Effect of the Combined Use of Beauveria bassiana (Balsamo) Vuillemin and Diatomaceous Earth for the Control of Tribolium castaneum (Herbst) and Trogoderma granarium Everts

Muhammad Yasin1, Azhar Abbas Khan2*, Sana Rubab2 and Sumaira Maqsood3 1Deprtment of Entomology, The Islamia University, Bahawalpur, Pakistan. 2College of Agriculture, Bahauddin Zakariya University, Bahadur Sub Campus Layyah, Pakistan. 3Department of Environmental Sciences, Faculty of Mountain Agriculture and Environmental Sciences, Kohsar University, Murree, Pakistan. Article Information Received 20 November 2021 Revised 11 August 2022 Accepted 08 September 2022 Available online 05 January 2023 (early access)


INTRODUCTION
T he stored commodities are attacked by a number of primary and secondary insect pests which may cause 10-40% damage, and in cases of severe infestation this may exceed up to 50% (Upadhyay and Ahmad, 2011). Primary insect pests have capability to attack whole or unbroken kernels, while secondary pests attack only already damaged grains, frass and milled products. Among stored product insect pests, the khapra beetle Trogoderma granarium (Everts) (Coleoptera: Dermestidae) and the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) have been primarily found to be of economic importance that ravage stored products (Boyer et al., 2012). Trogoderma granarium is one of the most destructive insect pest and pest of quarantine importance globally which is considered as one of the 100 worst invasive species in the world (Athanassiou et al., 2018). The larvae are damaging stage which causes both quantitative and qualitative losses in stored cereals. Also, contaminated food with barbed hairs and molted skin of larvae pose serious threat to human health (Borzoui et al., 2015). Tribolium castaneum is another important cosmopolitan insect pest, both larvae and adult of this pest feed on grain frass and already damage or broken grains. Beetles secrete quinone excretions which are carcinogenic in nature (Karunakaran et al., 2004;Michalaki et al., 2006).
The control of these insect pests is currently based on two broad categories of insecticides: Residual chemical insecticides and fumigants. Injudicious use of these insecticides causes hazardous effects on human health and environment, most importantly the insect pests has developed resistance against these insecticides (Finkelman O n l i n e F i r s t A r t i c l e et al., 2002). These issues had lead researchers to evaluate alternative grain protectant methods which are compatible to environment and safer for human health, and leave no residues on grains. Insect pathogenic fungi are potent alternative to the conventional grain protectant, as they have low mammalian toxicity and effective against a variety of stored product insect pests (Moore et al., 2000;Lord, 2001). Several entomopathogenic fungi have been tested against stored product pests with contradictory results (Moore et al., 2000). Among these, Beauveria bassiana (Balsamo) Vuillemin (Deuteromycotina: Hyphomycetes) found to be very effective against stored grain insect pests and self-perpetuating (Lord, 2001(Lord, , 2005Akbar et al., 2004). The desiccant dusts, particularly diatomaceous earths (DEs) are another potential alternative to the chemical insecticides to control stored product pests (Subramanyam and Roesli, 2000;Arthur, 2004;. DEs can be removed easily from the grains, and have low mammalian toxicity (Korunic et al., 1996(Korunic et al., , 1998. Also, resistance is unlikely to develop against DEs, as their action is strictly mechanical and abrasive through which body fluid ooze out of the body and ultimately insect die due desiccation (Athanassiou et al., 2005;Vayias et al., 2006). Integrated use of both agents can be very effective and could be a mean of reducing application rates (Lord, 2001;Akbar et al., 2004;Vassilakos et al., 2006;Wakil et al., 2011). Akbar et al. (2004) reported that the combined application of B. bassiana and the DE Protect-It act synergistically against larvae of Tribolium castaneum. Similar findings were also reported by Wakil et al. (2011) for DEBBM and B. bassiana against Rhyzopertha dominica F. (Coleoptera: Bostrychidae). Additionally, both agents persist on the grains which may provide long term are persistent inertion against stored product insect pests (Wakil et al., 2011).
The present study was aimed to evaluate the insecticidal properties of B. bassiana and DE against Trogoderma granarium and Tribolium castaneum alone and through combined use of both substances. Additionally, the progeny emergence for both insects was also checked on the treated grains.

MATERIALS AND METHODS
Populations of both insects were collected from different grain storage facilities of Layyah, Punjab (Pakistan) and further colony was established in the IPM laboratory, College of Agriculture, BZU Bahadur Sub-Campus, Layyah. The adults of Tribolium castaneum were reared on wheat flour+5% brewery yeast at 27±2 °C and 65±5% r.h. Mass production of Trogoderma granarium was carried out on whole wheat and conditions were maintained at 30±2 °C, relative humidity of 65±5%, and an approximate photoperiod of 14:10 (L: D) h, as described by Seifi et al. (2015). The laboratory culture which was reared in the laboratory since 2014s served as control.
Enhanced DE formulation Inert-PMS ® and entomopathogenic fungi B. bassiana were taken from Microbial Control Laboratory, Dept. Entomology University of Agriculture, Faisalabad (Pakistan). The laboratory culture was sub-cultured on Potato Dextrose Agar (PDA) by incubating at 20±5 ᵒC and 70% r.h., and the conidia were harvested using a sterilized scalpel after 14 days. The conidia were suspended in sterile 0.05% Tween-80, enumerated with a haemocytometer and adjusted to achieve concentrations of 1×10 6 and 1×10 7 conidia/ml. Prior to the initiation of the tests, the germination rate of B. bassiana conidia was determined as > 90%.
Untreated, clean and uninfested grains with very little dockage wheat grains were taken from the market. The moisture contents of the grains prior to the experiment were 11% measured by moisture meter. Grains then kept for 7 days at 25 °C and 75% r.h. to equilibrate the moisture contents. The moisture contents measured by moisture meter after 7 days were 13.8%. Beauveria bassiana and Inert-PMS ® were applied at the dose rate of 1×10 6 and 1×10 7 conidia/ml, DE at the dose rate of 50 and 100 ppm alone and their possible combinations. For each treatment, lots of 1kg wheat grains were taken and treated with respective doses of fungi and DE. Jars were shaken manually for 5 min to achieve an equal distribution of the fungal spores and dusts on the entire grain mass. There was an additional untreated grain lot which served as a control.
Twenty larvae of Trogoderma granarium and Tribolium castaneum were introduced into each treated jar separately. The jars were set at 27±2 ºC and 65±5% r.h. in an incubator for the experimental period. The numbers of dead individuals were counted after 5, 10 and 15 days post exposure. After 15 days the boxes were set for next 50 days for progeny emergence.
Mortality for each treatment was corrected for control mortality using Abbott's formula (Abbott, 1925). The data was subjected to one way Analysis of Variance (ANOVA) in Statistix 8.1 using Tukey's Kramer test (HSD) at 1% significance level (Sokal and Rohlf, 1995).  (Table I).

RESULTS AND DISCUSSION
After 5 days of exposure significant difference in mortality of Trogoderma granarium was found among all the treatments tested. Highest mortality was recorded for combined treatments of B. bassiana and DE (1×10 7 conidia/ml + 100 ppm), while the lowest was recorded for B. bassiana (1×10 6 conidia/ml). After 10 days of exposure, also significant differences in mortality levels were recorded for all the treatments. 100% mortality was recorded in combined treatments of B. bassiana and DE (1×10 7 conidia/ml + 100 ppm). Similar trend in mortality was recorded after 10 days as at 5 days. After last count 100% mortality was recorded for highest dose rate of B. bassiana and both DE concentrations (1×10 7 conidia/ml + 50 ppm and 1×10 7 conidia/ml + 100 ppm).
After 5 days of exposure significant difference in mortality of Trogoderma granarium was found among all the treatments. Highest mortality (71.47±2.09) was recorded for combined treatments of B. bassiana and DE (1×10 7 conidia/ml + 100 ppm), while the lowest (4.91±0.63%) was recorded for B. bassiana (1×10 6 conidia/ ml). After 10 days of exposure also significant difference in mortality levels were recorded for all the treatments. Maximum (97.48±1.21%) mortality was recorded in combined treatments of B. bassiana and DE (1×10 7 conidia/ml + 100 ppm). After last count 100% mortality was recorded for highest dose rate of B. bassiana and DE (1×10 7 conidia/ml + 100 ppm).
Highest progeny emergence of Tribolium castaneum was recorded in control treatments, while the lowest was recorded for highest dose rate of B. bassiana and DE in combination (1×10 7 conidia/ml + 100 ppm) (Fig. 1). Highest progeny emergence of Trogoderma granarium was recorded in control treatments, while the lowest was recorded for highest dose rate of B. bassiana and DE in combination (1×10 7 conidia/ml + 100 ppm) (Fig. 1).
The findings of our study suggested that combined use of both B. bassiana and DE can be an effective mean to control Tribolium castaneum and Trogoderma granarium than their sole application. The main reason of enhanced effect is their physical mode of action, and most importantly no need to ingest to show effectiveness (Lord, 2001(Lord, , 2005. Lord (2001) first time reported the insecticidal effect using DE formulation Protect-It against R. dominica, and Oryzophilus surinamensis (L.) (Coleoptera: Silvanidae). Similar findings were also reported by Akbar et al. (2004) against larvae of Tribolium castaneum. Similar results were also observed by Batta (2004), who used both Metarhizium anisopliae (Metschinkoff) Sorokin (Deuteromycotina: Hyphomycetes) and dust composed of several inert ingredients such as oven ash and charcoal. He subjected that the addition of these ingredients, increased the efficacy of fungus against Sitophilus oryzae (Linnaeus) (Coleoptera: Curculionidae) and suggested that these materials show a desiccant action. It is also suggested that the addition of DE may remove epicuticular lipid layers from the insect body that help fungal conidia to germinated fungal conidia to enter more easily into the insect body (Lord, 2001).  Moreover, the degree of conidial attachment also increase when entomopathogenic fungi is integrated with DE. Akbar et al. (2004) reported more B. bassiana conidial attachment to the larvae of Tribolium castaneum when the fungus was applied with DE. Athanassiou and Steenberg (2007) declared that the application of B. bassiana alone is less effective and the toxicity increased when combined with PyriSec ® , SilicoSec ® and Insecto ® DE formulations. It is also manifest from the results that DE enhanced the toxicity of fungal isolates against Tribolium castaneum, R. dominica and O. surinamensis. This is in conformity with the results of Kavallieratos et al. (2006); that the accumulation of DE Protect-It synergized the efficiency of M. anisopliae. Keeping in view the synergistic effect of both substances several inert resources such as clays or silicas, have been used to enhance the conidial viability of the Metarhizium flavoviride, Gams and Rozsypal (Deuteromycotina: Hyphomycetes) (Moore et al., 1996;Moore and Higgins, 1997;Horaczek and Viernstein, 2004). Desiccant dusts are being used in the fungal formulations in order to dry conidia up to the levels of 4-5% r.h., which makes conidia robust and capable of long storage (Hedgecock et al., 1995;Moore and Higgins, 1997). From commercial production point of view there is increasing need to control the permanence of entomopathogenic fungal formulations (Moore et al., 2000) but the influence of these extracts on the insecticidal effect of the fungus has not been assessed in detail.
In our case presence of both fungus and DE gave higher mortality as compare to the application of fungus or DE, and increased with the increase in in doe rates of both fungal spores and DE. This fact may suggest that fungal conidia benefit the DE efficacy only when conidial concentration exceeds. In our case mortality was high because experiment was conducted on sound grains instead of wheat flour presence of flour particles decrease the attachment of DE particles on the insect's cuticle or remove from their bodies. Contrarily, Moore and Higgins (1997) suggested that the presence of the certain clays had a harmful effect on the germinating conidia of M. flavoviride but in our case a positive influence in conidial viability have occurred.

CONCLUSION
It is concluded that B. bassiana and DEs can be successfully used against stored product pests for longer period of time, and both agents can be a successful alternate to the conventional grain protectants, and could be an effective component of IPM in stored grains.

ACKNOWLEDGMENT
We are thankful to the Department of Entomology, University of Agriculture Faisalabad-Pakistan for providing DE and entomopathogenic fungi.

Funding
No funds was provided for this study.

Ethical statement
No animals were involved for this study.

O n l i n e F i r s t A r t i c l e
Beauveria bassiana and Diatomaceous Earth Tribolium castaneum and Trogoderma granarium O n l i n e