Aptness of entomogenus fungi with diatomaceous earth against various stored grain insect pests

Background: Numerous biotic and abiotic factors are responsible for losses of grains quality and quantity during storage. Insecticides used to control stored grain insect pests are not only hazardous to mammals and environment but also induce resistance in insect pests towards these synthetic chemicals. A current trial was conducted, during 2020, in a stored grain laboratory at the College of Agriculture, BZU, Bahadur Sub Campus, Layyah, Punjab, Pakistan. A diatomaceous earth (DE) formulation enhanced with bitterbarkomycin (DEBBM) and combined with Beauveria bassiana (Balsamo) Vuillemin was evaluated against Cryptolestes ferrugineus, (Stephens) (Coleoptera: Laemophloeidae), Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) and Tribolium castaneum (Coleoptera: Tenebrionidae) under laboratory conditions. Results: DEBBM was applied at the rate of 50 mg/kg, DE (150 mg/kg) and Beauveria bassiana (1.5 × 10 and 1.5 × 10 conidia/kg) alone as well as in combination with wheat, rice and maize. Treated adult mortality was recorded at 7, 14 and 21 days after exposure. Results of the current study showed that insect pest mortality was maximum in the case of combined application of DEBBM with B. bassiana (high concentration) at prolonged exposure time as compared to their alone application. Mortality of C. ferrugineus was maximum in wheat and rice (100%) over maize (97%), while, R. dominica exhibited high mortality in wheat (100%) followed by rice (97%) and maize (94%) at combined application of DEBBM with B. bassiana (high concentration) after 21 days. Regarding T. castaneum mortality was high in wheat (100%) followed by rice (93%) and maize (88%) in case of combined application of DEBBM with B. bassiana (high concentration) at prolonged exposure time (21 days). Conclusion: In crux, the current trial showed that a mixture of DEBBM and B. bassiana is helpful in controlling tested


Background
Wheat (Triticum aestivum L.) (Family: Poaceae) is considered as a chief staple food among all cereal grains and Pakistan stands production and consumption wise at the 8th level (Shuaib et al. 2007). Wheat covers 37% of the total cultivated area in the country (Jilani 2007). Rice is the second chief food of Pakistan that covers 11% of the food production area and is cultivated on more than 2.5 million hectares (Chaudhry 1994). Maize (Zea mays L.) is also considered a food crop after wheat and rice. The Annual cultivation of maize is more than one million hectares (GOP 2003).
Page 2 of 6 Hanif et al. Egyptian Journal of Biological Pest Control (2022) 32:14 Rhyzopertha dominica (F.) (Coleoptera: Bostrichidae) female lays eggs externally on wheat kernel, while, larvae feed and pupate inside the kernel. (Athanassiou et al. 2011). Cryptolestes ferrugineus (Stephens) (Coleoptera: Laemophloeidae) is primary stored grain pest which attack germs of seed. This insect has white color having size 3 mm in length (Mason 2003). Tribolium castaneum (Coleoptera: Tenebrionidae) is secondary pest of stored grain. Both larval and adult stages cause damage and complete life cycle on grain (Karunakaran et al. 2004).
Numerous biotic factors are responsible for losses of grains quality and quantity during storage (Ahmed et al. 2008). Therefore, grain storage losses due to insects may increase 10-20% in Pakistan (Khan et al. 2010). In extreme conditions, losses may occur up to 25.5% (Irshad et al. 1988). Stored grains or products are attacked by the most important 23 species and 75% from all these species are belong to only one order Coleoptera (Viñuela et al. 1993).
For the preservation of stored grains since the 1950s, synthetic residual insecticides have been used to control stored grain insect pests. Continuous use of these insecticides had made the insect pest resistant to these chemicals. Additionally, these chemicals are also toxic to animal and hazardous to the environment (Pacheco et al. 1990;Arthur 1996). Now a days the use of new chemistry products is prevailing all over the world. Because these have lower mammalian toxicity and can control the insects effectively. Different insect pest of field crops can be effectively controlled by the use of bio-nicotine pesticides (Ye et al. 2014).
Fungi are considered biocontrol agents against insects and produce spores that cause insect mortality by penetration into the body of insects (Goettel et al. 2005). B. bassiana can be used as a component in integrated pest management (IPM). For birds, plants and mammals B. bassiana is considered ecologically non-toxic (El-Sharabasy 2015). B. bassiana is found in soil and decaying plant debris. Its conidia penetrate the body of the insect, by stimulating its growth and it generates a strong contact with the body layer of the host insect (Holder and Keyhani 2005). So, the fungus is a good choice in IPM, as insects have gained resistance against chemicals and other substances. The B. bassiana is tested against various pests on cereal crops as a biological agent (Bounechada and Doumandji 2004). B. bassiana has the advantage that it controls diseases by disease causing vectors of various arthropods and serves as entomopathogenic fungus (Stricker et al. 2006). Different scientists worked on the mutual effect of DE and entomopathogenic fungus , or chemical insecticides and entomopathogenic fungus (Srivastava et al. 2009), DE combined with various chemical insecticides (Kavallieratos et al. 2009).
In current study, the efficacy of certain control agents that affected by different parameters like concentration rate, insect species, grain type, time for storage and application method of combined form was studied.

Methods
The current study was conducted at the College of Agriculture, Bahauddin Zakariya University, Bahadur Sub-Campus Layyah Punjab Pakistan. DE, DEBBM and fungus (Beauveria bassiana) with different concentrations against Cryptolestes ferruginous, Rhyzopertha dominica and Tribolium castaneum.

Collection of insects
Insect species (C. ferruginous, R. dominica and T. castaneum) were collected from different godowns of stored grains in district Layyah.

Grain commodities
The grains used in this trial were free of chemicals and pests infestation from Wheat (var. galaxy-2013), Rice (var. Basmati-2011) and Maize (var. DK-6142). By using the Dickey-John moisture meter we measured the percentage of moisture present in the grains. The moisture present in wheat grains was 10.95% and in rice was 9.97% and 11. 17% in maize.

Diatomaceous earth and DEBBM formulation
The diatomaceous earth (DE) formulation used in the tests was DEBBM (Diatom Research and Consulting Inc., Guelph, ON, Canada). The DE constituent, which is 90% of the formulation, is from the group of fresh water DEs and is composed of 89% amorphous SiO 2 ,4% Al 2 O 3 , 1.7% Fe 2 O 3 , 1.4% CaO, > 1% MgO + K 2 O and 3% H 2 O. The remaining 0.05% active ingredient is Chinese plant extract (bitter barkomycin). Formulation of DE (150 mg/ kg) and DEBBM (50 mg/kg) was used against test insect.

Collection of Beuveria bassiana
Pure culture of B. bassiana was taken from the first fungal culture bank of PIAS, University of the Punjab, Lahore, Pakistan. Where the pathogenicity of fungi was tested against C. ferruginous as described by Freeman et al. (1998) and Twizeyimana et al. (2013) in laboratory and culture was maintained in the Plant Pathology Laboratory of College of Agriculture, BZU, Bahadur campus Layyah.

Collection of spores of B. bassiana
One liter distilled water and 40 g PDA (Potato Dextrose Agar) was boiled in a flask till dissolved entirely. The solution was autoclaved at 120 °C for 30 min. Spores of fungi were inoculated on PDA medium aseptically, and kept for incubation at 26 ± 1 °C. On culture after five days Mycelium was developed. By using Whatman Filter paper No.1, the culture was filtered. Obtained mycelium grew on filter paper. In bioassay after 25 days, the conidia collected from this mycelium were used using Hemocytometer (Murali et al. 2012).

Mortality effect of B. bassiana, DE and DEBBM alone and combined against the tested insects
One kilo gram of each grain commodities viz. wheat, rice and maize were prepared and kept in plastic jars separately. Two concentrations of B. bassiana (1.5 × 10 8 and 1.5 × 10 10 conidia kg −1 of grains), with DE (150 mg/ kg and 50 mg/kg) and (50 mg/kg) of DEBBM alone or in combinations. One untreated lot was kept as control. Data were taken after 7, 14 and 21 days to check insect mortality. The corrected mortality was calculated by using Abbott's formula (1925).

Statistical analysis
The analysis was done with Minitab 13.2 (Minitab, 2002 Software Inc., Northampton, MA). The Tukey-Kramer (HSD) test at the 5% significance level was used to compare the means of adult mortality (Sokal and Rohlf 1995).

Results
Applied treatments along with their exposure time exhibited maximum mortality of C. ferrugineus on wheat, followed by maize. Mortality percentages were ranged between 45 and 72% where DE alone was applied, at the  (Table 1). The mortality of R. dominica was maximum on wheat as compared to maize and rice. In all cases, sole application of DE caused mortality between 39 and 67%, after 21 days of treatment, and DEBBM exhibited a high mortality between 50 and 84%. Application of DE and DEBBM with a high concentration of B. bassiana (1.5 × 10 10 conidia kg −1 of grains) exerted more impact on mortality as compared to their sole application. The combined application of DEBBM with B. bassiana (high concentration) exhibited high mortality (68-100%), while DE with a high concentration of B. bassiana showed mortality between 60 and 96% after 21 days of treatments (Table 2).
Maximum mortality of T. castaneum was assessed on wheat followed by rice and maize. Regarding alone treatment application, DE exhibited mortality between 32 and 61% and DEBBM showed mortality between 45 and 78%. Among all cases except one, B. bassiana application with its high concentration exhibited maximum mortality as compared to lower dose application. Maximum mortality rate (56-100%) was observed where DEBBM was applied with a high concentration of B. bassiana as compared to B. bassiana (high concentration) alone. DE application with B. bassiana (high concentration 1.5 × 10 10 conidia kg −1 of grains), after 21 days post treatment, showed mortality between 74 and 87%, while B. bassiana (high concentration) with DEBBM exhibited mortality between 88 and 100% (Table 3).   Overall, all the DEs have similar method of action (Korunić 2013). Therefore, the substances present in the viable DEs formulations reduced to observe efficacy changes among several formulations. In the present study, 2 DEs were tested because of their differences in composition: the Protect-It comprises silica-aerogel and the DEBBM formulation of DE is improved with the bitterbarkomycin (BBM). The DEBBM showed to be much successful as to Protect-It, at least at the lowest concentration of 50 ppm, contrary to all targeted insect species present in each product type. The efficiency of DEBBM to the R. dominica (Wakil et al. 2011), C. ferrugineus, T. castaneum andS. zeamais (Athanassiou et al. 2009) have been described previously. These insect dead bodies might be served as a proper substrate in the application of fungal growth. BBM retains such antifeedant features (Wu et al. 2017) consequently, boosting mutual action of these 2 ingredients are also armored with poisonous
The influence of combined treatments (B. bassiana + Biopesticide) may be accredited in a way that showed no negative impact regarding conidial germination, vegetative growth and B. bassiana conidiogenesis (Neves et al. 2001). Alizadeh et al. (2007) documented that biopesticide (high concentration) significantly impacted mortality when used with B. bassiana. Similarly, Sheeba et al. (2001) observed the maximum S. oryzae mortality in a case where B. bassiana (high concentration) was used in bioassay. A high dosage of B. bassiana and Biopesticide significantly enhanced mortality when used against C. ferrugineus and R. dominica. Various insecticides viz. pyrethroids (Athanassiou et al. 2004) and microbial sprays ) impacted mortality concerning their concentration.
The current study revealed that mortality is directly proportional to the prolonged exposure time. Results of current trials are also in accordance with previous studies (Wakil et al. 2012). Various factors viz. surface to volume ratio, concentrations, application time and exposure length impacts insect pest susceptibility (Mewis and Ulrichs 2001). Enhanced application of biopesticides increases the strength of B. bassiana, after 14 days, and impact is enhanced more with prolonged time of exposure against insect pests (Michalaki et al. 2007).

Conclusions
It was concluded that DEBBM along with B. bassiana high concentration showed maximum impact regarding to mortality of the tested insect pests. While the present trial signifies the outcomes only in-vitro studies. Moreover, future studies are essential as in-vivo environment for the expansion of an effective, non-hazardous and IPMcompatible control measure of the tested insect pests in stored commodities.