Two are better than one: the combinations of Beauveria bassiana, diatomaceous earth, and indoxacarb as effective wheat protectants

The current study evaluates the efficacy of the entomopathogenic fungus Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Hypocreales: Cordycipitaceae), diatomaceous earth (DE) (Protect-It), and the oxadiazine indoxacarb, at single or combined applications on wheat kernels, for the management of the rusty grain beetle, Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae), the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), the khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae), and the lesser grain borer, Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae). The study was conducted between November 2020 and August 2021 in Faisalabad under a complete randomized block design. The combination of DE + indoxacarb was the most efficient as it caused higher overall mortalities ranging between 59.34 and 100%, and lower overall progeny production ranging between 8.35 and 33.70 individuals per vial, than all other treatments. Beauveria bassiana alone exhibited the lowest mortality rates ranging between 22.33 and 47.76%, and the highest offspring emergence, ranging between 51.33 and 78.55 individuals per vial. Similar pattern was observed when persistence bioassays were conducted. For a period of 120 days, the DE + indoxacarb was the most powerful combination against all tested species, providing overall mortality rates between 17.06 and 63.80%. The overall progeny production was lower for the insect individuals exposed on wheat treated with the DE + indoxacarb combination, ranging between 13.66 and 52.23 individuals per vial, and higher for those exposed to B. bassiana alone, ranging between 44.03 and 107.67 individuals per vial, for the entire duration of storage. However, the efficacy of all treatments decreased gradually during the course of storage. The findings of the current study indicate that the combinations of entomopathogenic fungi, DE, and indoxacarb can be used for the prolonged protection of stored wheat from the tested noxious insect species of stored products. Further research, which will include other inert dusts in combination with entomopathogenic fungi and indoxacarb, may provide additional knowledge towards an effective management of noxious species occurring in storages.

Introduction the infested stored products, ranging between 20 and 40% of the annual global crop yield (Hill 2003;Arthur et al. 2012;FAO 2022). Trogoderma granarium and R. dominica are primary pests that initiate the infestation, while C. ferrugineus and T. castaneum are secondary pests that continue the losses of the already damaged kernels (Hill 2003;Rees 2004;Robinson 2005;Hagstrum and Subramanyam 2009;Kumar 2017;Athanassiou et al. 2019;Galecki et al. 2019). All aforementioned species are serious pests in storages of Pakistan (Wakil and Schmitt 2015;Wakil et al. 2021a).
Through an extended search of the published literature, even though different B. bassiana isolates and DE formulations have been used alone, in combination, or in combination with chemical insecticides, their insecticidal activity with indoxacarb has yet to be evaluated since there are no available data. Thus, the objectives of this study are to assess for the first time the efficacy and persistence of binary combinations of indoxacarb with B. bassiana and DE against adults of C. ferrugineus, T. castaneum, T. granarium, and R. dominica on infested wheat, in terms of mortality and progeny production.

Insects
The unsexed C. ferrugineus, T. castaneum, T. granarium, and R. dominica adult individuals were acquired from local storage facilities of Faisalabad and reared since 2009 at the Department of Entomology, University of Agriculture Faisalabad Pakistan. The rearing medium for the development of C. ferrugineus was wheat flour, for T. castaneum was wheat flour with extra 5% brewer's yeast, and for R. dominica and T. granarium was wheat, at 30 °C, 65% relative humidity (RH), and total absence of light (Ganesan et al. 2021;Wakil et al. 2021b). The tested individuals of R. dominica, T. castaneum, and C. ferrugineus were < 2 weeks old, whereas the tested individuals of T. granarium were < 24 h old (Wakil and Schmitt 2015;Wakil et al. 2021b).

Commodity
Soft wheat, Triticum aestivum L. (var. Lasani-2008) without pests, debris, and pesticides was used in the bioassays. The kernel moisture content was calculated at 12.8% with the utilization of a moisture meter (Dickey-John Multigrain CAC II; Dickey-John Co., USA).

Entomopathogenic fungus culture
The inoculation of B. bassiana was conducted in Petri dishes (100 mm) covered with Sabouraud Dextrose Agar (SDA), wrapped with a piece of parafilm. Dishes were incubated at 25 °C, 70% RH, and 14 light:10 dark h photoperiod in a MIR-254 incubator (Panasonic, Japan), for a period of 10 days (Usman et al. 2020). Fungal conidia were collected with the utilization of a sterile scalpel from the SDA surface and then conveyed in a 50 ml conical tube containing 30 ml of sterile 0.05% Tween 80 (Merck, Kenilworth, NJ, USA) solution. Thereinafter, the suspension of conidia was agitated with a vortex (Classic Vortex Mixer, Velp Scientifica Srl, Usmate Velate, Italy) for 5 min, along with eight sterile glass beads to aim the agitation. The determination of the conidia concentration (1 × 10 7 conidia/ml suspension) was conducted with a Neubauer-improved hemocytometer (Marienfeld, Lauda-Königshofen, Germany) under a microscope. The germination of conidia was taken place by the inoculation of a 0.1 ml solution containing 1 × 10 6 conidia/ml, on two 60 mm dishes with SDA + Yeast (SDAY). Then, these dishes were covered with parafilm and incubated for 16 h, set at 25 °C and 14 light:10 dark h. After incubation, dishes were covered with a cover slip that was sterilized. In each dish, 200 conidia were counted and regarded germinated when conidia < germ tube (Inglis et al. 2012;Usman et al. 2020;Gulzar et al. 2021). The countings were conducted under a microscope (Euromex BB.1152-PLi, Euromex Microscopen bv, Arnhem, The Netherlands) with 400× total magnification. Before the beginning of the bioassays, > 94% of conidia was viable.

Laboratory bioassays
For the bioassays, six treatments and control were applied on wheat. The treatments were B. bassiana alone at 1 × 10 7 conidia/kg wheat (Bb), indoxacarb alone at 5 ppm (5 mg a.i./kg wheat), DE alone at 150 ppm (150 mg DE/kg wheat), B. bassiana + indoxacarb combination, B. bassiana + DE combination, indoxacarb + DE combination, and control (0.05% Tween 80). Wheat lots of 1 kg were laid in slim layers on separate trays for each treatment. The B. bassiana, indoxacarb, and the control solutions were applied on the wheat lots as liquids with a separate airbrush (Master Multipurpose Airbrush, USA), while the DE formulation was applied as dust. The wheat portions were sprayed using 1 ml of the suspension of conidia or with 1 ml of aqueous solution containing the analogous volume of indoxacarb. Thereafter, the treated wheat portions were conveyed to separate 3-l glass containers for a manual shake of 10 min total duration, to assure the equal distribution of each killing agent. As far as the combinations are concerned, the first application was B. bassiana followed by the DE (Bb + DE). In the combinations with indoxacarb, this insecticide was applied first and then the conidia of B. bassiana (Bb + indoxacarb) or the DE (DE + indoxacarb). Subsequently, the treated portions of wheat were shaken as above in different 3-l containers. From each treated wheat lot, three samples of 100 g were weighted with a balance (ELB 300 Shimadzu compact balance, Kyoto, Japan) on separate layers. The weighted samples were put into plastic containers of 11 cm height and 6.5 cm diameter. Polytetrafluoroethylene (60 wt % dispersion in water) (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was applied on the inside necks of the plastic containers to impede the insects from escaping. Next, fifty C. ferrugineus adults were put into each plastic vial. The adequate aeration of these vials was achieved by cyclic holes of 1.5 cm diameter on the lids, cladded with muslin cloth. The prepared vials were incubated at 30 °C and 65% RH for 7 and 14 days to assess the mortality with a stereomicroscope (Leica Wild M3B, Heerbrugg, Switzerland) under 40× total magnification. For each exposure interval, we prepared new series of vials. Upon the end of the 14-day experimental period, all tested insects were separated from the wheat lots. Afterwards, wheat was returned into the vials and into the incubators at the aforementioned abiotic conditions to document progeny production. All the above procedure was copied three more times with new vials, wheat portions, and insects. The same process was replicated for T. castaneum, T. granarium, and R. dominica. The emergence of offspring was documented after 62 days (Wakil et al. 2021c), 46 days (Kavallieratos et al. 2017a), and 62 days (Wakil et al. 2021c) for T. castaneum, T. granarium, and R. dominica, respectively. Progeny of R. dominica was adults as the immature stages of this species are into the grains (Aitken 1975), while for all the other species were adults and their immature stages. The bioassays were conducted between November 2020 and August 2021.

Persistence bioassays
The insecticidal effects of B. bassiana, DE, and indoxacarb, their binary combinations (Bb + DE, Bb + indoxacarb, and DE + indoxacarb), and control were recorded for a period of 120 days, 7 or 14 days after the initial exposure, against C. ferrugineus, T. castaneum, T. granarium, and R. dominica. Every 30 days, mortalities of the exposed individuals of the aforementioned species and the offspring emergence were estimated as in the laboratory bioassays. The wheat lots were kept in glass containers in incubators set at 30 °C and 65% RH, for the whole duration of the experiment.

Laboratory bioassays
Mortality values were corrected by Abbott's formula (Abbott 1925). Data were transformed to log (x + 1) before the analysis to make the variance normal (Zar 2014;Scheff and Arthur 2018). Data for each species were analyzed with a two-way analysis of variance (ANOVA) with treatment and exposure interval being the main effects, while the response variable was mortality. Interactions of the main effects were taken into consideration in the analysis. Similarly, for the progeny production, data were analyzed with a two-way ANOVA, having treatment and species as main effects, while the response variable was the number of offspring. Interactions of the main effects were incorporated into the analysis. Minitab 17 software (Minitab 2010) was utilized for all the conducted analyses. To compare mortality and progeny means, the Tukey-Kramer (HSD) test at 5% importance was used (Sokal and Rohlf 1995).

Persistence bioassays
Mortality values were corrected by Abbott's formula (Abbott 1925). Data were transformed to log (x + 1) before the analysis to make the variance normal (Zar 2014;Scheff and Arthur 2018). Data for each species were analyzed with a two-way ANOVA with treatment and period of storage being the main effects, while the response variable was mortality. Interactions of the main effects were taken into consideration in the analysis. The two-way ANOVA was conducted separately for each species and initial exposure (7 or 14 days). For the progeny production, data were analyzed with a twoway ANOVA, having treatment and period of storage as main effects, while the response variable was the number of offspring, per species. Interactions of the main effects were incorporated into the analysis. Minitab 17 software (Minitab 2010) was utilized for all the conducted analyses. To compare mortality and progeny means, the Tukey-Kramer (HSD) test at 5% importance was used (Sokal and Rohlf 1995).

Mortality and progeny in the laboratory
Concerning C. ferrugineus, main effects and their interaction were found significant ( Table 1). The lowest mortality rates were observed for the individuals exposed on wheat treated with B. bassiana, not exceeding 31.21 and 43.08%, 7 and 14 days post-exposure, respectively ( Table 2). The combination of DE + indoxacarb caused the highest mortality (93.88%) among all other tested insecticidal treatments, followed by Bb + indoxacarb (82.35%) and Bb + DE (71.47%), after 14 days of the initial exposure. Regarding T. castaneum, main effects and their interaction were significant (Table 1). The most efficient treatment was the combination of DE + indoxacarb that caused 66.13 and 87.42% mortality, while the least efficient was B. bassiana alone causing 28.46 and 39.65% mortality, after 7 and 14 days of initial exposure respectively ( Table 2). As far as T. granarium is concerned, main effects and their interaction were significant (Table 1). When the insecticides were tested alone, mortality rates ranged between 22.33 (B. bassiana) and 33.52% (indoxacarb) after 7 days of exposure and between 31.86 (B. bassiana) and 47.76% (indoxacarb) after 14 days of initial exposure ( Table 2). The mortality levels of the combinations were higher than the insecticides alone, with the treatment of DE + indoxacarb combination exhibiting the highest rates (59.34 and 81.67%, 7 and 14 days post-exposure, respectively), followed by Bb + indoxacarb (52.23 and 74.52%, after 7 and 14 days of exposure, respectively) and Bb + DE (40.67 and 56.26%, 7 and 14 days post-exposure, respectively). Main effects and their interaction were significant in the case of R. dominica (Table 1). Complete (100.00%) mortality was achieved for the adults exposed on the combination of DE + indoxacarb 14 days post-exposure, while 73.58% of the exposed R. dominica were dead after 7 days of initial exposure to the same combination (Table 2).
When B. bassiana was tested alone, mortality levels were the lowest, not exceeding 38.63 and 47.42%, after 7 and 14 days of exposure. The combination of Bb + indoxacarb was the second most efficient insecticidal combination that killed 65.77 and 92.18% of the exposed R. dominica individuals, after 7 and 14 days.
Regarding progeny production of all the tested species, the main effects and their interaction were significant  (Table 3). The DE + indoxacarb combination was the most successful towards all tested insect species, ranging from 8.35 to 33.70 individuals, and B. bassiana alone was the least efficient for controlling the progeny production, ranging from 51.33 to 78.55 individuals (Table 4). All tested insecticidal treatments caused significant reduction to the progeny production in comparison with control. Rhyzopertha dominica had the lowest offspring emergence while T. granarium had the highest progeny at all tested insecticidal treatments.

Persistence in the laboratory
Regarding C. ferrugineus, main effects and their interaction were significant after 7 days (Table 5) and 14 days (Table 7) of initial exposure. The highest mortality was observed for the DE + indoxacarb combination at 0 day of trial, reaching 61.07% 7 days (Table 6) and 95.22% 14 days (Table 8) postexposure. After 7 days of exposure, at the 120 days trial, indoxacarb exhibited the lowest mortality (8.53%), while the DE + indoxacarb combination caused the highest mortality 38.58% (Table 6). The B. bassiana alone had the lowest mortality rates during all trials, 14 days post-exposure (Table 8).
The DE + indoxacarb combination was the most efficient at all trials, after 14 days of initial exposure. Main effects and their interaction regarding T. castaneum were significant 7 days (Table 5) and 14 days (Table 7) postexposure. The highest and the lowest mortalities were noted for the DE + indoxacarb combination and B. bassiana alone treatments respectively, ranging between 27.30 and 56.32% (DE + indoxacarb) and 6.13 and 22.18% (B. bassiana alone) at all trials, 7 days post-exposure (Table 6). After 14 days of initial exposure, the DE + indoxacarb combination and B. bassiana alone treatments killed the most and least T. castaneum exposed adults at all trials (ranges: 43.67-84.31% and 13.31-27.65%, respectively) ( Table 8).
Concerning T. granarium, main effects and their interaction were significant when the individuals were exposed for 7 days (Table 5) and 14 days ( Table 7). The B. bassiana alone and indoxacarb alone did not kill any T. granarium adult after 7 days of initial exposure, at the 120 days trial ( Table 6). The most efficient treatment against T. granarium individuals was the DE + indoxacarb combination at  all trials, 7 days post-exposure, with mortality ranging from 17.06 (120 days) to 46.40% (0 days). Indoxacarb caused the lowest mortality rates (7.20%) among all other treatments at the 120 days trial, 14 days post-exposure (Table 8). The combination of DE + indoxacarb achieved the highest mortalities at all trials, demonstrating a range from 78.83% at the 0 days trial and 33.42% at the 120 days trial. As far as R. dominica is concerned, main effects and their interaction were significant after 7 days (Table 5) and 14 days (Table 7) of initial exposure. The DE + indoxacarb combination killed the most exposed individuals, reaching 72.37% mortality after 7 days of exposure, at the 0 days trial (Table 6). This combination was the most efficient against R. dominica adults at all trials. Beauveria bassiana alone caused the lowest mortality levels at all trials, ranging from 15.68 to 32.74%, 7 days post-exposure. Complete mortality was documented for the DE + indoxacarb combination, followed by the Bb + indoxacarb combination (94.18%) at the 0 days trial, 14 days post-exposure (Table 8). The B. bassiana alone treatments resulted to the lowest mortalities, at all trials (21.81-46.76%) after 14 days of exposure. The combination of DE + indoxacarb killed the most individuals at all trials with the 120 days trial exhibiting the lowest mortality of 63.80%.
Regarding progeny production, main effects and their interaction were significant for all the tested insect species (Table 9). The emergence of C. ferrugineus, T. castaneum, T. granarium, and R. dominica individuals at all treatments and trials was significantly lower than the control (Table 10). Among the treatments, B. bassiana alone was the least efficient and the DE + indoxacarb combination the most efficient at all trials, against all insect species. The lowest progeny production was recorded for R. dominica, not exceeding 13.66 individuals at the 0 days trial, on wheat treated with the DE + indoxacarb combination. The highest emergence was noted for the B. bassiana alone treatment, at the 120 days trial (107.67 T. granarium individuals per vial).

Discussion
The findings of our study suggest that all examined combinations resulted to higher mortality rates of C. ferrugineus, T. granarium, R. dominica, and T. castaneum adults than single applications. This phenomenon has been documented before with the binary combinations of B. bassiana, DE plus bitterbarkomycin (DEBBM), and imidacloprid causing higher mortality rates than each insecticide alone (Wakil and Schmitt 2015). Similarly, Hanif et al. (2022) showed that the 1.5 × 10 10 B. bassiana conidia/kg + 50 mg/kg DEBBM or the 1.5 × 10 10 B. bassiana conidia/kg grain + 150 mg DE /kg grain caused higher mortalities to C. ferrugineus, R. dominica, and T. castaneum adults than the entomopathogenic fungus and DE separately. We found that the most susceptible species was R. dominica followed by C. ferrugineus, T. castaneum, and T. granarium. More specifically, R. dominica was the only insect species that reached 100% mortality, when exposed on wheat treated with the DE + indoxacarb combination for 14 days. This phenomenon has been previously documented for R. dominica which was to be more susceptible than T. castaneum and T. granarium, when B. bassiana was combined with the phenyl pyrazole fipronil (Wakil et al. 2022). The knowledge of the order of stored-product insects' susceptibility to certain insecticides, triggers the early applications so as to restrain further population growth and concomitant damage of the products (Colunga Garcia et al. 2013;Yemshanov et al. 2014;Douma et al. 2016;Kavallieratos et al. 2017b;Papanikolaou et al. 2018;Skourti et al. 2022). Whether this order remains the same when insect species coexist merits further investigation.
One of the most important findings is that the mortality levels of T. castaneum were moderate to high, ranging from 48.80 to 66.13% and from 63.03 to 87.42% for the tested combinations, after 7 and 14 days of exposure, respectively. Tribolium castaneum adults are hard to manage (Kavallieratos et al. 2021a, b, c, d); thus, their suppression is crucial because they consist reproductive vehicles (Arthur 1998a(Arthur , b, 2008Sehgal et al. 2013;Kavallieratos et al. 2017cKavallieratos et al. , 2020. Since the mortality rates reached 87.42% at the DE + indoxacarb combination 14 days post-exposure, this insecticidal treatment could be considered for the effective management of T. castaneum. The most efficient treatment against all tested species included the DE + indoxacarb combination. Even though the DE and the indoxacarb alone provided low to moderate mortalities and reduction of offspring emergence, their combination resulted to elevated efficacy at all laboratory  (Ebeling 1971;Korunic 2013;Shah and Khan 2014;Losic and Korunic 2018), rendering by this way the insects sensitive and prone to indoxacarb. Several studies have documented that DE enhanced the efficacy of synthetic insecticides (Wakil et al. 2013(Wakil et al. 2021c(Wakil et al. , 2022Wakil and Schmitt 2014;Machekano et al. 2017) or entomopathogenic fungi (Akbar et al. 2004;Lord 2005;Kavallieratos et al. 2006;Batta 2008;Sabbour et al. 2012;Ashraf et al. 2017;Wakil et al. 2021d). In the case of entomopathogenic fungi, DE increases the ability of entomopathogenic fungi conidia to attach the cuticle of the insect, and therefore advances its efficacy (Akbar et al. However, it should be noted that the combinations of DE and entomopathogenic fungi do not always lead to increased mortality of exposed insects, but they may result to detrimental effects (Vassilakos et al. 2006). One other important factor that determines the efficacy of the combination of B. bassiana and DE is commodity. For example, 1.5 × 10 10 B. bassiana conidia/kg grain treated on maize and rice killed less C. ferrugineus than the combination of 1.5 × 10 8 B. bassiana conidia/kg grain + 50 ppm DE, while on wheat the results were reversed (Wakil et al. 2021d).
Our results revealed that the B. bassiana + indoxacarb combination caused higher mortality rates than when each insecticide was applied separately. This issue could be attributed to the stress caused by the synthetic insecticide that weakens the insects' immune system, allowing the entomopathogenic fungi to easily infect the insects (Hiromori and Nishigaki 2001). Numerous studies confirm that the efficacy of entomopathogenic fungi is enhanced by other insecticides (Ashraf et al. 2017;Wakil et al. 2012Wakil et al. , 2022Gad et al. 2020). However, the efficacy of a certain combination is affected by many factors (e.g., insect species, type of insecticide, entomopathogenic fungus species/ isolate) that should be taken into consideration before a large-scale treatment takes place (Batta and Kavallieratos 2018).
The persistence efficacy of the tested combinations followed similar pattern as the laboratory bioassays. Beauveria bassiana provided the lowest mortalities and reduction of progeny, while the combination DE + indoxacarb the highest, against all species. All treatments lost gradually (from 0 to 120 days) their effectiveness to kill the tested insect species and suppress the offspring emergence. Each combination has residual efficacy that results to long term protection. In a previous study, Miliordos et al. (2017) found that indoxacarb alone exhibited elevated effectiveness for 180 days against S. oryzae and R. dominica on maize and wheat. Wakil and Schmitt (2015) documented that the dual combinations of DEBBM, B. bassiana, and imidacloprid protected effectively wheat from C. ferrugineus, R. dominica, T. castaneum, and L. paeta, for 6 months. In a recent study, Wakil et al. (2021c) proved that the combinations of DEBBM + B. bassiana or imidacloprid provided adequate protection of stored wheat when it was infested with C. ferrugineus, R. dominica, T. castaneum, and L. paeta for 180 days.
To conclude, this study suggests that B. bassiana, DE, and indoxacarb combinations are effective tools for the management of C. ferrugineus, T. castaneum, T. granarium, and R. dominica, when applied to stored wheat. However, the tested species suffered different levels of mortality as follows: R. dominica > C. ferrugineus > T. castaneum > T. granarium, at both laboratory and persistence bioassays. More studies that will include more parameters, like additional species and their developmental stages, commodities, and abiotic factors, are needed to assess the potential effect of the above promising insecticidal combinations for a sufficient protection of commodities in storage facilities. Last but not least, apart from DEs, other inert dusts such as zeolites or wood ashes exhibit elevated insecticidal properties against major stored-product insects (Bohinc et al. 2018(Bohinc et al. , 2020. Therefore, it would be interesting to further investigate their effectiveness in combination with B. bassiana and indoxacarb as additional armory to the perpetual fight with stored-product pests.
Author contribution WW and NGK conceived and designed research. WW, AA, TY, and MA conducted experiments. WW and NGK analyzed data. WW, NGK, and EPN wrote the manuscript. All authors read and approved the manuscript.
Funding Open access funding provided by HEAL-Link Greece. This study was partially funded by the Agricultural Linkages Program CS-097, Pakistan Agricultural Research Council (ALP-PARC), Islamabad, Pakistan.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations
Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest The authors declare no competing interests.
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