Evaluation of the Efficacy of Commercial and Local Entomopathogenic Fungi Metarhizium anisopliae Against Ephestia cautella (Lepidoptera: Pyralida) under Lab Conditions

The Ephestia cautella (Walk) (Lepidoptera: Pyralida) is a crucial insect of storage products worldwide. The experiments were conducted to assess Entomopathogenic Fungi (EPFs) (two isolates) with commercial formulations, Met 52 EC (Metarhizium anisopliae strain F52) and local entomopathogenic fungal isolates against Ephestia cautella. Commercial formulations of entomopathogenic fungal M. anisopliae affected the main mortality levels in the E.cautella adult at all three sporal concentrations (2×104, 2×106, 2×108 spore/ml), compared to the local isolates of M. anisopliae. Corrected mortality of E.cautella adult caused by both Commercial and local M. anisopliae 7 days after exposure at 2 × 108 spore/ml was 32±1.3 and 48 ± 1.4 %, respectively. 2nd, 5th instar larvae had a higher mortality level than the adults of E. cautella. The results reveal that Entomopathogenic Fungi isolated from the UK and Iraq can be applied as the microbial bio-agent control against E. castella. Consequently, more studies are required to develop commercial Entomopathogenic Fungi of naturally isolated.


Introduction
Ephestia castella (Lepidoptera: Pyralidae) is a worldwide stored products pest that causes damage to the date palm, figs, cereals, fruit and nuts [1].Ephestia castella are multicultural insects that distribute in temperate environments [2].The damage of E. cautella is due to the decreased quantity and quality of food goods and loss of marketing costs [3].Pesticides are the primary methods of controlling and decreasing the damage of insect pests, including E. castella.Though, the wide use of synthetic pesticides produced numerous problems, such as insect resistance to the pesticides, and mental pollution, with adverse effects on human health and other organisms [4][5][6].Reducing the chemical insecticide problems in agriculture has encouraged searching for the development of alternative pest control methods in stored insects [7,8].Many entomopathogenic fungal fungi (EPFs) have been assessed on the stored grain insects, with I. fumosorosea, B. bassiana, Lecanicillium spp, and M. anisopliae [9,10].These entomopathogenic fungi have been used as commercial bio agents control against insects [11,12].Additionally, [13]  that 50 products of microbial application are recognized worldwide and 3 myconisecticides only have been applied against stored grain insects.There is poor knowledge about the ability of many Pathogenic known strains against E.cautella in Iraq.Consequently, this study aimed to evaluate two commercial formulations of the fungal EPFs against different developmental stages of E. cautella under laboratory conditions.

Rearing Ephestia cautella
Ephestia cautella was picked from the Agricultural Research Dept. of the Ministry of Science and Technology and reared in the Entomology lab at the School of Agriculture/ University of Kerbala (2021-2022).Using available identification key, a microscope recognised the insects.The E. castella were reared on a synthetic diet consists crushed grain wheat 81%, glasieren 12%, honey date 6% and yeast 1%.Moths were preserved in 1 litre of glass jars, protected by a muslin cloth, then tied using rubber bands, besides preserved in the incubator at a temperature of 25±2 °C and 60-70% R.H all steps of performing procedures and animal handling and the study was approved by the Ethical Committee [Approval letter No. UoM.Dent/A.L.66/21].No. Um.VET.2021.5.

Effect of Spore Concentrations of a Commercial Formulation and Local Fungal Isolate of M. anisopliae Against Adults of Insect
Five Petri dishes replicate, each with 10 insects, was tested for either local fungal isolate or commercial formulation of M. anisopliae.A handheld sprayer sprayed each replicate with 2 ml sporal suspension (2×104, 2×106, 2×108 spore/ml).By sterile 0.001% aqueous Tween 80, the control treatments were sprayed.When drying at room temperature for 20 min, Petri dishes cantina sterilized crushed grain (5 g) wrapped by Parafilm and kept at 27±2 °C with 65±2% RH in the incubator.After 24 hours.New caps of lids have ( 1-cm) holes were enclosed by nylon mesh and then set in the incubator for seven days.Dead Moths were removed from dishes, and mortality was recorded for 1, 3, 5, and 7 days after treatment.With 70% of ethanol, the surface of dead Moths was sterilized for 60 seconds and cleaned via distilled water, and then set on a container (3 grams of agar/L of water) in the Petri dishes (9 cm) for five days in order to check the infection of fungi [14].Insect's dead was observed as having died after infection by the fungi used if spores were recovered.

Effect of Ephestia cautella Developmental Stages on the Efficacy of Local and Commercial Formulation of M. anisopliae
In this test, a sporal concentration of 2×10 8 spore/ml of both local fungal isolate or commercial formulation of M. anisopliae was selected based on the mortality level of Ephestia cautella in the multiple-dose bioassays.The adult stages of E. cautella with different instar larvae ( 2nd and 5 th ) were obtained from the Entomology lab, as [15] mentioned.The treatment was replicated 5 times.

Statistical Analysis
By Abbott formula, the mortality was corrected [16].All experiments were statistically evaluated by using GenStat (version 16).The efficacy of each local fungal isolate or a commercial formulation of M. anisopliae on the life stage of E. cautella was analyzed using two-factor frequent measurement analysis ANOVA.In addition, the effect of all treatments on E. cautella mortality was analysed individually using ANOVA using the test of LSD at 5% significance levels (P < 0.05).

Effect of Different Sporal Concentrations of Local and a Commercial Formulation of M. anisopliae Against E. cautella Adults
Adult mortality varied significantly among sporal concentration and fungal isolates (P < 0.05).For example, the mortality levels of local isolates of M. anisopliae at 2×10 8 spore/ml reached 48±1.4 %, compared to 32±1.3% for commercial formulations of the same fungus, respectively (Table 1).Furthermore, in all treatments, the time effect after fungi application on the levels of mortality was significant (P < 0.05), which was achieved 10 days post-treatment as the highest level (Table 1).These results were similar to those [17], who reported that EPFs B. bassiana and M. anisopliae gave high levels of mortality in the storage products insects, including E. cautella.[17] also informed that B. bassiana and M. anisopliae were given significant mortality in the adults of Tribolium castaneum.[18] showed that the difference in the effectiveness of fungi might result in their varied proteins comprising most insect body wall cuticles.Thus, the degrading goings-on of proteases might expression a part in the rapid diffusion in the cuticle of insects, which reflects the virulence of EPFs [19,20].A highly solid implication among the activity and virulence of M. anisopliae isolates against Trogoderma granarium [21].
Table 1.The effect of different sporal concentrations of local and commercial formulations of M. anisopliae, on the corrected mortality (±SE) of E. cautella after ( 1, 3, 5 and 7) days of treatments.

Effect of E.cautella Developmental Stages on the Efficacy of Local or Commercial Formulation of M.anisopliae
The experiment results presented that the efficacy of a local fungal isolate and a commercial formulation of M. anisopliae varied among E. cautella developmental stages, with a mortality rate of 100% for 2 nd instar larvae tested with both local isolate, and a commercial formulation of M. anisopliae fungi (Table 2).Likewise, mortality levels for fifth-instar larvae reached 16±1.6 to 93±1.1%.In addition, a significant difference was in EPFs M. anisopliae to each beetle stage (P <0.05).While the mortality ranged from 2-4% in the control.The results were similar to the study of [22], who stated that the increased mortality rate of the Khapra Beetle affected by M. anisopliae fungus reached 100% in the larvae stage and 83.9% in the adults.Therefore, [23] indicated that the difference in results of E. cautella treated by Met 52 EC may defined by biochemical variations over host cuticle growth or difference sporal gaining of E. cautella larvae (2 nd instars), compared to the adult stages.[24] reported high mortality of whitefly using Nanparctclase.However, more and more investigations are required to evaluate these issues.