, biological alternatives are often sought as
a possible solution to the existing problem (Hussaini, 2002).
In particular, P. cus has innate defences against chemical
pesticides, such as its high reproductive rate, which allows
for an increase in the pace of development of its resistance to
pesticides (Daane et al., 2008), while both its cryptic choice
of environment (typically beneath raised grapevine bark)
and the waxy laments that it produces serve as barriers to
pesticide contact, post-application (Berlinger, 1977).
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Adjuvants to Improve EPN Foliar Application
Entomopathogenic nematodes (EPNs) are soil-
based pathogens of insects, mainly from the families
Steinernematidae and Heterorhabditidae, which are widely
used as biocides against the soil-based insect life stages
(Campos-Herrera, 2015). EPNs encounter their prey
by means of exhibiting behaviour on a continuum from
stationary, ‘ambushing’ (which is better for active prey) to
mobile, ‘cruising’ behaviour (which is more suited to passive
and/or cryptic prey) while in a free-living infective juvenile
(IJ) life stage (Lewis, 2002; Campbell et al., 2003; Grifn
et al., 2005). Once they encounter prey, the IJs enter the pest
insect’s body cavity through the natural openings, thereby
killing the insect, in conjunction with its symbiotic bacteria
species, and undergoing several generations within the cavity
of the insect (Grifn et al., 2005). EPNs are an attractive
potential biological control agent, due to their initial
virulence to the target pest, their ability to actively seek out
insect pests, and their relatively low persistence within the
environment (Smits, 1996; Wilson & Gaugler, 2004). The
use of EPNs to control insect pests is common and effective
against soil-based insect pests (Wilson & Gaugler, 2004).
The application of EPNs to control above-ground insect
life stages is less common, mostly due to abiotic factors that
affect nematode survival, particularly temperature (Grewal
et al., 1994), humidity (Lello et al., 1996; De Waal et al.,
2013), and ultraviolet radiation (Gaugler & Boush, 1978).
EPNs make use of any water lm on the leaves in humid
environments to infect their insect prey, making the EPN
application more useful in tropical and/or rainy environments
than in dryer ones, or at the time of day in which relative
humidity is highest (Mráček, 2002).
Arthurs et al. (2004) performed a review of 136 published
trials, each investigating the potential of EPNs against above-
ground pests. They found that nematode efcacy depended on
the target habitat, with most successful application occurring
against hole-boring insect pests, followed by insects that
select protected, cryptic habitats, with insect pests found in
exposed habitats proving most difcult to control. Various
studies have assessed the ability of EPNs to control insect
pests in the laboratory, glasshouse and eld, with the general
trend being: the more similar a target insect’s habitat is to
soil (in terms of temperature, humidity and shelter from
abiotic stresses such as UV exposure), the more successful
EPN application will be.
One possible means of increasing EPN efcacy on
foliage involves the improvement of EPN formulations by
means of adjuvants, which are chemical additives that alter
the physical properties of formulations. The formulation
of EPN solutions with adjuvants has proven promising in
their use against boring insect pests, whose bored tunnel
environments are shielded from environmental stresses.
Shapiro-Ilan and Cottrell (2006) assessed the efcacy of
EPNs against the lesser peach tree borer, Synanthedon
pictipes (Grote and Robinson), and found Steinernema
carpocapsae (Weiser, 1955) Wouts, Mráček, Gerdin &
Bedding, 1982 to be the most effective EPN. Further trials
tested the effects of several adjuvant compounds on the
survival and efcacy of S. carpocapsae. Shapiro-Ilan et al.
(2010) found that Barricade® re gel improved EPN activity
when applied as a post-application treatment, with further
trials (Shapiro-Ilan et al., 2016) nding that the formulation
of S. carpocapsae with Barricade® re gel gave control
equivalent to the application of chlorpyrifos.
The anti-transpirant Folicote® has been used to increase
the lifespan of S. carpocapsae on beans, improving IJ
viability from 38% to 60%, at 60% RH over 6 h in an exposed
foliage environment (Glazer, 1992). Baur et al. (1997)
investigated the application of several adjuvant-nematode
preparations for efcacy against the diamondback moth,
Plutella xylostella (Linnaeus) (Lepidoptera: Plutellidae),
and concluded that, while such preparations probably did not
justify their commercial application against P. xylostella, the
addition of adjuvants improved the persistence and efcacy
of the EPNs tested. Head et al. (2004) found that the addition
of either of the two surfactants, Agral® and Triton X-100®, to
formulations of Steinernema feltiae (Filipjev, 1934) Wouts,
Mráček, Gerdin & Bedding, 1982 signicantly increased the
latter’s efcacy against the foliage-dwelling life stages of the
tobacco whitey, Bemicia tabaci (Gennadius) (Homoptera:
Aleryodidae), on tomato and verbena plants, with no
adverse effects occurring on EPNs or in terms of host plant
phytotoxicity.
The main objective of this study was to test the effect
of adjuvants on the efcacy of above-ground applications
of EPNs to control P. cus on grapevine. Bioassays were
performed to assess the ability of adjuvants to improve EPN
deposition, and efcacy against P. cus on grapevine foliage.
These bioassays were performed in the growth chamber
(a highly-controlled, low-variance environment) and in
the glasshouse (a medium-controlled, medium-variance
environment).
MATERIALS AND METHODS
Source of nematodes
The nematode species used in the current study, Steinernema
yirgalemense Nguyen, Tesfamariam, Gozel, Gaugler
& Adams, originated from samples that were collected
locally, and maintained and cultured at Stellenbosch
University (Malan et al., 2011). Infective juveniles (IJs)
were cultured in vivo by infecting larvae of the mealworm
beetle Tenebrio molitor L. (Tenebrionidae: Coleoptera) with
IJs. Dead infected mealworms were kept at 25°C in a petri
dish lined with moist lter paper and sealed with Paralm
until IJ emergence, before being transferred to White traps
(White, 1927). The IJs harvested from the White traps were
transferred to vented culture asks, where they were kept
at 14°C, in keeping with the guidelines set out by Kaya and
Stock (1997). These asks were gently agitated once a week
to improve aeration. IJs for the experiment were used within
one week of emergence. The experiment was repeated on a
different test date, with a fresh batch of nematodes.
Source of insects
A laboratory culture of P. cus was established to ensure
reliable access to female individuals. The culture, which
originated at the Agricultural Research Council (ARC)
Infruitec-Nietvoorbij in Stellenbosch, South Africa, was
propagated on butternut squash (Cucurbita moschata
(Duchesne ex Lam.) Duschesne ex Poir.) in a Perspex cage
under ambient conditions. The cage was vented with mesh
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Adjuvants to Improve EPN Foliar Application
netting to allow for air circulation, although it was otherwise
kept sealed to prevent the escape of any mealybug nymphs.
A fresh butternut was added once every three weeks to
allow the individuals to migrate from the older butternut,
which was then removed once rot set in. Females were
removed for testing with a ne paintbrush and a pair of
tweezers. Individuals were removed only if they were not
currently feeding, as damage to mouthparts can impact the
survivability of the insect.
Adjuvant deposition
An experiment was set up to test the efcacy of two adjuvants,
Zeba® [starch-g-poly (2-propenamideco-2-propenoic acid)
potassium salt, Tongaat Hulett Starch, Germiston, South
Africa] and Nu-Film-P® (poly-1-p-menthene, spreader,
sticker; Hydrotech, Pretoria, South Africa), in applying
S. yirgalemense to grapevine leaves. Four nematode 250 ml
suspensions were prepared, each containing 1 000 IJs/ml,
with one containing Zeba® (0.3 g/L), one containing Nu-
Film-P® (0.6 ml/L), one containing equal parts Zeba® and
Nu-Film-P® at 0.3 g/L and 0.6 ml/L respectively, and one
with a control of IJs in water alone. The adjuvants were
applied according to the recommendations on the labels.
A handheld sprayer was used to apply the above-
mentioned formulations to the grapevine leaves. Each
suspension was applied to a green grapevine leaf (harvested
no longer than 24 hours prior to use) that was suspended
from a line, from a distance of 20 cm, until runoff (≈ 5 ml).
The procedure was repeated, using ve leaves per treatment.
Each leaf was allowed 3 min post-application to allow any
excess formulation to run off, after which two 4 cm2 discs
were cut from each leaf, giving 10 discs per suspension.
Each leaf disc was then rinsed with ≈ 5 ml of tap water,
holding the disk with a tweezer and using a wash bottle. The
IJs from each disc were concentrated by settling a 10 ml
cylinder for 30 min, after which the supernatant pipetted off
and 1 ml was transferred to the individual wells (3 ml) of a
bioassay tray (24-well, at bottom, Nunce Cat. No. 144530,
Sigma-Aldrich Pty. Ltd, Johannesburg, South Africa). The
nematodes present in the rinsed water were counted and
compared between treatments. The experiment was repeated
on a different test date with a fresh batch of nematodes.
Growth chamber bioassay
To simulate greenhouse conditions, large plastic containers
were lled with water and placed at the bottom of growth
chambers to increase humidity. Grapevine leaves obtained
from Welgevallen Experimental Farm were washed in
a solution of water and 0.01% household bleach, rinsed
thoroughly in tap water, and left to dry before use (to
ensure that the leaves were free of remnants of previous
unknown applications). Eight mealybugs were transferred to
each of eight leaves (eight replicates, 64 insects) for each
treatment. The leaves were cut to t Petri dishes of 13 cm
in diameter lined with moist lter paper. Treatments were
water only; S. yirgalemense in water; S. yirgalemense +
Zeba® in water; and S. yirgalemense + Zeba® + Nu-Film-P®
in water. Zeba® and Nu-Film-P® were used in the treatments
at a concentration of 0.03% and 0.06% respectively. The
treatment formulations were prepared 1 h before each trial.
The IJs were applied to the leaves with the aid of a calibrated
handheld spray applicator at a concentration of 3 000 IJs/ml.
The leaves were left for 3 min after treatment to eliminate
excess runoff. They were then placed in small pockets made
of ne-mesh netting. The pockets were hung in the growth
chamber in a complete randomised design. After 48 h, the
mealybugs were removed from the leaves and mortality was
assessed. The mealybugs were then washed to remove surface
nematodes, placed in Petri dishes lined with moistened lter
paper, and incubated for a further 48 h at 25°C (to ensure
nematode development), after which mortality by infection
was conrmed by dissection. The temperature and humidity
were monitored using iButtons, which were placed inside the
growth chambers. The experiment was repeated on a later
date with a fresh batch of nematodes.
Greenhouse trial
The leaf disc pockets, mealybugs and nematode/adjuvant
solutions were prepared as for the growth chamber bioassay,
with the same treatments and number of replicates per
treatment. After preparation, each of the 40 pockets
containing the treated mealybugs was hung on Chenin Blanc
potted grapevines located in a glasshouse. The temperature
(22 ± 5°C) and relative humidity (75 ± 8%) in the glasshouse
were monitored using dataloggers. The experiment was
repeated at a later date, with the results being pooled for
analysis.
Data analysis
The analysis of all trial data was conducted using
STATISTICA statistical analysis software version 13
(TIBCO Inc., 2017). The data were analysed using variance
estimation, precision and comparison (VEPAC) and analysis
of variance (ANOVA), using Bonferroni’s method for the
post-hoc comparison of means. Results from the adjuvant
deposition were analysed using a one-way ANOVA, while
for the growth chamber and greenhouse bioassays a two-way
ANOVA was used. Signicant differences were calculated to
the 95% probability level.
RESULTS
No signicant difference was recorded between the two sets
of data in each bioassay, and consequently the data from the
two test dates of each experiment were pooled for analysis.
Adjuvant deposition
The number of live IJs retrieved from the grapevine leaves
indicate a signicant difference between treatments (F (3, 76)
= 11.548, p < 0.01). The combination of Nu-Film-P® and
Zeba® was seen to result in the deposition of a signicantly
higher number (p = 0.01) of IJs (30.8 ± 4 IJs/4 cm2) compared
to the control (14.8 ± 2 IJs/4 cm2) and that of Nu-Film-P®
alone (23.3 ± 2 IJs/4 cm2). However, the combination of
Nu-Film-P® and Zeba® did not result in signicantly more
nematodes being deposited (p = 0.59) than did the Zeba®
alone (29.2 ± 3 IJs/4 cm2) (Fig. 1).
Growth chamber bioassay
The analysis of the results shows that mortality for each
treatment differed signicantly from all others (F (3, 120) =
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Adjuvants to Improve EPN Foliar Application
241.52, p = < 0.01). The combination of Zeba® and Nu-
Film-P® was found to be the most effective (84% ± 5%
mortality). The aforementioned combination was followed
by Zeba® alone (47% ± 3%), and then by the nematodes
alone (26% ± 2%), compared to the water control (9% ± 2%)
(Fig. 2).
Greenhouse bioassay
The analysis of the results for mortality, after 48 h, showed
each treatment differed signicantly from the others (F (3,
120) = 207.42, p = < 0.01). The combination of Zeba® and
Nu-Film-P® was the most effective (88% ± 3% mortality),
followed by Zeba® alone (56% ± 5%), and then by the
nematodes alone (30% ± 3%), compared with the water
control (13% ± 2%) (Fig 3).
DISCUSSION
Although P. cus was found to be highly susceptible to EPNs
in the laboratory bioassays (Le Vieux & Malan, 2013; Platt
et al., 2018), special challenges are encountered under above-
ground environmental conditions, as soil is the natural habitat
of EPNs. This study is a stepping-stone from very articial
conditions to less articial conditions, with the next step
being to move on to eld trials. Lack of moisture/humidity
is the most signicant challenge. One option for overcoming
the problem of humidity is the addition of adjuvants to the
nematode suspension, assisting in the ability of nematodes to
stick onto the leaves and prolonging the lm of water on the
leaves that is required for nematode movement.
The addition of Zeba® to EPN formulations resulted
in signicantly higher deposition of S. yirgalemense IJs,
both alone and in combination with Nu-Film-P®. The
application of Zeba® and Nu-Film-P® gave signicantly
more IJs deposited than did Nu-Film-P® alone, although all
the treatments resulted in signicantly higher deposition of
S. yirgalemense IJs onto grapevines leaves than did water
alone. The ability to double the number of IJs deposited onto
grapevine leaves makes Zeba® (and, to a lesser extent, Nu-
Film-P®) an attractive addition to suspensions for nematode
application. The nding follows a similar trend to the research
of Van Niekerk and Malan (2015), who assessed the use of
Nu-Film-P® and Zeba® for the deposition of Heterorhabditis
zealandica Poinar, 1990 onto citrus leaf discs. In that study,
only the combination of Nu-Film-P® and Zeba® signicantly
increased the nematode deposition on citrus leaves compared
to the control, due to the waxy (water-repellent) coating on
the citrus leaves. This study indicates that Zeba® and Nu-
Film-P® can effectively be used when targeting plants
without waxy coatings, such as grapevine leaves.
The results of the growth chamber bioassay showed that
S. yirgalemense was most effective when applied to female
P. cus in a combination of Zeba® and Nu-Film-P®, with 84%
mortality having been caused after 48 h. The above indicates
that, despite the fact that the addition of Nu-Film-P® did
not signicantly improve the deposition of nematodes onto
grapevine leaves over the application of Zeba® alone, its
status as a spreader and stick can still improve the control
of P. cus in formulation with S. yirgalemense. Van Niekerk
and Malan (2015) performed a similar bioassay, assessing
the mortality of P. citri, post-application of H. zealandica and
S. yirgalemense in suspension with distilled water, xanthan
gum or Zeba®. They found that the addition of Zeba® caused
a signicant increase in the mortality of P. citri, improving
the H. zealandica-induced mortality by 22% and the
S. yirgalemense mortality by 27% at 80% relative humidity.
The greenhouse bioassay sought to assess the impact of
1
1
Control Nu-Film Zeba NuFilm + Zeba
Adjuvant treatment
0
5
10
15
20
25
30
35
40
Mean num ber of S. y irgalem ense
a
ab
b
c
FIGURE 1
Mean percentage (95% condence interval) deposition of Steinernema yirgalemense infective juveniles (IJs) onto grapevine
leaves, applied with a handheld sprayer at a concentration of 1 000 IJs/ml. After rinsing the leaves with tap water, the nematodes
in the runoff were counted (one-way ANOVA: F (3, 76) = 11.548, p = < 0.01). Means of bars sharing a letter are not signicantly
different from one another.
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Adjuvants to Improve EPN Foliar Application
a less-controlled environment on treatments from the growth
chamber bioassay. However, unlike the growth chamber
bioassay, the average temperature and humidity were
much lower over the course of the experiment – closer to
conditions that would be expected in the eld. Interestingly,
these conditions did not appear to lower the overall P. cus
mortality, following the trend set by the growth chamber
bioassay, in which the most effective treatment was also that
of the combination of Zeba® with the IJ. The mortality of
the control mealybugs was higher in the greenhouse bioassay
than it was in the growth chamber bioassay, although only
by 4%, making this a promising indication that Zeba® and
Nu-Film-P® can be used in conjunction to control P. cus
on grapevines under sheltered, or covered, conditions. The
results are mirrored by the ndings of Van Niekerk (2012),
who emulated greenhouse conditions by performing a growth
chamber bioassay at 22°C and 75 ± 8% RH. However, the
authors found that the addition of the both adjuvants, Zeba®
and Nu-Film-P®, to S. yirgalemense resulted in higher
mortality in P. citri.
In conclusion, the results obtained indicate the potential
for S. yirgalemense to be used to control P. cus on foliage
under controlled conditions, which is a key step in developing
methods to apply S. yirgalemense to P. cus in the eld. Zeba®,
1
Control IJs only Zeba Zeba + Nu
Treatment
20
40
60
80
100
% Mortality
d
c
b
a
1
FIGURE 2
Mean percentage (95% condence interval) mortality of Planococcus cus on grapevine leaves treated with Steinernema
yirgalemense infective juveniles (IJs) after 48 h exposure in a glasshouse environment. IJs were applied to leaves with a
handheld sprayer at a concentration of 3 000 IJs/ml (one-way ANOVA: F (3,120) = 241.52; p = < 0.01). Means of bars sharing a
letter are not signicantly different from one another.
FIGURE 3
Mean percentage (95% condence interval) mortality of Planococcus cus on grapevine leaves kept in a greenhouse
environment, post-treatment with Steinernema yirgalemense. Infective juveniles (IJs) were applied to leaves with a handheld
sprayer at a concentration of 3 000 IJs/ml. Means of bars sharing a letter are not signicantly different from one another.
1
Control IJs only Zeba Zeba + Nu
Treatment
0
20
40
60
80
100
%P. ficus m ortality
d
c
b
a
1
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Adjuvants to Improve EPN Foliar Application
a polysaccharide starch, improves nematode deposition and
infectivity when compared to Nu-Film-P®. The use of EPN
suspensions containing Nu-Film-P® (a spreader and sticker)
alone showed much lower improvement in P. cus mortality
when compared to the use of suspensions containing
Zeba® alone. However, combinations of both adjuvants
offered signicantly higher mortality, indicating that both
adjuvants work synergistically to promote EPN survival
and infectivity on foliage. When assessing adjuvants for use
in EPN suspensions going forward, attention must be paid
to the qualities of each constituent and how they interact.
In addition, the ability of suspensions of S. yirgalemense,
Zeba® and Nu-Film-P® to achieve 88% mortality in P. cus
in the glasshouse warrants future research into the ability
of S. yirgalemense to control other insect pests in indoor
environments.
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