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Article

Entomopathogenic Fungus and Enhanced Diatomaceous Earth: The Sustainable Lethal Combination against Tribolium castaneum

by
Waqas Wakil
1,2,*,
Nickolas G. Kavallieratos
3,*,
Erifili P. Nika
3,
Tahira Riasat
1,4,
Muhammad Usman Ghazanfar
5,
Khawaja G. Rasool
6,
Mureed Husain
6 and
Abdulrahman S. Aldawood
6
1
Department of Entomology, University of Agriculture, Faisalabad 38040, Pakistan
2
Senckenberg German Entomological Institute, D-15374 Müncheberg, Germany
3
Laboratory of Agricultural Zoology and Entomology, Department of Crop Science, Agricultural University of Athens, 75 Iera Odos str, 11855 Athens, Greece
4
Department of Zoology, Government College University, Faisalabad 38000, Pakistan
5
Department of Plant Pathology, College of Agriculture, Sargodha University, Sargodha 40100, Pakistan
6
Department of Plant Protection, College of Food and Agriculture Sciences, King Saud University, Riyadh 11451, Saudi Arabia
*
Authors to whom correspondence should be addressed.
Sustainability 2023, 15(5), 4403; https://doi.org/10.3390/su15054403
Submission received: 2 February 2023 / Revised: 20 February 2023 / Accepted: 21 February 2023 / Published: 1 March 2023
(This article belongs to the Special Issue Biocontrol for Sustainable Crop and Livestock Production)
Versions Notes

Abstract

:
This study determined the efficacy of the Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Hypocreales: Cordycipitaceae) alone or combined with the diatomaceous earth DEA (a mixture of DE + abamectin) against adults and larvae of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). DEA was evaluated at 50 ppm while the fungi at 1.6 × 105, 1.6 × 106, and 1.6 × 107 conidia/kg wheat). Mortalities were assessed after 7 or 14 days of exposure, while progeny reduction in adults after 30, 60, 90, or 120 days. The radial fungus growth was significantly affected by the dose of DEA. Mortalities were higher in the combined treatments compared to the application of DEA or B. bassiana alone for both larvae and adults. Larvae were more susceptible than adults in all treatments and exposure intervals. Insect survival and progeny production were recorded for four months. Significantly fewer progeny was noted on wheat treated with DEA + B. bassiana treatments compared with control. The maximum number of mycosed cadavers and the rate of sporulation were observed at the lowest dose of B. bassiana alone, followed by the higher doses gradually. These findings indicate that the DEA + B. bassiana combinations can efficiently control T. castaneum, providing long-term protection of wheat.

1. Introduction

Global food security is threatened by losses caused by storage insect pests, which are lower in developed countries than in developing countries [1,2]. Numerous insect pest species constitute a serious threat to stored products leading to elevated losses [3,4,5,6]. There are two foremost eradication chemical methods that are used worldwide to manage stored-product insect pests: contact insecticides and fumigation [7,8,9]. Even though these application methods have advantages (e.g., comparatively low cost and enhanced effectiveness), they face health and ecological concerns [7,10]. Furthermore, the problem of resistance is linked with the continuous exposure of storage pests to chemicals [9,11,12]. Therefore, to delay the development of the resistance of insects and avert the losses of quantity and degradation of quality of stored commodities, integrated pest management (IPM) approaches have focused on the use of insecticides applied at low doses in combination with biocontrol agents [13,14,15].
Due to diverse insecticidal effects, entomopathogenic fungi can be potentially incorporated in the management of stored-product insects. These pathogenic agents are considered as appropriate alternatives to synthetic insecticides because they are easily combined with other killing agents [13,14,15,16,17,18] and have high efficacy according to their geographical origin [19,20]. Other benefits making entomopathogenic fungi desirable for stored-product protection are short life cycles, being naturally available, being easy to produce due to less nutrient requirements, having a high reproductive potential, and having less chances of resistance development [21,22,23].
The efficacy of several entomopathogenic fungi, e.g., Beauveria bassiana (Balsamo-Crivelli) Vuillemin, Isaria fumosorosea Wize (Hypocreales: Cordycipitaceae), Metarhizium anisopliae (Metschnikoff) Sorokin (Hypocreales: Clavicipitaceae), as grain protectants has been well documented in former studies [13,24,25,26]. Beauveria bassiana has been demonstrated to be effective against Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae) [13,27], Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) [19,28], Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae), Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) [29], and Sitophilus oryzae (L.) (Coleoptera: Curculionidae) [30]. The infection of B. bassiana includes a series of steps: the attachment of the asexually produced conidia with the cuticle of the host, their germination, insect penetration, and proliferation of fungus on cadaver surface [21,22,23].
Diatomaceous earths (DEs) consist of fossilized phytoplankton (diatoms), which are unicellular eukaryotic enriched silicon dioxide [31,32,33]. The main activity of DE is abrasion of insect cuticle and sorption of wax layer [31,33]. Death occurs after the excessive loss of the water content of insects [31,33,34,35]. They have extensively been considered as proficient insecticides against a broad spectrum of stored-product pests [13,29,36]. However, the reduced bulk density of grains is negatively influenced by the application of DEs [36]. To overcome this disadvantage, DEs have been combined with other killing agents, such as entomopathogenic fungi [13,29], insecticides [13,37,38,39], and essential oils and their emulsions [40,41]. Interestingly, the combination of DEs with entomopathogenic fungi has been proven to enhance conidial attachment and their virulence [28,42]. Although, there are numerous reports about the combination of DEs and entompathogenic fungi [13,18,29,43,44], there is no published information regarding the efficacy of DE enhanced with abamectin and B. bassiana as stored-grain protectants. Therefore, the objectives of this study deal with the evaluation of DEA (DE + abamectin) combined with B. bassiana at different doses, to assess the radial fungus growth, mycosis, and sporulation, as well as the pesticidal activity of the aforementioned combinations against T. castaneum (larvae and adults), progeny reduction in adults on wheat after prolonged storage periods.

2. Materials and Methods

2.1. Insect Culture

A laboratory strain of T. castaneum from Faisalabad was reared on wheat flour plus 5% brewer’s yeast at 30 °C, 65% RH, in darkness. Adults and larvae, younger than 14 days old and from the 3rd-4th larval instar, respectively, participated in the trials.

2.2. Grains

Non-infested wheat (variety Faisalabad 2008) was cleaned of dockage and insecticides. Before trials, moisture content of wheat was 11.9%, as estimated by a moisture meter (Dickey-John Multigrain CAC II; Dickey-John Co., Lawrence, KS, USA).

2.3. DE Formulation

A fresh water DE (90%) [45] enhanced with 0.25% active ingredient (a.i.) abamectin (DEA) was used at 50 ppm, against larvae and adult stages of T. castaneum. Abamectin consisted of an avermectin mixture, i.e., 20% B1b and 80% B1a avermectins, derived from the Streptomyces avermitilis (ex Burg et al.) Kim and Godfellow (Actinomycetales: Streptomycetaceae) soil bacterium.

2.4. Entomopathogenic Fungus Culture

Beauveria bassiana isolate (WG-25) was maintained in test tubes with Sabouraud Dextrose Agar (SDA) slopes, stored in refrigerator at 4 °C. It was mass-reared on Petri dishes (10 cm diameter) with SDA, closed with parafilm, preserved for 10 days at 25 °C and 10:14 h (dark:light). Conidia were acquired from dishes with a germ-free scalpel and conveyed into falcon tubes of 50 mL that contained 30 mL of uncontaminated 0.05% solution of Tween 80 (Merck, Kenilworth, NJ, USA). The conidia suspension was subsequently submitted for 5 min to a vortex (Classic Vortex Mixer, Velp Scientifica Srl, Usmate Velate, Italy), along with eight uncontaminated beads made of glass. A Neubauer-improved hemocytometer (Marienfeld, Lauda-Königshofen, Germany) and a microscope (BB.1152-PLi, Euromex Microscopen bv, Arnhem, The Netherlands) were utilized to determine the fungi concentrations at 1.6 × 105, 1.6 × 106, and 1.6 × 107 conidia/mL. The germination of the conidia was estimated on two dishes (6 cm diameter) that contained yeast plus SDA, by inoculation of 0.1 mL solution (1 × 106 conidia/mL). The dishes were sealed with parafilm and kept for 16 h at 25 °C and 10:14 h (dark:light). An uncontaminated cover slip was placed onto the dishes at the end of incubation. One counting of 200 conidia was performed for each dish. Germinated conidia were inspected under 400× magnification, having conidia smaller than the germ tube [46]. The viability of conidia was >93% before assays.

2.5. Radial Growth Test

The SDA was used as a fungal growing medium. The conical flasks containing SDA medium was autoclaved at 121 °C for 15 min, and after cooling four different concentrations of DEA (25, 50, 75, 100 ppm) were added. The same SDA medium without DEA was used as control. After gently shaking the containers for 2–3 min, each dish (9 cm diameter) was filled with 15 mL SDA + DEA. Three dishes per concentration were used in the bioassays. To check the impact of DEA on the radial growth of B. bassiana, 3 mm (diameter) cores of the fungus from new non-sporulated cultures were inverted and inserted separately in the center of the dish. The radial colonies were estimated after 2, 4, 7, 10, and 15 days using two radii that were sketched at the bottom part of each dish at the correct angles [47]. The entire experiment was repeated thrice.

2.6. Bioassays

In total, seven treatments and the control were tested on stored wheat. Three doses of B. bassiana: 1.6 × 105 (F1), 1.6 × 106 (F2), and 1.6 × 107 (F3) conidia/kg wheat were treated alone or combined with 50 ppm (mg/kg wheat) DEA. The control consisted of water containing 0.05% Tween 80. The applications took place on disks, with 1 kg of wheat laid as a slim layer. DEA was treated as dust, while B. bassiana and control were liquids, therefore, applied with different airbrushes (Master Multipurpose Airbrush, US Art Supply, San Diego, CA, USA). Concerning sprayings, 1 mL of each conidia suspension or control was applied on 1 kg of wheat (different), then transferred to a 3-L glass vase (different) and shaken by hand for 10 min to further distribute equally each insecticide or control. For DEA, 1 kg of wheat was conveyed into a 3 L glass vase, and subsequently, DEA was added to be fully shaken as above. Regarding the combinations, B. bassiana was applied first, and then DEA was added. Three samples weighing 100 g each (estimated with an ELB 300 Shimadzu balance (Kyoto) onto layers) from all treated lots were conveyed into separate plastic vials (6.5 cm diameter; 11 cm height). Different scoops and layers were used for each treatment. Before the bioassays, the top internal sides of the vials were polished with polytetrafluoroethylene (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) to impede the insect individuals to leave the vials. Caps bore a 1.5 cm diameter hole to aerate the vials, covered with a cloth. Thereafter, 50 T. castaneum larvae of adults were conveyed into the vials and kept at 30 °C/65% RH. Mortalities were calculated 7 and 14 days after the initial exposure, under a Wild M3B stereomicroscope (Leica, Heerbrugg, Switzerland). Each exposure interval consisted of new vials. After the 14-day stay into the vials, all tested insects were taken out from the vials. The wheat lots used in the bioassays remained into the vials to collect progeny data after a period of 62 days [13]. As progeny, immature stages and adults of T. castaneum were taken into account. The above practice was repeated in its entirety three times, using new vials, insects, and wheat. Mortality and progeny emergence data of all tested insecticides and control were collected for 120 days (7 and 14 days post-exposure). Countings were taken every 30 days.

2.7. Mycosis and Sporulation

Mycosed T. castaneum cadavers were maintained in uncontaminated dishes at 4 °C, after each mortality count of the laboratory bioassays. The surface of cadavers was sterilized using a 0.05% solution of sodium hypochlorite lasting for ~3 min. Subsequently, the cadavers were washed three times with sterilized distilled aqua. Afterwards, T. castaneum dead individuals were inserted on SDA plates at 25 °C and 75% RH for the following 7 days. A microscope was used to examine the white conidial spores emerging from the cadavers. Sporulation (i.e., number of conidia/mL) was estimated by mixing the mycosed T. castaneum individuals with 20 mL of water (distilled) + 1 drop Tween 80 [48]. After stirring for 10 min, a hemocytometer was utilized to count the number of conidia/mL [49].

2.8. Statistical Analysis

Radial growth data were analyzed with a two-way ANOVA with exposure interval and dose as the main effects, and radial growth as response variable. The formula of Abbott was utilized to adjust control mortalities for both adult and larval bioassays [50]. Values were transformed into log (x + 1) prior the conduction of analysis to normalize variance [51,52]. The mortalities of adults and larvae were analyzed with a four-way ANOVA. Treatment, developmental stage, storage period, and exposure interval were the main effects. Mortality was the response variable. Progeny was analyzed with a two-way ANOVA. Treatment and storage period were the main effects. Progeny was the response variable. Regarding mycosis and sporulation, a three-way ANOVA was conducted. Developmental stage, treatment, and storage period were the main effects whilst mycosis or sporulation was the response variable. In all analyses, the Tukey–Kramer (HSD) test (5% level of significance) was considered to compare means [53]. All interactions of the main effects in all groups of analyses were considered during the analysis. The entire analysis was conducted by Minitab 17 software [54].

3. Results

3.1. Radial Growth Test

The radial growth of B. bassiana was affected significantly by the doses of DEA at all tested exposure intervals (Table 1). The interaction dose × exposure interval was also significant. At all exposure intervals, the control was significantly different from the DEA doses with the exception of 25 ppm after 15 days of exposure (Table 2). None of the DEA doses were able to suppress the vegetative growth of B. bassiana, as it grew significantly during the experimental period.

3.2. Mortality Bioassays

All main effects and their associated interactions significantly affected mortality except exposure interval × developmental stage (Table 3). After 7 days of exposure, the combinations DEA + F2 and DEA + F3 provided 100% mortality at 0 days of storage, while only DEA + F3 killed all exposed larvae at 30 days of storage (Table 4). The least effective application was the lowest concentration of B. bassiana (F1) alone, followed by the other two concentrations of B. bassiana (F2 and F3) and DEA alone. The most effective application was DEA + F3, killing 58.7% of larvae 7 days post-exposure, at 120 days of storage. All three DEA + B. bassiana combinations suppressed larvae, 14 days post-exposure, at 30 days of storage (Table 5). At 60 days of storage, DEA + F2 and DEA + F3 caused 100% mortality. At the end of the storage period (120 days), 87.3% of T. castaneum larvae died after the exposure to DEA + F3 for 14 days. The least effective application was F1, followed by F2, F3, and DEA alone, throughout the storage periods. Concerning T. castaneum adults, only DEA + F3 was able to suppress the participating individuals after 7 days of exposure, at 0 days of storage (Table 4). The DEA + F1 and DEA + F2 combinations provided elevated mortalities, at 0 days of storage. Their efficacy was reduced drastically as storage periods were increased, reaching 40.2–49.7% at 120 days of storage, for all three DEA + B. bassiana combinations. F1, F2, F3, and DEA killed fewer individuals than all tested combinations, with F1 being the least effective. After 14 days of exposure, all three DEA + B. bassiana combinations caused the death of all exposed adults at 0 days of storage, while only DEA + F2 and DEA + F3 achieved their suppression at 30 days of storage (Table 5). The least effective treatment was F1, while the most effective was DEA + F3, at all storage periods. At 120 days of storage, mortalities ranged between 24.9% (F1) and 70.5% (DEA + F3).

3.3. Progeny

All main effects and their associated interaction were significant (Table 3). All treatments (alone and combined) led to significantly less progeny than controls, at all storage periods (Table 6). Among combinations, only DEA + F2 and DEA + F3 did not allow the production of offspring at 0 days of storage. However, at 30 days of storage, only DEA + F3 suppressed progeny emergence. The highest number of individuals was found in vials treated with F1 and the lowest number in vials treated with DEA + F3. At 120 days of storage, progeny production ranged from 10.7 individuals/vial (DEA + F3) to 46.6 individuals/vial (F1), while control bore 86.8 individuals/vial.

3.4. Mycosis and Sporulation

Concerning mycosis, the main effects and their associated interactions were significant (Table 7). The highest percentage of mycosis on larval cadavers was noted at F1, reaching 98.3%, at 0 days of storage, followed by F2 (92.4%) and F3 (85.7%) (Table 8). The lowest percentage was recorded at DEA + F3 (61.2%) at the same storage period. At 120 days of storage, mycosis ranged between 76.4% (F1) and 20.4% (DEA + F3). Throughout the bioassays, the combinations of DEA + B. bassiana provided lower mycosis rates than B. bassiana alone (F1, F2, and F3). Concerning mycosis on adult cadavers, all rates were lower than on larvae. F1 achieved the highest percentage of 91.5%, followed by F2 (87.3%) and F3 (81.5%), at 0 days of storage. All combinations of DEA + B. bassiana provided lower mycosed cadavers, at all storage periods. At 120 days of storage, the lowest mycosis was noted in DEA + F3 combination (16.6%) and the highest in F1 (69.3%).
Regarding sporulation, the main effects and their associated interactions were significant (Table 7). The sporulation on larvae received its highest number in the case of F1 (301.5 conidia/mL) at 0 days of storage (Table 9). All applications of B. bassiana alone provided more conidia/mL than all DEA + B. bassiana combinations, at all storage periods. Among the combinations, DEA + F3 had the lowest sporulation. At 120 days of storage, the sporulation ranged between 129.7 conidia/mL (DEA + F3) and 213.3 conidia/mL (F1). As far as sporulation on adults is concerned, at 0 days of storage it ranged from 186.2 (DEA + F3) to 287.8 (F1). The highest and the lowest sporulation were documented in vials containing wheat treated with F1 and DEA + F3, respectively, throughout the storage periods. At 120 days of storage, the sporulation ranged between 114.6 conidia/mL (DEA + F3) and 194.8 conidia/mL (F1).

4. Discussion

Our study signified that the fungal growth was significantly affected by the doses of DEA at all exposure intervals. The mortality of T. castaneum larvae and adults differed significantly among treatments after each exposure interval. The maximum rate of mycosed larvae or adults and their sporulation were observed at 0 day, while they decreased gradually with the passage of time. The lowest dose of conidia alone provided the highest rates of both mycosis and sporulation. These parameters increased inversely with fungal concentration. Previously, Riasat et al. [48] described that lower conidial concentrations of B. bassiana increased the percentage of mycosis and sporulation on R. dominica adult cadavers. Similarly, the mycosis percentage and conidial germination were higher at lower doses of silicoSec + B. bassiana than at higher doses [29]. Furthermore, the presence of the DE enhanced the mycosis percentage and conidial germination [29]. Apart from the additive effect of DE to B. bassiana, it also enhanced the conidial ability to attach onto insects’ cuticle [28]. It should be noted that the mycosis and sporulation are also depended on the target insect species. In a recent study, M. anisopliae mycosed and sporulated more on cadavers of Liposcelis paeta Pearman (Psocoptera: Liposcelididae) than on Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae), R. dominica, or T. castaneum [55].
Earlier studies indicate that the impact of entomopathogenic fungi to T. castaneum depends on several factors, such as the susceptibility of the target instar, the virulence of the fungal species/strain, grain type, and environmental conditions. For instance, B. bassiana killed more T. castaneum adults at 30 °C than at 25 °C or 20 °C [37,38]. Interestingly, Wakil et al. [20] reported statistically significant different mortalities of T. castaneum when it was exposed to four and three different geographical isolates of B. bassiana and M. anosiopliae, respectively, at both species and isolate levels. The virulence of entomopathogenic fungi against different instars of a certain species is diverse, as in the case of T. castaneum. Baek et al. [56] reported that 72 h after initial exposure to the ERL1170-egfp B. bassiana isolate, all larvae were dead vs. 5% of adults. The virulence of entomopathogenic fungi is based on the adherence of conidia onto the host’s body, their germination, and finally, their penetration into the insect [21]. The fact that insect stages have functionally and morphologically different cuticular layers, as well as variable thickness and softness of the epicuticle [35,57,58], may partially explain the variable virulence of fungi. For example, Noh et al. [59] reported that the larval cuticle of T. castaneum is flexible and soft, while adult cuticle is highly sclerotized. Similarly, in this study, the larvae of T. castaneum were more susceptible than the adults at all bioassays. On the other hand, T. castaneum adults suffered high mortalities when exposed to the combination of DEA + B. bassiana, an issue that could be attributed to discontinuities of the insect cuticles provoked by the DE [31,33], resulting in fungal penetration.
The combination of DEs with natural products may enhance their DE’s efficiency and decrease their adverse effects [13,18,29,36,44]. For example, DEs plus entomopathogenic fungi provided sufficient control against several stored-product insects [18]. SilicoSec and conidia of B. bassiana resulted in additive and/or synergistic effects against C. maculatus and O. surinamensis adults [29]. Beauveria bassiana combined with Celite or Sayan DEs reduced the fecundity, egg hatching, oviposition period, and longevity of Trogoderma granarium Everts (Coleoptera: Dermestidae) adults, more than each formulation alone [44]. Metarhizium anisopliae plus Hudson DE were proved highly effective against Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) larvae vs. M. anisopliae or DE alone [43]. The findings of this study indicate that DEA + B. bassiana exhibit elevated efficacy against T. castaneum adults and larvae compared to each agent alone. One of the most important finding of this research is that the elevated mortalities of T. castaneum, caused by the combination of DEA + B. bassiana, were achieved by the lowest conidial concentrations. For example, even the lowest dose of B. bassiana (1.6 × 105 conidia/kg) + DEA killed all T. castaneum adults and larvae, 14 days post-exposure.
Progeny was significantly affected by treatments when compared to control. Similarly, Riasat et al. [48] ascertained that the integration of B. bassiana with DE Diafil led to the considerable reduction of R. dominica progeny production, 60 days post-application. Furthermore, the combination of two DEs (Sayan and Celite) with B. bassiana almost suppressed the progeny production of T. granarium during a period of eight weeks [60]. The combinations of B. bassiana and DE Protect-It, as well as each formulation alone, provided significantly lower progeny production than controls, even 120 days post-application [14]. The suppression of insect offspring production is a crucial parameter because it reduces grain damage [61,62]. For instance, three insecticides that inhibit chitin synthesis and spinosad (alone and combined) applied on wheat caused the reduction of offspring production of S. oryzae and grain weight loss [61].
The performance of DEs and entomopathogenic fungi is slow in comparison to numerous chemical insecticides, thus, insects continue damaging grains and/or move to other treated or untreated areas of the grain mass [14,55]. The persistence of entomopathogenic fungi depends on their inoculum and the contagion from the mycosed dead insects [63]. Actually, mycosis and sporulation were kept at high levels, 76.4% and 69.33% (mycosis for larvae and adults, respectively), as well as 213.33 conidia/mL and 194.8 conidia/mL (sporulation for larvae and adults, respectively), after 120 days. Apparently, these persistence indicators led to the recorded high levels of larval and adult mortalities, even after 120 days (i.e., 87.25% and 70.52% for larvae and adults, respectively). It should be noted that the extension of protection is crucial when insect management tactics are focused on stored-product pests, given that stored products are maintained for several months in facilities before they are further proceeded [13,64].

5. Conclusions

In conclusion, the combinations of B. bassiana and DEA resulted in positive effects in terms of both direct mortality and reduction of progeny production against T. castaneum compared to B. bassiana or DEA alone. This dual effect is a desirable characteristic when killing agents are applied as grain protectants, thus advancing B. bassiana plus DEA to a sustainable tool for long-term storage protection. More research efforts are needed containing several combinations of alternative substances for the protection of stored-product pests, over long storage periods.

Author Contributions

Conceptualization, W.W., N.G.K. and E.P.N.; methodology, W.W. and N.G.K.; software, W.W., N.G.K. and E.P.N.; validation, W.W., N.G.K., E.P.N., T.R., M.U.G., K.G.R., M.H. and A.S.A.; formal analysis, W.W., N.G.K. and E.P.N.; investigation, W.W., N.G.K., E.P.N., T.R. and M.U.G.; resources, W.W.; data curation, W.W., N.G.K., E.P.N. and M.U.G.; writing—original draft preparation, W.W., N.G.K., E.P.N., T.R., M.U.G., K.G.R., M.H. and A.S.A.; writing—review and editing, W.W., N.G.K., E.P.N., T.R., M.U.G., K.G.R., M.H. and A.S.A.; visualization, W.W., N.G.K., E.P.N., T.R., M.U.G., K.G.R., M.H. and A.S.A.; supervision, W.W. and N.G.K.; project administration, W.W.; funding acquisition, W.W. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Pakistan Science Foundation (PSF/AGR-381), Islamabad, Pakistan and Researchers Supporting (Project RSPD2023R721), King Saud University, Riyadh, Saudi Arabia.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Available upon request.

Acknowledgments

We thank Zlatko Korunic (Diatom Research and Consulting Inc., Toronto, Canada) who provided us the DEA that was used in this study.

Conflicts of Interest

The authors declare no conflict of interest.

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Table
Table 1. ANOVA parameters for radial growth of Beauveria bassiana on SDA (total DF = 224).
Table 1. ANOVA parameters for radial growth of Beauveria bassiana on SDA (total DF = 224).
SourceDFFp
Dose4584.2<0.01
Exposure interval42441.6<0.01
Dose × exposure interval164.2<0.01
Table
Table 2. Radial growth rate (mm/days ± SE) of Beauveria bassiana on SDA amended with 0 (control), 25, 50, 75, and 100 ppm of DEA. Within each row, means followed by the same lowercase letter are not significantly different (in all cases DF = 4, 44, Tukey–Kramer test at p = 0.05). Within each column, means followed by the same uppercase letter are not significantly different (in all cases DF = 4, 44, Tukey–Kramer test at p = 0.05).
Table 2. Radial growth rate (mm/days ± SE) of Beauveria bassiana on SDA amended with 0 (control), 25, 50, 75, and 100 ppm of DEA. Within each row, means followed by the same lowercase letter are not significantly different (in all cases DF = 4, 44, Tukey–Kramer test at p = 0.05). Within each column, means followed by the same uppercase letter are not significantly different (in all cases DF = 4, 44, Tukey–Kramer test at p = 0.05).
Dose2 Days4 Days7 Days10 Days15 DaysFp
25 ppm15.2 ± 0.6 Bd18.6 ± 0.8 Bd23.6 ± 0.9 Bc31.4 ± 0.7 Bb40.5 ± 1.3 ABa128.0˂0.01
50 ppm13.4 ± 0.5 Bd17.5 ± 0.8 Bcd22.3 ± 0.7 Cc29.4 ± 0.5 Cb38.4 ± 1.0 Ba121.0˂0.01
75 ppm10.3 ± 0.6 Cd15.6 ± 0.6 Cd18.8 ± 0.7 Cc25.2 ± 0.8 Db36.6 ± 1.1 Ca106.0˂0.01
100 ppm6.4 ± 0.4 Dd9.3 ± 0.3 Dd11.2 ± 0.4 Dc18.6 ± 0.5 Db29.6 ± 0.8 Da118.0˂0.01
Control16.7 ± 0.6 Ad20.5 ± 0.9 Ac24.2 ± 0.8 Ac33.3 ± 1.1 Ab41.4 ± 1.2 Aa115.0˂0.01
F60.059.863.770.068.4--
p˂0.01˂0.01˂0.01˂0.01˂0.01--
Table
Table 3. ANOVA parameters of main effects and associated interactions for mortality levels (larvae and adults) (total DF = 1259) and progeny emergence (total DF = 359) of Tribolium castaneum on wheat.
Table 3. ANOVA parameters of main effects and associated interactions for mortality levels (larvae and adults) (total DF = 1259) and progeny emergence (total DF = 359) of Tribolium castaneum on wheat.
EffectMortalityProgeny
DFFpDFFp
Treatment64592.17<0.01714,831.9<0.01
Exposure interval17734.65<0.01---
Developmental stage1774.40<0.01---
Storage period43941.58<0.0142035.62<0.01
Treatment × exposure interval628.17<0.01---
Treatment × developmental stage63.28<0.01---
Treatment × storage period2411.29<0.012870.70<0.01
Exposure interval × developmental stage11.390.23---
Exposure interval × storage period48.82<0.01---
Developmental stage × storage period44.52<0.01---
Treatment × exposure interval × developmental stage62.88<0.01---
Treatment × exposure interval × storage period2429.70<0.01---
Treatment × developmental stage × storage period246.05<0.01---
Exposure interval × developmental stage × storage period415.93<0.01---
Treatment × exposure interval × developmental stage × storage period243.07<0.01---
Table
Table 4. Mean mortality (% ± SE) of Tribolium castaneum larvae and adults exposed for 7 days on wheat treated with DEA (50 ppm) and Beauveria bassiana (F1: 1.6 × 105; F2: 1.6 × 106; F3: 1.6 × 107 conidia/kg) alone or in combination for five storage periods, from 0 to 120 days after treatment. For each developmental stage, within each treatment, means followed by the same lowercase letter are not significantly different (in all cases DF = 4, 44, Tukey–Kramer test at p = 0.05). For each developmental stage, within each storage period, means followed by the same uppercase letter are not significantly different (in all cases DF = 6, 62, Tukey–Kramer test at p = 0.05).
Table 4. Mean mortality (% ± SE) of Tribolium castaneum larvae and adults exposed for 7 days on wheat treated with DEA (50 ppm) and Beauveria bassiana (F1: 1.6 × 105; F2: 1.6 × 106; F3: 1.6 × 107 conidia/kg) alone or in combination for five storage periods, from 0 to 120 days after treatment. For each developmental stage, within each treatment, means followed by the same lowercase letter are not significantly different (in all cases DF = 4, 44, Tukey–Kramer test at p = 0.05). For each developmental stage, within each storage period, means followed by the same uppercase letter are not significantly different (in all cases DF = 6, 62, Tukey–Kramer test at p = 0.05).
Host StageTreatmentStorage Period
0 Days30 Days60 Days90 Days120 DaysFp
LarvaeDEA71.1 ± 1.4 Ca66.8 ± 1.4 Da54.4 ± 1.1 Db42.2 ± 1.1 Cc36.6 ± 2.1 Dc100.0˂0.01
F143.3 ± 1.5 Fa36.3 ± 1.6 Fb29.7 ± 1.4 Fc18.3 ± 1.0 Ed13.8 ± 0.6 Fd90.9˂0.01
F248.6 ± 1.3 Ea41.5 ± 0.9 Fb33.4 ± 1.2 Fc24.5 ± 1.1 Ed19.6 ± 1.6 EFd102.1˂0.01
F363.7 ± 1.5 Da56.4 ± 1.8 Eb43.7 ± 1.1 Ec30.8 ± 1.3 Cd25.1 ± 1.4 Ed130.0˂0.01
DEA + F188.5 ± 1.4 Ba79.5 ± 1.5 Cb67.7 ± 1.0 Cc53.1 ± 1.1 Bd43.8 ± 1.0 Ce226.5˂0.01
DEA + F2100.0 ± 0.0 Aa91.3 ± 1.1 Bb74.8 ± 1.3 Bc57.8 ± 1.4 Bd51.7 ± 1.2 Be343.4˂0.01
DEA + F3100.0 ± 0.0 Aa100.0 ± 0.0 Aa83.8 ± 1.7 Ab64.8 ± 1.1 Ac58.7 ± 1.5 Ad275.0˂0.01
F371.0330.0263.2242.0140.0--
p˂0.01˂0.01˂0.01˂0.01˂0.01--
AdultsDEA62.9 ± 1.0 Da54.3 ± 1.4 Db50.7 ± 1.7 Cb39.9 ± 1.5 Cc28.3 ± 1.2 Cd92.2˂0.01
F135.5 ± 2.2 Fa30.0 ± 1.5 Fa22.9 ± 1.5 Eb17.7 ± 1.0 Eb9.5 ± 0.9 Fc59.1˂0.01
F240.9 ± 1.6 Fa33.3 ± 1.5 Fb25.5 ± 1.4 Ec21.4 ± 1.4 Ec15.2 ± 1.0 Ed52.0˂0.01
F352.7 ± 1.6 Ea45.5 ± 1.3 Eb34.8 ± 1.9 Dc28.7 ± 1.1 Dd21.4 ± 1.1 De78.7˂0.01
DEA + F181.8 ± 1.7 Ca72.4 ± 1.6 Cb61.3 ± 1.9 Bc49.3 ± 1.2 Bd40.2 ± 1.8 Be103.0˂0.01
DEA + F292.8 ± 1.4 Ba84.7 ± 1.4 Bb70.9 ± 1.6 Ac54.4 ± 1.4 Bd47.6 ± 1.4 Ae176.0˂0.01
DEA + F3100 ± 0.0 Aa92.8 ± 2.0 Ab78.2 ± 1.9 Ac61.2 ± 1.5 Ad49.7 ± 1.4 Ae190.0˂0.01
F288.0261.3169.0190.0150.0--
p˂0.01˂0.01˂0.01˂0.01˂0.01--
Table
Table 5. Mean mortality (% ± SE) of Tribolium castaneum larvae and adults exposed for 14 days on wheat treated with DEA (50 ppm) and Beauveria bassiana (F1: 1.6 × 105; F2: 1.6 × 106; F3: 1.6 × 107 conidia/kg) alone or in combination for five storage periods, from 0 to 120 days after treatment. For each developmental stage, within each treatment, means followed by the same lowercase letter are not significantly different (in all cases DF = 4, 44, Tukey–Kramer test at p = 0.05). For each developmental stage, within each storage period, means followed by the same uppercase letter are not significantly different (in all cases DF = 6, 62, Tukey–Kramer test at p = 0.05).
Table 5. Mean mortality (% ± SE) of Tribolium castaneum larvae and adults exposed for 14 days on wheat treated with DEA (50 ppm) and Beauveria bassiana (F1: 1.6 × 105; F2: 1.6 × 106; F3: 1.6 × 107 conidia/kg) alone or in combination for five storage periods, from 0 to 120 days after treatment. For each developmental stage, within each treatment, means followed by the same lowercase letter are not significantly different (in all cases DF = 4, 44, Tukey–Kramer test at p = 0.05). For each developmental stage, within each storage period, means followed by the same uppercase letter are not significantly different (in all cases DF = 6, 62, Tukey–Kramer test at p = 0.05).
Host StageTreatmentStorage Period
0 Days30 Days60 Days90 Days120 DaysFp
LarvaeDEA92.3 ± 1.1 Ba87.3 ± 1.4 Ba80.7 ± 1.1 Cb66.6 ± 1.4 Dc54.2 ± 1.5 Dd141.0˂0.01
F165.5 ± 1.4 Da58.5 ± 1.0 Eb51.6 ± 1.1 Fc34.9 ± 1.3 Fd28.4 ± 1.1 Fe173.2˂0.01
F278.7 ± 1.0 Ca69.5 ± 1.2 Db56.3 ± 1.1 Ec41.7 ± 1.1 Ed35.9 ± 1.5 Ee235.5˂0.01
F389.3 ± 1.0 Ba78.7 ± 1.1 Cb61.3 ± 1.0 Dc53.3 ± 1.1 Ed41.8 ± 1.6 Ed303.0˂0.01
DEA + F1100.0 ± 0.0 Aa100.0 ± 0.0 Aa86.7 ± 1.2 Bb73.5 ± 1.3 Cc65.7 ± 1.5 Cd232.4˂0.01
DEA + F2100.0 ± 0.0 Aa100.0 ± 0.0 Aa100.0 ± 0.0 Aa89.4 ± 1.1 Bb76.5 ± 1.9 Bc114.0˂0.01
DEA + F3100.0 ± 0.0 Aa100.0 ± 0.0 Aa100.0 ± 0.0 Aa96.1 ± 1.2 Ab87.3 ± 1.6 Ac37.4˂0.01
F241.0352.0537.1379.3201.0--
p˂0.01˂0.01˂0.01˂0.01˂0.01--
AdultsDEA90.3 ± 1.0 Ba81.3 ± 1.5 Cb69.3 ± 1.3 Cc56.5 ± 1.4 Cd45.67 ± 1.4 De183.0˂0.01
F159.2 ± 1.4 Ea55.3 ± 1.1 Fa47.6 ± 1.1 Eb33.9 ± 1.2 Ec24.86 ± 1.0 Gd148.0˂0.01
F267.6 ± 1.5 Da62.8 ± 1.3 Eb51.7 ± 1.4 DEc37.5 ± 0.9 Ed31.73 ± 0.9 Fe180.0˂0.01
F380.5 ± 1.3 Ca69.2 ± 1.3 Db55.8 ± 1.3 Dc46.4 ± 1.5 Dd38.22 ± 1.3 Ee162.0˂0.01
DEA + F1100.0 ± 0.0 Aa93.8 ± 1.3 Bb76.2 ± 2.0 Bc64.2 ± 1.6 Bd52.49 ± 1.6 Ce204.0˂0.01
DEA + F2100.0 ± 0.0 Aa100.0 ± 0.0 Aa89.6 ± 1.2 Ab77.7 ± 1.9 Ac61.18 ± 1.5 Bd190.0˂0.01
DEA + F3100.0 ± 0.0 Aa100.0 ± 0.0 Aa95.4 ± 1.0 Ab83.4 ± 1.3 Ac70.52 ± 1.5 Ad165.0˂0.01
F287.0269.0195.0185.0161.0--
p˂0.01˂0.01˂0.01˂0.01˂0.01--
Table
Table 6. Mean number (± SE) of Tribolium castaneum individuals per vial, after a 14-day exposure interval on wheat grains treated with DEA (50 ppm) and Beauveria bassiana (F1: 1.6 × 105; F2: 1.6 × 106; F3: 1.6 × 107 conidia/kg) alone or in combination, and control for five storage periods, from 0 to 120 days after treatment. Within each row, means followed by the same lowercase letter are not significantly different (in all cases DF = 4, 44, Tukey–Kramer test at p = 0.05). Within each column, means followed by the same uppercase letter are not significantly different (in all cases DF = 7, 71, Tukey–Kramer test at p = 0.05.
Table 6. Mean number (± SE) of Tribolium castaneum individuals per vial, after a 14-day exposure interval on wheat grains treated with DEA (50 ppm) and Beauveria bassiana (F1: 1.6 × 105; F2: 1.6 × 106; F3: 1.6 × 107 conidia/kg) alone or in combination, and control for five storage periods, from 0 to 120 days after treatment. Within each row, means followed by the same lowercase letter are not significantly different (in all cases DF = 4, 44, Tukey–Kramer test at p = 0.05). Within each column, means followed by the same uppercase letter are not significantly different (in all cases DF = 7, 71, Tukey–Kramer test at p = 0.05.
TreatmentStorage Period
0 Days30 Days60 Days90 Days120 DaysFp
DEA5.4 ± 0.4 Ce8.7 ± 0.5 Ed17.5 ± 0.7 Dc24.3 ± 0.6 Db31.5 ± 1.3 Da223.0˂0.01
F118.3 ± 0.7 Be23.1 ± 0.8 Bd31.4 ± 0.9 Bc38.6 ± 1.2 Bb46.6 ± 1.0 Ba154.0˂0.01
F215.7 ± 0.7 Bd17.5 ± 0.7 Cd23.5 ± 0.9 Cc32.6 ± 1.2 Cb37.7 ± 1.1 Ca107.0˂0.01
F37.4 ± 0.5 Ce13.3 ± 0.7 Dd19.1 ± 0.8 Dc27.7 ± 0.8 Db34.4 ± 1.1 CDa182.0˂0.01
DEA + F11.2 ± 0.3 Ed3.5 ± 0.4 Fd11.8 ± 0.7 Ec18.3 ± 0.9 Eb23.6 ± 0.8 Ea239.0˂0.01
DEA + F20.0 ± 0.0 De2.6 ± 0.3 FGd7.6 ± 0.4 Fc10.4 ± 0.7 Fb15.4 ± 0.8 Fa133.0˂0.01
DEA + F30.0 ± 0.0 Dd0.0 ± 0.0 Gd2.2 ± 0.4 Gc6.5 ± 0.6 Fb10.7 ± 0.7 Ga96.9˂0.01
Control84.6 ± 1.9 Ab89.5 ± 1.5 Aab91.7 ± 1.3 Aa87.5 ± 1.3 Aab86.8 ± 0.9 Aab3.68˂0.01
F1282.01586.01216.0717.0635.0--
p˂0.01˂0.01˂0.01˂0.01˂0.01--
Table
Table 7. ANOVA parameters of main effects and associated interactions for mycosis (total DF = 539) and sporulation (total DF = 539) of Beauveria bassiana on larvae and adults of Tribolium castaneum on wheat.
Table 7. ANOVA parameters of main effects and associated interactions for mycosis (total DF = 539) and sporulation (total DF = 539) of Beauveria bassiana on larvae and adults of Tribolium castaneum on wheat.
EffectMycosisSporulation
DFFpDFFp
Treatment52237.8<0.0158115.4<0.01
Developmental stage13976.2<0.0112174.4<0.01
Storage period43245.1<0.0146610.5<0.01
Treatment × developmental stage51582.1<0.0155.4<0.01
Treatment × storage period2017.0<0.0120315.5<0.01
Developmental stage × storage period428.8<0.014163.8<0.01
Treatment × developmental stage × storage period207.7<0.0120235.5<0.01
Table
Table 8. Mycosis (% ± SE) in cadavers of larvae and adults of Tribolium castaneum treated with Beauveria bassiana (F1: 1.6 × 105; F2: 1.6 × 106; F3: 1.6 × 107 conidia/kg) and DEA (50 ppm) alone or in combination for five storage periods, from 0 to 120 days after treatment. For each developmental stage, within each treatment, means followed by the same lowercase letter are not significantly different (in all cases DF = 4, 44, Tukey–Kramer test at p = 0.05). For each developmental stage, within each storage period, means followed by the same uppercase letter are not significantly different (in all cases DF = 5, 53, Tukey–Kramer test at p = 0.05).
Table 8. Mycosis (% ± SE) in cadavers of larvae and adults of Tribolium castaneum treated with Beauveria bassiana (F1: 1.6 × 105; F2: 1.6 × 106; F3: 1.6 × 107 conidia/kg) and DEA (50 ppm) alone or in combination for five storage periods, from 0 to 120 days after treatment. For each developmental stage, within each treatment, means followed by the same lowercase letter are not significantly different (in all cases DF = 4, 44, Tukey–Kramer test at p = 0.05). For each developmental stage, within each storage period, means followed by the same uppercase letter are not significantly different (in all cases DF = 5, 53, Tukey–Kramer test at p = 0.05).
Host StageTreatmentStorage Period
0 Days30 Days60 Days90 Days120 DaysFp
LarvaeF198.3 ± 0.7 Aa95.5 ± 0.7 Ab90.3 ± 1.5 Ac84.2 ± 1.6 Ad76.4 ± 1.0 Ae190.3˂0.01
F292.4 ± 1.0 Ba88.1 ± 1.2 Bb82.4 ± 1.6 Bc74.5 ± 1.4 Bd67.4 ± 0.7 Be233.7˂0.01
F385.7 ± 1.3 Ca80.7 ± 1.2 Cb73.4 ± 1.7 Cc66.5 ± 1.7 Cd59.6 ± 1.6 Ce263.2˂0.01
DEA + F175.7 ± 1.7 Da71.3 ± 1.7 Db63.7 ± 2.1 Dc50.6 ± 1.4 Dd34.5 ± 1.4 De715.0˂0.01
DEA + F269.5 ± 1.7 Ea64.6 ± 1.6 Eb55.3 ± 1.4 Ec43.1 ± 1.6 Ed31.3 ± 1.1 Ee563.5˂0.01
DEA + F361.2 ± 1. 5 Fa54.6 ± 1.7 Fb43.6 ± 1.7 Fc32.7 ± 1.5 Fd20.4 ± 1.5 Fe719.0˂0.01
F431.0488.2775.01131.41300.0--
p˂0.01˂0.01˂0.01˂0.01˂0.01--
AdultsF191.5 ± 1.3 Fa87.3 ± 1.2 Fa81.2 ± 0.8 Fb78.5 ± 0.6 Fc69.33 ± 0.4 Fd352.0˂0.01
F287.3 ± 1.2 Aa82.8 ± 2.1 Aa74.5 ± 1.7 Ab65.7 ± 1.1 Ac54.8 ± 1.3 Ad74.4˂0.01
F381.5 ± 0.8 Ba76.6 ± 1.6 Bb67.8 ± 1.0 Bc59.2 ± 0.9 Bd48.3 ± 0.6 Be161.0˂0.01
DEA + F172.8 ± 0.7 Ca67.8 ± 0.9 Cb56.1 ± 1.1 Cc47.5 ± 0.9 Cd30.5 ± 0.7 Ce368.0˂0.01
DEA + F266.3 ± 1.0 Da60.4 ± 1.1 Db49.5 ± 0.9 Dc40.1 ± 0.7 Dd26.3 ± 0.9 De304.0˂0.01
DEA + F359.6 ± 1.1 Ea51.3 ± 0.9 Eb38.5 ± 0.9 Ec29.4 ± 0.8 Ed16.6 ± 0.9 Ee317.0˂0.01
F185.0112.0226.1431.0449.0--
p˂0.01˂0.01˂0.01˂0.01˂0.01--
Table
Table 9. Sporulation (conidia/mL ± SE) of cadavers of larvae and adults of Tribolilum castaneum treated with Beauveria bassiana (F1: 1.6 × 105; F2: 1.6 × 106; F3: 1.6 × 107 conidia/kg) and DEA (50 ppm) alone or in combination for five storage periods, from 0 to 120 days after treatment. For each developmental stage, within each treatment, means followed by the same lowercase letter are not significantly different (in all cases DF = 4, 44, Tukey–Kramer test at p = 0.05). For each developmental stage, within each storage period, means followed by the same uppercase letter are not significantly different (in all cases DF = 5, 53, Tukey–Kramer test at p = 0.05).
Table 9. Sporulation (conidia/mL ± SE) of cadavers of larvae and adults of Tribolilum castaneum treated with Beauveria bassiana (F1: 1.6 × 105; F2: 1.6 × 106; F3: 1.6 × 107 conidia/kg) and DEA (50 ppm) alone or in combination for five storage periods, from 0 to 120 days after treatment. For each developmental stage, within each treatment, means followed by the same lowercase letter are not significantly different (in all cases DF = 4, 44, Tukey–Kramer test at p = 0.05). For each developmental stage, within each storage period, means followed by the same uppercase letter are not significantly different (in all cases DF = 5, 53, Tukey–Kramer test at p = 0.05).
Host StageTreatmentStorage Period
0 Days30 Days60 Days90 Days120 DaysFp
LarvaeF1301.5 ± 1.3 Aa291.2 ± 1.3 Ba270.3 ± 1.0 Ca242.4 ± 1.1 Da213.33 ± 1.1 Ea979.0˂0.01
F2292.4 ± 1.2 Ab280.2 ± 1.2 Bb261.4 ± 1.1 Cb229.1 ± 1.2 Db198.8 ± 1.1 Eb1091.2˂0.01
F3275.3 ± 1.3 Ac264.2 ± 1.2 Bc248.5 ± 1.1 Cc218.4 ± 1.1 Dc186.4 ± 1.2 Ec937.5˂0.01
DEA + F1223.2 ± 1.3 Ad212.7 ± 1.2 Bd201.3 ± 1.1 Cd184.5 ± 1.1 Dd160.2 ± 1.1 Ed543.0˂0.01
DEA + F2208.8 ± 0.9 Ae199.3 ± 1.1 Be186.7 ± 1.1 Ce171.2 ± 1.3 De152.1 ± 1.2 Ee423.0˂0.01
DEA + F3195.1 ± 1.1 Af187.2 ± 1.0 Bf173.1 ± 1.4 Cf158.7 ± 1.0 Df129.7 ± 1.2 Ef493.1˂0.01
F1476.31490.01320.0890.0761.0--
p˂0.01˂0.01˂0.01˂0.01˂0.01--
AdultsF1287.8 ± 2.0 Aa279.7 ± 1.7 Ab251.1 ± 1.2 Bc229.6 ± 1.2 Ad194.8±1.7 Ae576.1˂0.01
F2284.7 ± 1.6 Aa267.8 ± 1.9 Bb232.6 ± 1.3 Cc216.7 ± 1.2 Bd164.2±1.9 Be892.0˂0.01
F3260.8 ± 1.8 Ba252.1 ± 1.4 Cb225.5 ± 1.4 Dc202.2 ± 1.4 Cd159.3±1.6 Be720.3˂0.01
DEA + F1212.1 ± 1.3 Cb203.4 ± 1.0 Fe186.5 ± 1.4 Aa161.2 ± 1.4 Dc142.8±1.3 Cd2918.6˂0.01
DEA + F2201.4 ± 1.5 Da182.1 ± 1.8 Db170.1 ± 1.2 Ec154.5 ± 1.6 Ed128.6±1.4 De336.0˂0.01
DEA + F3186.2 ± 2.2 Ea173.6 ± 1.8 Eb154.3 ± 1.6 Fc143.7 ± 1.5 Fd114.6±1.3 Ee263.0˂0.01
F641.01761.01358.0684.0340.0--
p˂0.01˂0.01˂0.01˂0.01˂0.01--
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Wakil, W.; Kavallieratos, N.G.; Nika, E.P.; Riasat, T.; Ghazanfar, M.U.; Rasool, K.G.; Husain, M.; Aldawood, A.S. Entomopathogenic Fungus and Enhanced Diatomaceous Earth: The Sustainable Lethal Combination against Tribolium castaneum. Sustainability 2023, 15, 4403. https://doi.org/10.3390/su15054403

AMA Style

Wakil W, Kavallieratos NG, Nika EP, Riasat T, Ghazanfar MU, Rasool KG, Husain M, Aldawood AS. Entomopathogenic Fungus and Enhanced Diatomaceous Earth: The Sustainable Lethal Combination against Tribolium castaneum. Sustainability. 2023; 15(5):4403. https://doi.org/10.3390/su15054403

Chicago/Turabian Style

Wakil, Waqas, Nickolas G. Kavallieratos, Erifili P. Nika, Tahira Riasat, Muhammad Usman Ghazanfar, Khawaja G. Rasool, Mureed Husain, and Abdulrahman S. Aldawood. 2023. "Entomopathogenic Fungus and Enhanced Diatomaceous Earth: The Sustainable Lethal Combination against Tribolium castaneum" Sustainability 15, no. 5: 4403. https://doi.org/10.3390/su15054403

APA Style

Wakil, W., Kavallieratos, N. G., Nika, E. P., Riasat, T., Ghazanfar, M. U., Rasool, K. G., Husain, M., & Aldawood, A. S. (2023). Entomopathogenic Fungus and Enhanced Diatomaceous Earth: The Sustainable Lethal Combination against Tribolium castaneum. Sustainability, 15(5), 4403. https://doi.org/10.3390/su15054403

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