Introduction

Chemicals and fumigants that are currently being used in stored grains have uncertain future because they cause environmental issues, are not safe and many stored grain insects have developed resistance against many of these grain protectants specially to organophosphates (Daglish 2008). So, for the successful effectiveness of these chemicals higher doses are required. Nowadays, people are demanding products that are free from pesticides residues which have led to evaluating alternative controls like use of pyrethroids (Athanassiou et al. 2004). The use of inert dusts, microbial insecticides, bio-control agents and botanical based insecticides have been evaluated in this regard.

Spinetoram is based on spinosyn L and J derived synthetically from the metabolism of soil actinomycete Saccharopolyspora spinosa Mertz and Yao. It has been studied against many stored product species and has been found effective for their control (Vassilakos and Athanassiou 2012). It is a nerve poison that attacks on receptors of nicotine acetylcholine and it acts via contact and ingestion (Dripps et al. 2011) and is more active than spinosad (Sparks et al. 2008). Abamectin, belonging to the avermectin family is another bacterial insecticide derived from a soil bacterium, Streptomyces avermitilis (Pozo et al. 2003). This insecticide has a vast range which leads to its effective use for managing various insect pests (Kavallieratos et al. 2009).

Use of inert dusts is another alternative control tactic in stored grain insect management. Diatomaceous earths (DEs) are inert dusts that are natural in origin and are made up from the fossil remains of diatoms (Subramanyam and Roesli 2000). Zeolites, among other naturally occurring silica dusts are known and classified in the same group as the diatomaceous earths because of their silica content (Eroglu et al. 2017). Both diatomaceous earths and zeolites have a physical mode of action, i.e., act via cuticle of insect causing desiccation and death (Andric et al. 2012). The difference between zeolites and DEs are of the structure. DEs are fossilised remaining of diatoms but zeolites are crystalised alumina silicates having amorphous silica (Golob 1997; Andric et al. 2012).

Certain abiotic factors of the environment are of great significance for the development of insects in the storage ecosystem such as temperature, R.H. and composition of gases (Muir 2000). In case of insecticides various studies with contradictory results have been done depicting the role of temperature on action of various insecticides (Nayak and Collins 2008; Golic et al. 2016). In case of inert dusts, their efficacy has been found to be affected by insect species (Fields & Korunic 2000), physical properties (Vayias et al. 2009b), type of dust, temperature, R.H. and exposure time (Athanassiou et al. 2005; Vardeman et al. 2006).

These inert dusts adhere to the grains that may cause negative impact on the physical characteristics of the grains reducing their value commercially (Korunic et al. 1996). A possible solution of this problem is to apply these dusts (at low doses) in combination with other control methods. This combination could be useful in applying inert dusts widely as a grain protectant (Arthur 2003). Combined use of inert dust, especially of DEs, with other control methods have been tested previously like the combination of DEs with entomopathogenic fungi (Athanassiou and Steenberg 2007), with plant extracts (Athanassiou et al. 2008a), with pyrethroids (Vayias et al. 2006), or with insecticidal dusts like spinosad (Chintzoglou et al. 2008a).

In the present study combined efficacy of inert dusts with bacterial based insecticides was evaluated and the effect of other factors like temperature and grain commodities was also studied against Trogoderma granarium.

Materials and methods

Test products

Inert dusts used were diatomaceous earth “SilicoSec” (Biofa GmbH, Münsingen, Germany) which is a fresh water originated diatomaceous earth and zeolite “ZeoFeed” (Zeocem, Bystre, Slovakia) which is registered in its country of origin as a feed additive.

Insecticides used in the bioassay were spinetoram (Radiant) and abamectin (Vertimec) that were obtained from Dow Agro Chemical Company and Syngenta Pakistan Ltd., respectively.

Test insects and grain commodities

Experiment was conducted at Grain Research Training and Storage Management Cell, department of Entomology, University of Agriculture, Faisalabad, Pakistan during the year 2018–19. The insect used in studies was T. granarium. Insects were obtained from different storages and grain market of Faisalabad and were reared in laboratory at 30 ± 2 °C and 60 ± 10% R.H. For bioassays, T. granarium larvae of third instar were used.

Tested grain commodities were wheat, rice and maize which were clean and free of infestation. Moisture content of commodities was measured by moisture meter (Dickey-John Multigrain CAC II; Dickey-John Co., U.S.A) which was 11.25% (wheat), 10.50% (rice) and 11.65% (maize).

Bioassays

Five lots of 1-kg of each grain type were prepared in plastic containers. From each grain type, four lots were taken and two lots of each type of grains were separately treated with diatomaceous earth (DE), other two lots with zeolite at the rate 750 mg/kg by admixing. Then these were shaken for 5 min by hand to equally distribute inert dusts particles with grains. Two DE-treated lots of each commodity were treated with spinetoram and abamectin at the rate of 1 ppm separately. Two zeolites treated lots of each grain commodity were also treated separately with abamectin and spinetoram at 1 ppm. Grains were spread in the trays for the uniform application of the insecticides. Using a hand sprayer, 5 ml of each of insecticide or water (in control) was sprayed on each grain type and then grains were placed in the incubator at 25 °C and 65% R.H. for two days so that moisture could equilibrate (Kavallieratos et al. 2009). These doses were selected from the previous experiments on the basis of mortality of insects. One untreated lot of each commodity served as control. From each grain lot 50-g samples were taken out to serve as experimental units and placed in small jars (11 cm × 6.5 cm). Jars were covered with muslin cloth for aeration. Fifty larvae of T. granarium were placed in each jar. Experiment was conducted at three different levels of temperature (15, 25 and 35 °C) and two levels of R.H. (55% and 75%) so there were six different combinations of temperature and relative humidity. Jars were placed in the incubator that was set at each tested level of temperature and relative humidity. Saturated salt solutions recommended by Greenspan (1977) were used for the maintenance of desired relative humidity levels during the whole experimental period. Whole experiment was repeated three times. Data of mortality was taken at 1, 3, 7 and 14 days of post treatments.

Data analysis

Control mortalities were adjusted by using Abbott’s formula (Abbot 1925). Factorial analysis under Completely Randomized Design was used. Means for mortality were compared by using Tukey–Kramer (HSD) test at α = 0.05 (Sokal and Rohlf 1995). The Statistix 8.1 program was used for statistical analyses.

Results

Mortality

In combined treatment of DE + abamectin, for all exposure intervals all main effects were significant but their associated interactions were non-significant with the exception of Temperature × R.H. after 1 day, Commodity × R.H. and Temperature × R.H. after 7 days and Commodity × Temperature and Temperature × R.H. after 14 days (Table 1). As temperature and exposure interval increased mortality increased but it was opposite in case of R.H. (Table 2). Generally, mortality was more in wheat than in rice or maize (Table 2). After the 14 days of exposure period at 35 °C + 55% R.H. 100% of mortality was found in all three tested grain commodities (Table 2).

Table 1 ANOVA for the mortality of T. granarium at different exposure intervals after the combined treatment of DE + abamectin at different temperature and relative humidity (R.H.) levels on three grain commodities
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Table 2 Percentage mean mortality (± SE) of T. granarium at different exposure intervals after the combined treatment of DE + abamectin at different temperature and relative humidity (R.H.) levels on three grain commodities. Within each exposure interval, means following same letters are non-significant; HSD test at P < 0.05
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In case of DE + spinetoram, at each exposure interval all main effects were significant but their interactions were non-significant except Commodity × Temperature, Commodity × R.H. and Temperature × R.H. after 7 days and Commodity × Temperature and Temperature × R.H. after 14 days of exposure (Table 3). In most of the cases, mortality was higher as compare to combined treatment of DE + abamectin. More insects were died on wheat than on rice or maize in most of the cases (Table 4). Increased R.H. decreased the mortality while increased temperature and exposure period increased the mortality (Table 4). On all grain commodities 100% mortality was achieved at 35 °C + 55% R.H. at 14 days of post treatment (Table 4).

Table 3 ANOVA for the mortality of T. granarium at different exposure intervals after the combined treatment of DE + spinetoram at different temperature and relative humidity (R.H.) levels on three grain commodities
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Table 4 Percentage mean mortality (± SE) of T. granarium at different exposure intervals after the combined treatment of DE + spinetoram at different temperature and relative humidity (R.H.) levels on three grain commodities. Within each exposure interval, means following same letters are non-significant; HSD test at P < 0.05
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In combined application on zeolite + abamectin, all main effects were found significant at all exposure periods while their associated interactions were significant only for Temperature × R.H. after 1 day, Commodity × Temperature after 3 days and Commodity × Temperature and Temperature × R.H. after 14 days of exposure (Table 5). Effect of temperature was positive on the mortality of the insects but in case of R.H. mortality decreased with increase in R.H. (Table 6). As exposure period increased mortality was also increased (Table 6). Generally, wheat found to be more susceptible as compared to the other grain types (Table 6). Complete mortality was obtained after 14 days of treatment at 35 °C + 55% R.H. in all tested grain types (Table 6).

Table 5 ANOVA for the mortality of T. granarium at different exposure intervals after the combined treatment of zeolite + abamectin at different temperature and relative humidity (R.H.) levels on three grain commodities
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Table 6 Percentage mean mortality (± SE) of T. granarium at different exposure intervals after the combined treatment of zeolite + abamectin at different temperature and relative humidity (R.H.) levels on three grain commodities. Within each exposure interval, means following same letters are non-significant; HSD test at P < 0.05
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For combination of zeolite + spinetoram, at all exposure durations main effects were found to be significant while their associated interactions were non-significant with the exception of Commodity × R.H. after 3 days, Commodity × Temperature and Temperature × R.H. after 7 days and Commodity × Temperature and Temperature × R.H. after 14 days of exposure (Table 7). Overall, in this combination mortality was more as compared to the combination of zeolite + abamectin. High temperature and low R.H. caused more mortality (Table 8). Mortality was tended to increase at increased exposure (Table 8). Among grain types, wheat was generally most susceptible as compared to rice or maize (Table 8). 100% mortality of insects in all grain commodities occurred at 35 °C + 55% R.H. after 14 days of exposure (Table 8).

Table 7 ANOVA for the mortality of T. granarium at different exposure intervals after the combined treatment of zeolite + spinetoram at different temperature and relative humidity (R.H.) levels on three grain commodities
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Table 8 Percentage mean mortality (± SE) of T. granarium at different exposure intervals after the combined treatment of zeolite + spinetoram at different temperature and relative humidity (R.H.) levels on three grain commodities. Within each exposure interval, means following same letters are non-significant; HSD test at P < 0.05
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Discussion

Results of current study suggest that inert dusts can be used in combination with microbial insecticides against T. granarium but many factors influence their efficacy like temperature, R.H. and exposure periods. In present study increased exposure caused more mortality of the insects. Nwaubani et al. (2014) also reported increased mortality by increasing interval of exposure. Longer exposure period is required for diatomaceous earth in order to kill the insects (Athanassiou et al. 2006, 2007, 2008a, b) because at longer exposure more dust is attached to the body of insect leading to more water loss (Arthur 2000a, b, 2001). Previous studies are also in the support of this fact that effectiveness of the inert dusts is increased by increasing the exposure duration (Kljajić et al. 2010a, b; Perisic et al. 2018). Regarding the insecticides, Kavallieratos et al. (2009) noted more mortality of T. confusum with increasing exposure interval from 7 to 14 days by using abamectin.

It was obvious that temperature was positively co related with the mortality of insects while the effect of R.H. was negative. These results are in the light of pervious reports depicting that mortality of stored grain insects was more at higher temperature and lower relative humidity using various inert dusts (Vayias and Athanassiou 2004; Kjajic et al. 2010a, b, Vassilakos and Athanassiou 2013; Athanassiou et al. 2014; Bohinc et al. 2018 and Eroglu et al. (2019). At increased temperature food uptake and mobility of the insects is increased due to which more particles of the inert dusts are attached to their bodies. High level of temperature also caused increased water loss and more respiration in the insects (Arthur 2000b; Subramanyam and Roesli 2000). Vayias et al. (2009b) noted high mortality of T. confusum with increased temperature by the combined treatment of DE + spinosad which is similar to this study. In this study 55% R.H. caused more insect mortality. This may be due to the fact that at lower levels of R.H. more water is lost from the insect body as reported in previous studies by using the diatomaceous earth (Vayias and Athanassiou 2004). Similar effect of these factors in case of various insecticides has been previously reported by Athanassiou et al. (2007), Kavallieratos, et al. (2009, 2010) and Vassilakos et al. (2012).

More insects died on wheat than on rice and maize in all combinations but difference between the mortalities on commodities was not so strong. Kavallieratos et al. (2017) and Perisic et al. (2018) have also reported that effectiveness of DEs was more on wheat as compared to other grain commodities against various stored grain insects. Perisic et al. (2018) reported less adherence of zeolite particles to maize grains was less than rice or barley. In case of insecticides, effect of commodity has been studied by many authors. Similarly, Vayias et al. (2009a) found less mortality of S. oryzae on maize. Chintzoglou et al. (2008b) noted less action of dust of spinosad on maize than on wheat. Vassilakos et al. (2015) also showed similar results depicting that efficacy of spinetoram was lower in the maize while it was highest in the hard wheat regarding the mortality of stored grain insects but effect of commodity on the efficacy of insecticide was not so strong similar to the present study. Vayias et al. (2009b) reported that combined treatment of DE + spinosad was more effective against T. confusum on wheat. Similarly, Wakil et al. (2013) examined that more R. dominica died on wheat than on rice and maize with combined treatment of diatomaceous earth + thiamethoxam. Difference in the efficacy of dusts or insecticides to various grains may be due to physiochemical characters of grains.

Combination of inert dusts (DEs) with other control methods as an integrated control has been suggested by Arthur (2003). Combination of DE with betacyfluthrin (Athanassiou 2006), with spinosad (Vayias et al. 2009b), with plant extract (Athanassiou et al. 2008a), with thiamethoxam (Wakil et al. 2013) has been studied by many researchers indicating that these dusts can be combined with other control methods. In summary, results of present study suggested that it is possible to obtain complete mortality of T. granarium by combined treatment of inert dusts and bacterial based insecticides but their efficacy is dependent on various factors like temperature, R.H., doses and grain commodities. These factors should be kept in mind while planning IPM strategy.