Crop Protection 132 (2020) 105095

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Crop Protection
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Insecticidal effect of Dittrichia viscosa lyophilized epicuticular material

against four major stored-product beetle species on wheat
E. Lampiri a, b, P. Agrafioti a, E. Levizou b, C.G. Athanassiou a, *
a
Laboratory of Entomology and Agricultural Zoology, Department of Agriculture, Crop Production and Rural Environment, University of Thessaly, Phytokou str., Nea
Ionia, Magnesia, 38446, Greece
b
Laboratory of Weed Science, Department of Agriculture, Crop Production and Rural Environment, University of Thessaly, Phytokou str., Nea Ionia, Magnesia, 38446,
Greece

A R T I C L E I N F O A B S T R A C T

Keywords: We examined the insecticidal effect of lyophilized epicuticular material of the ruderal species Dittrichia viscosa in
Dittrichia viscosa four major stored-product beetle species. Furthermore, the potential of this material in progeny production
Lyophilized epicuticular material suppression was also evaluated. The water-soluble extract was derived from plants that had been harvested in
Botanicals
September 2016, through freeze-drying, in order to create a fine powder formulation. In our bioassays, the
Post-harvest control
Stored-product insects
powder was applied in four doses on wheat: 0 (control), 1000, 3000 and 5000 ppm and mortality of the exposed
individuals was measured after 1, 3, 7, 14 and 21 days of exposure, while progeny production capacity was
recorded 65 days later. Among the species tested, Oryzaephilus surinamensis was found to be the most susceptible,
followed by Tribolium confusum and Sitophilus oryzae, while Rhyzopertha dominica was not practically affected.
Progeny production was particularly reduced for all species relative to the controls. Indicatively, for
O. surinamensis, at the highest dose rate, there were only 0.2 adults per vial, while the respective figures for the
control exceeded 40 adults per vial. To our knowledge, this study is the first that examined the insecticidal effect
of epicuticular material of D. viscosa for the control of stored-grain insect species. Additional experimentation is
required to indicate the rationale of using this natural resource-based material under a non-chemical control
strategy at the post-harvest stages of agricultural commodities.

1. Introduction
The most common method that is currently in use for the control of
stored-product pests is chemical insecticides, which are mainly sepa­
rated into two major categories: contact insecticides and fumigants, such
as grain protectants and phosphine (Athanassiou and Arthur, 2018;
Daglish et al., 2018). Despite the positive effects that conventional in­
secticides can provide against stored-product pests, over the past few
decades, there are increasing concerns regarding their use for health and
environmental reasons, which has led to the withdrawal of many active
ingredients (Dubey et al., 2010; Nayak and Daglish, 2018; Stejskal et al.,
2018). Furthermore, a major issue resulting from the continuous use of
chemical control is the development of resistance of key stored-product
insect species to many of the currently used conventional insecticides.
For instance, the red flour beetle, Tribolium castaneum (Herbst) (Cole­
optera: Tenebrionidae) (Jagadeesan et al., 2012, 2013) and the lesser
grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae)
(Schlipalius et al., 2002; Kaur et al., 2013), were found to be resistant to
phosphine. Hence, it is essential to evaluate alternatives that are viable
and effective, and at the same time have reduced environmental impact
and human health risks.
Numerous papers have suggested insecticides of botanical origin for
use at the post-harvest stages of agricultural commodities (Prakash and
Rao, 1997; Weaver and Subramanyam, 2000; Athanassiou et al., 2014).
It is generally considered that plant derivatives fulfill the above re­
quirements, since, in majority, are non-toxic to mammals and have
natural origin. The most well-studied plant for this purpose is by far the
neem tree, Azadirachta indica A. Juss. (Sapindales: Meliaceae), and its
main active ingredient azadirachtin which has been evaluated with
success against a wide range of stored-product insect species (Weaver
and Subramanyam, 2000; Athanassiou et al., 2005). Nevertheless, aza­
dirachtin requires elevated dose rates to act, which are far higher than
those of the currently used conventional insecticides (Athanassiou et al.,
2005). Other well-studied plants for stored-product protection are the

* Corresponding author.
E-mail address: athanassiou@agr.uth.gr (C.G. Athanassiou).

https://doi.org/10.1016/j.cropro.2020.105095
Received 1 December 2019; Received in revised form 27 January 2020; Accepted 29 January 2020
Available online 29 January 2020
0261-2194/© 2020 Elsevier Ltd. All rights reserved.
E. Lampiri et al.
species of the genera Brassica, Citrus, Cymbopogon, Mentha and Ros­
marinus, which have been effective for both stored-product beetles and
moths (Prakash and Rao, 1997; Weaver and Subramanyam, 2000).
Indicatively, derivatives from Rosmarinus officinalis L. (Lamiales: Lam­
iaceae), Mentha microphylla C. Kock (Lamiales: Lamiaceae) and Mentha
viridis L. (Lamiales: Lamiaceae) were able to control various species,
such as Sitophilus spp. (Coleoptera: Curculionidae), Callosobruchus spp.
(Coleoptera: Bruchidae), the cigarette beetle, Lasioderma serricorne (F.)
(Coleoptera: Anobiidae), the bean weevil, Acanthoscelides obtectus (Say)
(Coleoptera: Bruchidae) (Huang et al., 2000), T. castaneum (Lee et al.,
2002), the confused flour beetle, Tribolium confusum Jacquelin du Val
(Coleoptera: Tenebrionidae) and the Mediterranean flour moth, Ephestia
kuehniella Zeller (Lepidoptera: Pyralidae) (Tunc et al., 2000). In this
context, one of the most important families that have been evaluated
towards this direction is Asteraceae, with several plants and plant sub­
stances to pose a noticeable insecticidal activity (Pascual-Villalobos and
Robledo, 1998; Torres et al., 2003; Pavela, 2004; Boussaada et al.,
2008). For instance, Anacyclus clavatus Pers (Asterales: Asteraceae) and
Asteriscus maritimus (L.) Less (Asterales: Asteraceae) were toxic to
T. castaneum (Pascual-Villalobos and Robledo, 1998). However, there is
still inadequate information for one of the Asteraceae species, the false
yellowhead, Dittrichia viscosa (L.) Greuter, despite the fact that there are
some initial works that show a potential insecticidal value (Allal-Ben­
fekih et al., 2011; Mamoci et al., 2012; Grauso et al., 2019; Rotundo
et al., 2019). This kind of plant is an evergreen, perennial shrub with
rich sticky foliage and intense odor, widely known for its medicinal use
(Parolin et al., 2014). The application of this plant, either as an extract
from plant tissues or as an essential oil, has shown antifungal activity
against Fusarium moniliforme J. Sheld. (Hypocreales: Nectriaceae),
Sclerotinia sclerotiorum (Lib.) de Bary (Helotiales: Sclerotiniaceae),
Rhizoctonia solani J.G. Kuhn (Cantharellales: Ceratobasidiaceae) and
Phytophthora capsici Leonian (Peronosporales: Peronosporaceae) (Yegen
et al., 1992). Moreover, D. viscosa has a repulsive or even an acaricidal
action against the carmine spider mite, Tetranychus cinnabarinus (Bois­
duval) (Prostigmata: Tetranychidae) (Topakci et al., 2005), and is
effective for the control of certain plant parasitic nematodes, such as
Meloidogyne javanica (Treub) Chitwood (Tylenchida: Meloidogyne) (Oka
et al., 2001). In addition, its application as a plant extract to larvae of
Tuta absoluta (Meyrick) (Lepidoptera: Gelenchidae) (Allal-Benfekih
et al., 2011) and to Myzus persicae (Sulzer) (Hemiptera: Aphididae)
(Mamoci et al., 2012) have shown positive results.
Most of the published works are focused on D. viscosa shoot (Qasem
and Abu Blan 1995) and extracts from different aerial parts of the plant
or applications of its essential oil (Rotundo et al., 2019; Grauso et al.,
2019). Specifically, hexane and ethanolic extracts of D. viscosa have
shown antifeedant activity against M. persicae, Rhopalosiphum padi (L.)
(Hemiptera: Aphididae) and Spodoptera littoralis (Boisduval) (Lepidop­
tera: Noctuidae) (Mamoci et al., 2012; Grauso et al., 2019), while high
constant toxicity was found when tested against the granary weevil,
Sitophilus granarius (L.) (Coleoptera: Curculionidae) (Rotundo et al.,
2019). However, there is another plant part that has never been evalu­
ated towards this direction: its epicuticular material. The epicuticular
material of D. viscosa is a water-soluble mixture of flavonoids and ses­
quiterpenes, which is attached to the waxy surface of the cuticle (Stav­
rianakou et al., 2004). Many eco-physiological roles have been ascribed
to this exudate, such as the prevention of excessive water loss via
transpiration and the increased protection against ultraviolet radiation.
Moreover, the allelopathic potential of the epicuticular material is
well-documented, which successfully inhibits the germination of the
neighboring plants seeds and/or interferes with their radicle growth and
seedling development, whilst is not toxic for its own seeds and seedlings
(Stephanou and Manetas, 1997; Levizou et al., 2002).
Considering the lack of information regarding the epicuticular ma­
terial of D. viscosa, we have carried out laboratory bioassays to examine
its insecticidal effect for the control of four major stored-product beetle
species, two primary colonizers, R. dominica and the rice weevil,
2
Crop Protection 132 (2020) 105095
Sitophilus oryzae (L.) (Coleoptera: Curculionidae) and two secondary
colonizers, T. confusum and the saw-toothed grain beetle, Oryzaephilus
surinamensis (L.) (Coleoptera: Silvanidae). Apart from parental mortal­
ity, suppression on progeny production capacity was also examined.
2. Materials and methods
2.1. Test insects
All species were reared at the Laboratory of Entomology and Agri­
cultural Zoology, Department of Agriculture, Crop Production and Rural
Environment, University of Thessaly, at 25 � C, 65% relative humidity (r.
h.) and continuous darkness. Rhyzopertha dominica and S. oryzae were
reared on whole wheat kernels, T. confusum on wheat flour and
O. surinamensis on oat flakes. Adult beetles, <1 month-old, were used in
the tests. All rearings have been kept in laboratory conditions for more
than 20 years.
2.2. Plant epicuticular material
Aerial parts of D. viscosa were collected during September 2016 from
rural areas of Volos (Thessaly, Central Greece) and soaked in water for 3
h. Then, the aqueous solution (containing the epicuticular material) was
collected, centrifuged and its absorbance at 290 nm (OD290) was
measured using a spectrophotometer (UV-1900, Shimatzu, Japan) after
the appropriate dilutions. This procedure resulted to a three-fold more
concentrated extract (OD290 ¼ 25) in comparison with the concentration
naturally found on leaves. Then, freeze-drying was followed in a vacuum
concentrator (Azbil Telstar Technologies, S. L. U.) at a temperature of
50 � C and pressure of 0.02 mBar. Finally, the epicuticular material,
formed as a powder, was collected, weighed and stored at 4 � C until
used.
2.3. Commodities
Untreated, clean and uninfected soft wheat was used in the tests.
Before the experiments, the wheat was kept in cold storage ( 20 � C) for
at least two weeks to eliminate possible insect infestation.
2.4. Bioassays
Lots of 500 g of wheat were placed in glass jars of 1000 ml in capacity
and treated with the powder formulation in 4 doses rates: 0 (control),
1000, 3000 and 5000 ppm. An additional series of lots, in which the
formulation was not applied, was used as a control. The jars were then
shaken for 5 min to ensure that the formulation was equally distributed
throughout the wheat mass. For the tests, we used plastic cylindrical
vials (3 cm in diameter, 8 cm high, Rotilabo Sample tins Snap on lid, Carl
Roth, Germany), with the top one quarter of the inside “neck” covered
with Fluon (polytetrafluoroethylene; Northern Products, Woonsocket,
USA) to avoid insects’ escape. Each vial contained 20 g of treated or
untreated wheat, and then 20 adults were placed into each vial, using
different vials for each insect species and dose. All vials were maintained
in incubators set at at 26 � C, 55% r. h. and continuous darkness. The
mortality of the exposed beetles was recorded after 1, 3, 7, 14 and 21
days. For each species-dose combination there were three vials, while
the entire procedure was repeated three times, i.e. there were three
replicates with three sub-replicates, and new lots of treated and un­
treated grains each time (3 jars X 3 vials each ¼ 9 vials for each com­
bination). At the end of this interval, all adults (dead and alive) were
removed from the vials and the vials remained in the same conditions for
an additional period of 65 days. Then, the vials were opened for the last
time and the number of progeny was recorded.
E. Lampiri et al. Crop Protection 132 (2020) 105095

2.5. Data analysis Table 2

Mean mortality (% � SE) of R. dominica adults after 1, 3, 7, 14 and 21 days of
Control mortality was generally low for all species, and did not exposure on wheat treated with different concentrations of the D. viscosa
exceed 10% in the vast majority of the cases. Since the same vials were formulation (in all cases df ¼ 3.35).
examined for mortality at the different exposure intervals (1–21 days), Treatment Exposure interval
mortality data were analyzed by using a Multivariate Analysis of Vari­ 1d 3d 7d 14 d 21 d
ance (MANOVA-Fit with Wilk’s Lambda) with concentration and
Control 0.6 � 0.6 0.6 � 0.6 1.6 � 1.1 2.2 � 1.2 3.9 � 1.1
exposure time as the main effects, by using the JPM 8 software (SAS 1000 ppm 0.6 � 0.6 0.6 � 0.6 1.1 � 1.1 9.4 � 5.8 11.6 � 7.4
Institute Inc., Cary, North Carolina, USA). In the case of progeny pro­ 3000 ppm 2.2 � 1.2 3.3 � 2.2 5.0 � 2.7 5.0 � 2.7 5.6 � 2.7
duction, the data were analyzed by using an Analysis of Variance 5000 ppm 2.2 � 2.2 3.3 � 2.2 3.8 � 2.3 5.0 � 2.7 7.2 � 3.2
(ANOVA), with dose rate as main effect. Means were separated by using F 0.52 0.99 0.86 0.69 0.60
P 0.66 0.40 0.47 0.56 0.61
the Tukey-Kramer Honestly Significant Difference (HSD) test, at a level
of 0.05 (Sokal and Rohlf, 1995). Within each column, means followed by the same letter are not significantly
different (Tukey-Kramer HSD test at 0.05; where no letters exist, no significant
3. Results differences were noted).

3.1. Adult mortality

Table 3
Mean mortality (% � SE) of S. oryzae adults after 1, 3, 7, 14 and 21 days of
Regarding parental mortality, MANOVA indicated that both dose exposure on wheat treated with different concentrations of the D. viscosa
and its interaction with exposure were significant for adults of formulation (in all cases df ¼ 3.35).
O. surinamensis, T. confusum and S. oryzae (Table 1). In contrast, only
Treatment Exposure interval
exposure was found to be significant in the case of R.dominica (Table 1).
For this species, regardless of the dose rate, adult mortality did not differ 1d 3d 7d 14 d 21 d

significantly from the control on any of the exposure intervals, and did Control 0.6 � 1.1 � 0.7 3.3 � 1.4 b 6.6 � 2.9 b 11.6 � 3.3
not exceed 12% (Table 2). Similarly, mortality of S. oryzae adults was 0.6 b b
1000 ppm 1.1 � 2.7 � 1.6 10.0 � 3.9 12.2 � 3.9 13.9 � 3.7
extremely low, and did not exceed 33% for any of the dose-exposure
0.7 ab ab ab ab
combinations tested (Table 3). However, the application of the formu­ 3000 ppm 0.0 � 8.3 � 1.6 a 25.0 � 5.3 a 26.6 � 5.2 32.2 � 5.3
lation resulted, at exposures that were longer than 1 day, in higher 0.0 a a
mortality levels than those in the control vials, for many of the combi­ 5000 ppm 0.6 � 5.6 � 2.4 23.3 � 6.8 a 24.4 � 7.1 26.6 � 6.8
nations tested. Furthermore, at exposures that were longer than 3 days, 0.6 ab ab ab
F 0.71 3.35 4.69 3.63 3.89
there were significant differences among treatments for adult mortality
P 0.55 0.03 <0.01 0.02 0.01
of T. confusum (Table 4). Still, despite any differences, mortality was low
and did not exceed 37%. Finally, O. surinamensis was proved to be the Within each column, means followed by the same letter are not significantly
different (Tukey-Kramer HSD test at 0.05; where no letters exist, no significant
most susceptible species from the ones tested, as mortality reached 87%
differences were noted).
after 21 days of exposure at the highest dose rate (Table 5). In fact, with
the exception of 1 day, for all other exposures there were significant
differences among treatments. At the 7, 14 and 21 days of exposure, Table 4
significantly more adults were dead in vials containing 3000 and 5000 Mean mortality (% � SE) of T. confusum adults after 1, 3, 7, 14 and 21 days of
ppm of the D. viscosa formulation, as compared with the respective exposure on wheat treated with different concentrations of the D. viscosa
figures of control and 1000 ppm. Moreover, at these exposures, 5000 formulation (in all cases df ¼ 3.35).
ppm gave significantly higher mortality levels than those in 3000 ppm. Treatment Exposure interval

1d 3d 7d 14 d 21 d
3.2. Progeny production
Control 0.0 � 0.0 0.6 � 0.6 0.6 � 0.6 b 2.7 � 1.2 c 2.7 � 1.2 c
1000 ppm 0.0 � 0.0 0.6 � 0.6 2.2 � 1.2 ab 3.3 � 1.1 c 6.1 � 1.3 c
Regarding progeny production counts, dose had a significant effect 3000 ppm 0.0 � 0.0 1.1 � 0.7 6.6 � 2.2 a 13.3 � 4.2 b 17.2 � 4.0 b
for all species, with the exception of S. oryzae (Table 6). For R. dominica, 5000 ppm 1.1 � 1.1 1.1 � 1.1 3.9 � 1.8 ab 26.1 � 2.4 a 35.6 � 3.2 a
the increase of dose decreased progeny production, but only 3000 and F 0.99 0.17 2.72 17.75 28.30
P 0.40 0.91 <0.01 <0.01 <0.01
5000 ppm gave fewer offspring than that in the control (Table 7).
Nevertheless, even in these doses, progeny production was high, and Within each column, means followed by the same letter are not significantly
exceeded 21 adults per vial. In contrast, for S. oryzae adults, the appli­ different (Tukey-Kramer HSD test at 0.05; where no letters exist, no significant
cation of the formulation on wheat did not significantly affect progeny differences were noted).
production capacity, despite the fact that fewer adults were recorded at

Table 1
Repeated Measures MANOVA parameters for mortality levels of the four species tested (total df ¼ 35).
df Species

R. dominica S. oryzae T. confusum O. surinamensis

F F F P F P F P

Between variables 3 0.37 0.77 4.33 0.01 17.08 <0.01 69.56 <0.01
Y- intercept 1 11.81 <0.01 56.67 <0.01 85.15 <0.01 264.68 <0.01
Dose 3 0.37 0.77 4.33 0.01 17.08 <0.01 69.56 <0.01
Within variables 12 0.97a 0.47 2.40a 0.01 8.00a <0.01 8.88a <0.01
Exposure 4 5.08 <0.01 23.74 <0.01 43.90 <0.01 61.19 <0.01
Exposure X Dose 12 0.97a 0.47 2.40a 0.01 8.00a <0.01 8.88a <0.01
a
Wilks’ Lamda approximate F value.

3
E. Lampiri et al. Crop Protection 132 (2020) 105095

Table 5 Similarly, mortality and progeny production control was rather low for
Mean mortality (% � SE) of O. surinamensis adults after 1, 3, 7, 14 and 21 days of S. oryzae. As both species are primary colonizers, and their immature
exposure on wheat treated with different concentrations of the D. viscosa development occurs within the grain kernel (Athanassiou and Arthur,
formulation (in all cases df ¼ 3.35). 2018), their larvae may remain unaffected by substances that have been
Treatment Exposure interval applied in the external kernel part. Still, the progeny production sup­
1d 3d 7d 14 d 21 d pression for R. dominica was higher than that of S. oryzae, despite that
the reverse was true for mortality. This could be attributed to the fact
Control 0.0 � 0.0 � 0.0 b 0.6 � 0.6 c 3.3 � 1.4 c 6.6 � 2.2 c
0.0
that, although immature development for R. dominica occurs within the
1000 ppm 0.6 � 3.9 � 1.6 5.6 � 1.5 c 7.7 � 1.6 c 11.6 � 2.9 kernel, oviposition occurs at the external kernel part, and newly-hatched
0.6 ab c larvae have to crawl to enter the kernel (Mayhew and Phillips, 1994;
3000 ppm 1.1 � 11.1 � 3.3 35.0 � 7.8 43.9 � 6.9 46.1 � 7.4 Batta, 2005). In contrast, for S. oryzae, females oviposit directly inside
0.7 a b b b
the kernels where egg hatching takes place (Haines, 1991; Rita Devi
5000 ppm 0.6 � 13.9 � 3.9 58.3 � 6.5 84.4 � 4.1 86.6 � 4.7
0.6 a a a a et al., 2017). For diatomaceous earths (DEs), Arthur and Throne (2003),
F 0.71 5.70 27.10 81.66 60.32 showed that there was no effect to larvae of S. oryzae when DEs were
P 0.55 <0.01 <0.01 <0.01 <0.01 applied on already infested grains. Similarly, probably for the same
Within each column, means followed by the same letter are not significantly reason, R. dominica is considered as one of the least susceptible species to
different (Tukey-Kramer HSD test at 0.05; where no letters exist, no significant DEs (Korunic, 1998). There are several paradigms on which primary
differences were noted). colonizers are more tolerant than secondary colonizers, i.e. species on
which immature development occurs outside of the kernel, to bota­
5000 ppm. For both T. confusum and O. surinamensis, the application of nicals/plant extracts. For instance, Hernandez- Lambrano et al. (2015)
the D. viscosa formulation at 3000 and 5000 ppm significantly sup­ confirmed that O. surinamensis was more susceptible than the maize
pressed progeny production in comparison with the control vials. Hence, weevil, Sitophilus zeamais Motschulsky (Coleoptera: Curculionidae)
there were less than 1 adult per vial at 3000 and 5000 ppm and at 5000 when exposed to essential oils produced by three plants of the genus
ppm for T. confusum and O. surinamensis, respectively (Table 7). Cymbopogon. In addition, the work of Tapondjou et al. (2005) showed
that T. confusum was more susceptible than S. zeamais when exposed to
4. Discussion cymol, the major component of essential oil of Eucalyptus saligna Sm.
(Myrtales: Myrtaceae). However, given that the mode of action of
To our knowledge, this is the first work that has examined the D. viscosa is not well-known, additional experimental work is required to
insecticidal efficacy of the lyophilized epicuticular material of D. viscosa. illustrate the basis of these differences among species that share the
This species has been the subject of many studies concerning the anti­ same habitat in the stored-grain ecosystem.
microbial effects of plant extracts as well as the allelopathic potential of In contrast with the primary colonizers, O. surinamensis and
its exudates, but there is still inadequate information regarding its T. confusum were found to be more susceptible to the material tested, as
insecticidal effects on stored product insects. Chromatographic separa­ progeny production was significantly reduced in the treated grains, in
tion of the epicuticular material of D. viscosa, carried out by Daniewski comparison with the respective figures for the untreated grains,
et al. (1986), isolated illicic acid, 2α-hydroxyisocostic acid and methyl regardless of the level of parental mortality. From the species tested,
ester of illicic acid. In that study, the authors tested the action of these O. surinamensis was found to be by far the most susceptible, as parental
components as food deterrents against adults and larvae of T. confusum mortality was high, even at short exposure intervals. Moreover, for this
and the khapra beetle, Trogoderma granarium Everts (Coleoptera: Der­ species, parental mortality was notably increased with the increase of
mestideae), as well as adults of S. granarius. The results shown that the the exposure interval, and would probably reach 100% at longer
2α-hydroxyisocostic acid and methyl ester of illicic acid were highly
effective as food deterrents for S. granarius, whereas illicic acid and Table 7
methyl ester of illicic acid were highly effective for T. confusum larvae. Mean number of progeny production (adults per vial � SE) for each species on
The least susceptible life stage examined was T. granarium larvae, as they treated and untreated wheat, 65 d after the removal of the parental adults (in all
were less affected by all components of the epicuticular material. The cases df ¼ 3.35).
above research showed that the component with the highest suppressive Treatment Species
effect was 2α-hydroxyisocostic acid, followed by methyl ester of illicic
O. surinamensis T. confusum S. oryzae R. dominica
acid and illicic acid. The above study is the only work that has examined
components of the epicuticular material of D. viscosa for the control of Control 40.0 � 10.6 a 4.6 � 1.4 a 73.9 � 8.7 107.4 � 22.9 a
1000 ppm 37.7 � 9.0 a 1.5 � 0.6 ab 77.0 � 15.2 69.0 � 15.9 ab
stored-product insects; still, that approach was related with behavioral
3000 ppm 4.9 � 2.6 b 0.9 � 0.6 b 84.7 � 20.2 21.1 � 4.2 b
aspects, i.e. antifeedant effects, and not the evaluation of this material as 5000 ppm 0.2 � 0.2 b 0.7 � 0.4 b 47.2 � 8.0 21.3 � 6.4 b
an insecticide/grain protectant. F 0.78 3.96 1.36 8.33
Our results clearly indicate that the efficacy of this material varies P <0.01 0.01 0.27 <0.01
among the species tested, and it is highly related with the dose rate. Within each column, means followed by the same letter are not significantly
Practically, this material has no effect in the case of R. dominica, despite different (Tukey-Kramer HSD test at 0.05; where no letters exist, no significant
the fact that there was some suppression in progeny production. differences were noted).

Table 6
ANOVA parameters for progeny production counts for each species (total df ¼ 32).
df Species

O. surinamensis T. confusum S. oryzae R. dominica

F P F P F P F P

Model 3 8.78 <0.01 3.96 0.01 1.36 0.27 8.33 <0.01

Y-Intercept 1 33.95 <0.01 18.46 <0.01 102.45 <0.01 57.28 <0.01
Dose 3 8.78 <0.01 3.96 <0.01 1.36 0.27 8.33 <0.01

4
E. Lampiri et al.
exposures. At the same time, progeny suppression was high at 3000 and
5000 ppm, which may be considered as a direct consequence of parental
mortality. Given that the immatures of the secondary colonizers develop
and move freely at the external kernel part, it is generally expected that
their contact with the D. viscosa material around the grain kernels would
be substantially increased, as compared with the immatures of the pri­
mary colonizers. Conversely, progeny production suppression of
T. confusum may not be linked with parental morality, as this was much
lower than that of O. surinamensis. It is well established that T. confusum
cannot develop easily in sound kernels (Aitken, 1975), which stands in
accordance with our findings, as progeny production in the untreated
wheat was extremely low. Still, progeny production was significantly
suppressed in the treated grains, which consists an additional indication
of the adverse effect of the lyophilized epicuticular material of D. viscosa
on immature development and longevity.
The dose rates that have been used here can be considered as high, at
least in comparison with traditional grain protectants that are usually
applied at concentrations that do not exceed 10 ppm (Arthur, 1996;
Weaver and Subramanyam, 2000; Athanassiou et al., 2014). Moreover,
the lyophilized epicuticular material of D. viscosa, at least at the con­
ditions tested here, can be considered as slow-acting towards its insec­
ticidal efficacy, as compared with the majority of the conventional grain
protectants. Nevertheless, there are some registered insecticides that are
applied directly on grains at dose rates that are comparable with the
ones tested here, such as DEs and zeolites (Rojht et al., 2010; Rumbos
et al., 2016; Athanassiou and Arthur, 2018). Other natural substances or
plant-derived insecticides, such as azadiractin, are also effective at
elevated concentrations that usually exceed 1000 ppm (Athanassiou
et al., 2005). In fact, other botanical extracts have been proved effective
as contact insecticides against stored product insects at even higher
doses. Indicatively, Tofel et al. (2017), who examined various plant
tissues of A. indica and Plectranthus glandulosus Hook. (Lamiales: Lam­
iaceae), found that satisfactory control of the cowpea weevil, Calloso­
bruchus maculatus (F.) (Coleptera: Bruchidae) and S. zeamais was
possible at doses that exceeded 10,000 ppm. At the same time, there are
some insecticides that have been proved particularly slow-acting against
stored product insect species due to their mode of action. For instance,
the insect growth regulator (IGR) S-methoprene affects only progeny
production, despite the fact that in some cases there is some parental
mortality (Daglish et al., 2013). Similarly, the pyrrole chlorfenapyr
causes a delayed mortality and no knockdown after exposure, since its
mode of action is sufficiently different than that of neurotoxic in­
secticides (Dagg et al., 2019). Worth noting here is that the epicuticular
material of D. viscosa, as many other botanicals, may be easily manip­
ulated concerning the initial concentration of the bioactive substances.
The present study used a three-fold concentrated material in respect to
the naturally occurring one on leaves but this would be augmented by
using higher amount of plant biomass in the same water volume,
resulting in more condensed and possibly more effective exudate.
There is extensive literature concerning flavonoid-phytophagous
insect interactions broadening our understanding on the influential
role of flavonoids in host selection, survival, growth, feeding behavior
and reproduction of insects (Simmonds, 2003). Certain leaf flavonoids
directly interact with hormone system, or function as feeding deterrents,
which is also the case of sesquiterpenes (Gonzales-Coloma et al., 2010).
Flavonoids on the leaf surface act as the first barrier that the herbivore
encounters after landing on the leaf and have been reported to reduce
the fitness of phytophagous insects, such as Euphydryas chalcedona
(Doubleday) (Lepidoptera: Nymphalidae) (Simmonds, 2003). In this
context, the flavonoids and sesquiterpenes present in D. viscosa epicu­
ticular material may act as antifeedant agents leading to starvation or
exhibit moderate but direct toxic effects on the stored product insects of
the present study, thus further experiments needed in order to clarify
their mode of action.
In summary, our results indicate that the lyophilized epicuticular
material of D. viscosa has some insecticidal value for the control of the
5
Crop Protection 132 (2020) 105095
species tested here, but this effect varies according to the target species.
In contrast with the majority of the studies that have evaluated plant
materials chiefly as formulated essential oils, the results of the present
work show that the D. viscosa material used provided some insecticidal
effect when admixed with the grain, suggesting that its mode of action
can be comparable with that of other contact insecticides. Further
research is needed to shed light to these parameters, including the mode
of action of D. viscosa epicuticular material, and to evaluate the basis of
its use in realistic scenarios in stored-product protection.
Declaration of competing interest
We would like to confirm that the authors have no conflict of
interest.
CRediT authorship contribution statement
E. Lampiri: Data curation, Methodology, Formal analysis, Investi­
gation, Writing - original draft, Writing - review & editing. P. Agrafioti:
Methodology, Data curation, Supervision, Writing - original draft. E.
Levizou: Conceptualization, Supervision, Writing - original draft. C.G.
Athanassiou: Conceptualization, Project administration, Supervision,
Validation, Writing - original draft, Writing - review & editing.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.cropro.2020.105095.
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