Comparison of insecticidal efficacy of four natural substances against granary weevil ( Sitophilus granarius [ L . ] ) adults : does the combined use of the substances improve their efficacy ?

Laboratory tests were carried out to evaluate the insecticidal efficacy of different natural inert dusts (diatomaceous earth, wood ash, quartz sand) and the leaf powder of Azadirachta indica A. Juss. against granary weevil (Sitophilus granarius [L.]) adults. The efficacy of the substances was tested individually and in combination with each other. The substances were applied at different concentrations, and bioassays were carried out at four different temperatures (20, 25, 30 and 35°C) and two different relative humidity (RH) levels (55% and 75%). The adult mortality was recorded after the 7th, 14th and 21st days of exposure. The progeny production of individuals exposed to different combinations was also assessed. Wood ash proved to be the most efficient inert dust in our research. We detected 100% mortality in the treatment exposed to a higher concentration (5 w%) of wood ash at 35°C and 55% RH after 7 days of exposure. A lower RH level had also a negative impact on the progeny production. We can conclude that wood ash can be efficient in controlling granary weevil adults as a single substance or in combination with other substances. Further surveys should focus on the impact of the wood ash dose rates. Due to the high percentage of area covered with forest in some European countries, the main ingredient is present locally, but additional surveys are needed to help improve the practical use of wood ash. Additional keywords: wood ash; inert dusts; botanical insecticide. Authors’ contributions: Designed the research and wrote the manuscript: TB and ST. Performed the counting of specimens, and statistically analysed the data: TB. Citation: Bohinc, T.; Trdan, S. (2017). Comparison of insecticidal efficacy of four natural substances against granary weevil (Sitophilus granarius [L.]) adults: does the combined use of the substances improve their efficacy? Spanish Journal of Agricultural Research, Volume 15, Issue 3, e1009. https://doi.org/10.5424/sjar/2017153-11172. Received: 07 Feb 2017. Accepted: 05 Jul 2017 Copyright © 2017 INIA. This is an open access article distributed under the terms of the Creative Commons Attribution (CC-by) Spain 3.0 License. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Correspondence should be addressed to Tanja Bohinc: tanja.bohinc@bf.uni-lj.si


Introduction
The granary weevil (Sitophilus granarius [L.]) is an important primary stored product insect pest of cereals, and its economic impact has been controlled with various methods in different regions of the world.The application of synthetic insecticides has proven to cause negative impacts on the environment, non-target organisms and people's health, which has stimulated the search for alternative ways of controlling pests (Hamza et al., 2016).For this reason, the control of stored product pests using inert dusts (diatomaceous earth, quartz sand, wood ash, zeolites) (Mohapatra et al., 2015) and plant preparations, such as plant extracts, essential oils and powdered parts of plants (Bohinc et al., 2013;Bouayad et al., 2013;Mohapatra et al., 2015) has been investigated.Diatomaceous earth stands out among the inert dusts by its extent of application (Korunic, 1998;Kavallieratos et al., 2015), while research on the insecticidal properties of wood ash has been most frequently undertaken in African countries and India (Demissie et al., 2008;Jean et al., 2015).Research on insecticidal dusts made of powdered plant leaves is common both in Europe (Bohinc et al., 2013) and elsewhere (Mkenda et al., 2015).
The purpose of our research was to investigate the insecticidal effects of three inert dusts and one plant powder applied for the suppression of granary weevils independently and in combinations.

Natural substances, commodity and insects
Four different natural substances were used in the tests: diatomaceous earth, wood ash, quartz sand, and neem leaf powder.As the diatomaceous earth, we used the commercial product SilicoSec® (manufacturer: Biofa, Germany); supplier: Metrob d.o.o., Slovenia), and wood ash (from European beech [Fagus sylvatica L.]) was obtained from a local household (location: Logatec, Slovenia).We used local quartz sand (location: Moravče, Slovenia), and neem leaf powder (product Neem listni prah®; supplier Azimut-Vester, Ltd., Slovenia).The tested wheat grain was infestation-and pesticide-free.
The granary weevils used in the tests were reared at the Laboratory of Entomology, Department of Agronomy, Biotechnical Faculty, University of Ljubljana.The age of the unsexed exposed granary weevils was unidentified.

Bioassay
Exposure studies were carried out in incubators set at 20°C, 25°C, 30°C and 35°C, 55% and 75% relative humidity (RH), and continuous darkness, and the mortality was assessed on the 7 th , 14 th and 21 st days after exposure.We tested the insecticidal properties of the four substances mentioned above, applied individually or in combination with each other.For each application treatment, 500 g lots were prepared.All treatments are presented in Table 1.The lots of wheat grain were placed into a plastic container of 2 L, and a specific powder or powder combination was added to each plastic container (Trdan & Bohinc, 2013).The preparation of each individual treatment (in a 100-mL vial) was done according to Bohinc et al., 2013).Untreated wheat served as control treatment.All bioassays were repeated three times, and after day 21, the progeny tests were performed.For each specific treatment exposed to specific parameters (temperature & RH), all 100 mL vials were stirred into 1000 mL vials.Then, after 56 days, the vials were opened, and then the numbers of progeny were counted.The number of progeny was expressed as an average number per treatment.

Chemical analysis of substances used in our research and their particle size
According to Rojht et al. (2010a), the diatomaceous earth that was used in our experiment, contained 87.47% SiO 2 .The SilicoSec® particle size was between 8 and 12 µm (Vayias & Athanassiou, 2004).Quartz sand contained 99.24% SiO 2 , and the other components present in quartz sand included 0.13% Al 2 O 2 , 0.06% Fe 2 O 3 , 0.02% CaO, and traces of MnO, P 2 O 5 , TiO 2 , Na 2 O and MgO that were less than 0.01% (Rojht et al., 2010b).The particle size of all quartz sands used in this study was 12 µm.According to Kranjc et al. (2009), wood ash contained 25-26 w% (as weight percent) SiO 2 .We sieved the wood ash and Neem listni prah® through a 1 mm mesh.The main active ingredient in neem is azadirachtin, which is a limonoid; it is available in up to 96% purity (Gahukar, 2014).Other limonoids in neem are melantriol, salannin, and nimbin (NRC, 1992).

Data analysis
The mortality counts were corrected according to Abbott's formula (Abbott, 1925).The data were analysed using repeated measure analysis (Statgraphics Centurion XVI, 2009) with the exposure interval set as the repeated factor, the insect mortality set as the response variable and the treatment, dose, temperature and RH set as the main effects.The associated interactions of the main effects were also included in the analysis.The means were separated using the

General mortality of S. granarius
All the main effects and associated interactions for the corrected mortality of S. granarius adults were significant at p<0.005 (Table 2).
The mortality of adults was significantly different within the exposure intervals, (F 2,2642 ) = 88.34,p<0.0001.All the main effects and associated interactions for the corrected mortality of weevil adults between exposure intervals (df = 880) are presented in Table 3.

Mortality of S. granarius after 7 days of exposure
After 7 days, more than 48% (48.52±1.49) of the weevil adults exposed to 2.5 w% wood ash at 20°C and 55% RH were dead (Table 4).The mortality rates of the granary weevils exposed to SilicoSec®, quartz sand and Neem listni prah® were significantly lower.The mortality of the individuals exposed to 75% and 20°C was significantly the highest in the treatments where 2.5 w% wood ash was used, and the mortality in vials exposed to 2.5 w% wood ash at 25°C and 55% RH reached > 90%.At 25°C and 55% RH, the mortality was significantly the highest for T11.At 25°C and 75% RH, the highest mortality was 24%, in T12.

Progeny production of S. granarius
We recorded no progeny production on wheat exposed to 35°C, and there was no impact of the RH value.Regarding temperatures 20°C, 25°C and 30°C number of individuals was the highest in control treatments (non-treated grain).If we compare number of weevils born in vials treated with SilicoSec® to number of vials treated with wood ash (applied alone), we can confirm that progeny production was influenced by wood ash (applied alone) All values are presented in Table 7.

Dicussion
The insecticidal effects of different inert dusts have been for the last decades a subject of numerous studies (Fields et al., 2001).These studies have focused primarily on the research of diatomaceous earth (Korunic, 1998;Vayias et al., 2009;Kavallieratos et al., 2015).A growing body of research now focuses on the study of the combined effects of inert dusts (diatomaceous earth, kaolin, etc.) and essential oils (Campolo et al., 2014), as well as the effects of environmentally more acceptable insecticides in combination with diatomaceous earth and entomopathogenic fungi (Athanassiou et al., 2016) on the mortality rates of stored product insect pests.In view of the presented information, our research tested the combined use of one or more inert dusts, since we have not come across any studies applying this approach.
After 7 days of a combined application of diatomaceous earth and wood ash at 30°C and 55% RH, we achieved 99.99% mortality of the granary weevil adults.The insecticidal effects of wood ash used independently were very pronounced.Our findings are congruent with those reported by Tadesse & Basedow (2005) Table 6.Mean corrected mortality (%) of S. granarius adults exposed for 21 days to different natural substances (lowercase letters present differences between treatments when exposed to one combination of temperature and relative humidity level).
Table 5. Mean corrected mortality (%) of S. granarius adults exposed for 14 days to different natural substances (lowercase letters present differences between treatments when exposed to one combination of temperature and relative humidity level).
the concentration of azadirachtin is also higher in trees growing in alkaline soil.
The mortality of the beetles was also, in our research, conditioned by the length of exposure to inert dusts, which was reported in different studies (Athanassiou et al., 2006 for other species from the genus Sitophilus, e.g., for the species S. oryzae.The great importance of the length of time that harmful pests are exposed to wood ash was also recognised in the study by Demissie et al. (2008), in which a longer exposure significantly increased the mortality of beetles of the species S. zeamais.We detected no influence of the amount of ash on the beetle mortality in relation to the length of exposure.The higher mortality of the beetles after treatments with wood ash was, in our research, influenced primarily by the higher temperature and longer exposure.
Quartz sand did not provide satisfactory insecticidal effects for the suppression of some stored product pests, as was already proven by Rojht et al. (2010b), who investigated on rice weevils.On the basis of the results of our study, we can confirm their findings.
The sizes of the individual particles in the dusts used in our research differ considerably.The concentration of SiO 2 was significantly the highest in quartz sand (Rojht et al., 2010a,b), yet its insecticidal effects also depend on its shape and size (Vayias et al., 2009).Diatomaceous earths contain SiO 2 in amorphous form, while the SiO 2 in quartz sand is also available in crystal form (Subramanyam & Roesli, 2000).The inert dusts used in our research all work in the same way.It is known that diatomaceous earth (Korunić, 1998;Rojht et al., 2012) and quartz sand (Rojht et al., 2010b) can affect harmful pests on the basis of their abrasiveness, dehydration or absorption of waterproof epicuticular was in vitro very effective in suppressing stored product pests.These authors proved the satisfactory insecticidal effects of wood ash on the species Sitophilus zeamais Motsch.
After 14 days at 55% RH at temperatures 25-35°C, we achieved 100% mortality of individuals in all treatments with wood ash.This proves that the temperature and relative air humidity are important factors in the beetle mortality rates, which has also been established by previous studies (Athanassiou et al., 2006;Bohinc et al., 2013;Throne & Waver, 2013).Like the above authors, we too proved that a combination of higher temperature and lower relative air humidity contributes to the higher mortality of beetles.The diatomaceous earth that we used in our research is one of the commercially available types of this product, which produces satisfactory insecticidal effects at concentrations below 1000 ppm (Kavallieratos et al., 2015), which was also the case in our research.
Using appropriate natural substances (dusts), we achieved 100% mortality after 21 days at lower relative air humidity (Athanassiou et al., 2006).In our research this holds true for wood ash and diatomaceous earth or preparations related to these substances.
Tadesse & Basedow (2005) reported a very high mortality of S. zeamais beetles that were exposed to leaf powder of the plant Azadirachta indica.Our research did not confirm the insecticidal effects of the leaf powder made of A. indica on the beetle species S. granarius.Several factors can influence the effectiveness of this leaf powder, among them the geographic origin of the tree and the tree part from which the preparation is made (Gahukar, 2014).The concentration of azadirachtin is the highest in the seeds of the trees (Gahukar, 2014), and waxes.The absorption of waterproof epicuticular waxes is also attributed to wood ash (Hakbijl, 2002), while dusts made of powdered parts of plants are primarily associated with repellent effects (Bohinc et al., 2013).Though Golob (1997) reports that the amount of wood ash applied is important in suppressing stored product pests, our research determined that satisfactory results can also be achieved by applying a lower amount of ash.
In our research, different substances influenced the progeny production differently, which was already confirmed by Kavallieratos et al. (2015).Wood ash has all the characteristics of an appropriate natural insecticide because it also reduced the progeny of granary weevils.Diatomaceous earth also diminishes the progeny production of stored product pests (Kavallieratos et al., 2015).Wood ash has been most frequently used for the suppression of stored product pests in Africa and Indonesia (Hakbijl, 2002;Tadesse & Basedow, 2005;Demissie et al., 2008), yet its significance in the protection of plants against harmful pests is today relatively small, despite the fact that it is one of the oldest known insecticides (Hakbijl, 2002).
Ash, a traditional insecticide, caused higher beetle mortality in all treatments in our research, which is encouraging for further research in which we would like to stimulate its use in the protection of stored cereals against harmful insects.By adding wood ash to quartz sand and the leaf powder of the plant A. indica, we only improved the effects of the latter.The usefulness of wood ash as fertiliser has been studied much more extensively (Brais et al., 2015) than its insecticidal effects.The results of some studies (Boiteau et al., 2012;Maltas & Sinaj, 2014) indicate that wood ash can also be used to adequately suppress the Colorado potato beetle.
Our research thus puts wood ash side by side with diatomaceous earths, which is today a standard alternative way of suppressing stored product pests.Its insecticidal properties are also satisfactory in combination with other dusts.Some recent studies have reported the negative effects of diatomaceous earth application on stored cereals (Korunić, 2016), which puts the independent use of wood ash into a new perspective.Due to the lack of research on the methods of application (concentration of dust, etc.) and the importance of the ash's chemical composition, researchers in this field will still have to do a lot of work.Wood ash, which is in Europe due to the large forest areas in some countries (Scandinavia, Slovenia etc.) a readily accessible raw material, thus undoubtedly represents a substance that will have to be researched more thoroughly in relation to plant protection, as the results of our and numerous other studies have shown that its significance in the suppression of stored product pests has been underrated.

Table 2 .
MANOVA parameters for main effects and associated interactions for mortality levels of S. granarius adults (df =2406).

Table 3 .
MANOVA parameters for main effects and associated interactions for mortality levels of S. granarius adults between exposure intervals (for all intervals, error df =880).

Table 4 .
Mean corrected mortality (%) of S. granarius adults exposed for 7 days to different natural substances (lowercase letters present differences between treatments when exposed to one combination of temperature and relative humidity level).

Table 7 .
Total number of progeny generation of S. granarius adults after 56 days.