Efficacy of some entomopathogens against Ephestia cautella (Walker) (Lepidoptera: Pyralidae) in stored date fruits

Ephestia cautella (Walker) (Lepidoptera:Pyralidae) is one of the most economically important date fruits insect pests, which causes major losses in quantity and quality of the date yield. The present study aimed to evaluate 3 of the entomopathogens: Beauveria bassiana (Basonym 1836), Metarhizium anisopliae (Metchnikoff 1879), and Bacillus thuringiensis (Berliner 1915) var. kurstaki individually and mixed with each other against E. cautella in stored date fruits. Four concentrations from spores' suspension of each entomopathogen were sprayed on 50 eggs of E. cautella (24:48 h. old) (for fungal pathogens) and 30 2nd instar larvae of E cautella in glass jars. Pathogenicity was evaluated at different time intervals post treatments 3, 5,7and 14 days. Obtained results showed that mortality percentages of E cautella stages increased by increasing the tested entomopathogen concentrations and increasing of the exposure time. Results indicated that the reduction percentages of E. cautella eggs hatchability after separately treated at the concentrations (3.0 × 105, 3.0 × 106, 3.0 × 107 and 3.0 × 108 spores/ml) were (27.50, 39.17, 48.33 and 62.50%) for B. bassiana, (35.83, 44.17, 50.83 and 69.17%) for M. anisopliae, respectively. After14 days of treatment, the recorded larval mortalities were (49, 62, 77, and 81%) for B. bassiana and (60, 66, 78, and 89%) for M. anisopliae at concentrations (3.0 × 105, 3.0 × 106, 3.0 × 107and 3.0 × 108 spores/ml), respectively. In case of treating E. cautella larvae with B. thuringiensis, the mortality percentage was (44, 52, 63, and 72%) at concentrations (2.0 × 108, 2.0 × 109, 2.0 × 1010 and 2.0 × 1011 spores/ml), respectively, after14 days of exposure. E. cautella larvae were more susceptible to B. bassiana and M. anisopliae than the eggs. M. anisopliae was more effective than B. bassiana. The combination of the entomopathogens LC50 (B. bassiana + M. anisopliae, B. bassiana + B. thuringiensis, M. anisopliae + B. thuringiensis and B. bassiana + M. anisopliae + B. thuringiensis) caused E. cautella larval mortality percentages of 67.00, 73.33, 63.33 and 86.29%, respectively, 14 days after exposure. The combination of the 3 tested entomopathogens (B. bassiana + M. anisopliae + B. thuringiensis) increased their efficacy for controlling E. cautella. The combination of the tested entomopathogens: B. bassiana, M. anisopliae and B. thuringiensis could be recommended for controlling E. cautella stages in stored date fruits.


Background
The date palm has great importance as a source of food and heritage symbols, especially in the Arab world, which is characterized by the breadth of its area and variety of its climate, which helped within the spread of date palms cultivation in many areas of it. Egypt occupies the primary of date production within the world since 2001, accounting 21.5% of the world's date production. Several pests attack all parts of the date palms, causing great losses affecting the yield quantity, and quality of the date fruits. One of the most important and economic date fruits insect pests is Ephestia cautella Walker (Lepidoptera: Pyralidae).
Ephestia cautella was a major worldwide insect of stored products. It founds in tropical and temperate zones and commonly infests date fruit, grains, nuts, dried fruits, and many varieties of other stored products (Horak 1994). Larval stage was the harmful stage and its feeding pollutes stored products with feces and webbings that caused products spoiling. Also, the almond moth (fig moth) E. cautella was recorded in Siwa Oasis and the Governorate of New Valley in Egypt (Kashif et al. 2002) in Iraq (Hameed et al. 2011) and in Tunisia (Ben Jemaa et al. 2012). The larvae of E. cautella infest dry and semi-dry fallen date fruits moreover, stored dates during storage.
Many chemical insecticides and fumigates have been used as protectants against insect infestation in stored grains and fruits. But their indiscriminate use and residual toxicity affects the non-target animals and human beings. The sustainable alternative to pesticides is biological control which could give a share in reducing the use of synthetic insecticides. The promotion and use of entomopathogens as classical, conservation, biological control agents have achieved successes and a few failures in the past 15 years. The long-run directions of insect-specific viruses, bacteria, fungi, and nematodes as components of integrated pest management strategies for control of crops arthropod pests of forests, urban habitats, and insects of the medical and veterinary field. The entomopathogenic bacteria are commercially developed for controlling the lepidopteran larval pests as B. thuringiensis var. kurstaki. These pathogens merge the advantages of chemical pesticides and MCAs: they're fast-acting, inexpensive to make, cheap to combine, have a protracted-time period, and could be delivered using traditional application equipment and systems (Lacey et al. 2015).
Fungi are common natural entomopathogens that frequently cause epizootics in their hosts and they have several beneficial characteristics that support their growth as MCAs. Some species such as: B. bassiana and M. anisopliae var. anisopliae that controlled a numerous numbers of pests' species have been effectively utilized on a wide scale as microbial control agents (Srinivasan 2012). Commercialized microbial pesticides based on entomopathogenic fungi (EPF) are now primarily confined to niche markets. EPF control invertebrate pests, according to Lacy et al. (2001). The efficacy of 4 fungal isolates against Indian meal moths: B. bassiana, Verticillium lecanii (Balazy), M. anisopliae var. anisopliae, and Paecilomyces farinosus (Brown &Smith) was tested. All of the fungal isolates tested were pathogenic, but to varying degrees (Būda and Pečiulytė 2008).
The aim of the present work was to evaluate the efficacy of 3 entomopathogens, 2 fungi: M. anisopliae and B. bassiana, and the bacterium, B. thuringiensis separately and on combinations against the stored date fruit pest E. cautella.

Rearing of tested insect
The tested insect pest, E. cautella, was collected from infested date fruits and was reared at Date Palm Pests and Diseases Department, Central Laboratory of Date Palm, Agricultural Research Center, Giza, Egypt. Adult insects were reared on semi-dry date fruits Siwi cultivar. The date fruits used in rearing culture were conserved at the freezer for 2 weeks before using them to kill potential contamination with other pests. The 2 stages of E. cautella eggs and larvae were separately evaluated. All insect's cultures and experiments were conducted at 26 ± 2 °C and 65 ± 5% (RH), with 16 h of light and 8 h. of darkness (Abd El-Aziz et al. 2012).

Entomopathogens tests Isolation and identification of fungi
Two isolates of the EPF were isolated at Bio-insecticide Production Unit, Plant Protection Research Institute, Agricultural Research Center (ARC), Giza, Egypt. The first isolate, Beauveria Bassiana (AUMC 9808) was isolated from diseased larvae of the cotton leaf worm Spodoptera littoralis (Boisduval 1833) collected from a sugar beet field and was identified at the Mycological Center, Faculty of Science, Assiut University (Sahar and Moharram 2014). The second isolate, Metarhizium anisopliae was isolated from red palm weevil, Rhynchophorus ferrugineus (Olivier 1790). Eggs were obtained from the Department of Date Palm Pests and Diseases, Central Laboratory for Date Palm, ARC, Giza, Egypt, covered with fungi placed on Czapek-dox agar (CZA) medium and incubated at 27 ± 2 °C for 7-10 days to allow growth of infecting fungus. Pure slant culture of fungus was identified in Plant Pathology Research Institute, ARC, Giza, Egypt.

Fungi culture
The isolates were cultured on (CZA) medium with 1% yeast extract plates in several Petri dishes (9 cm in diameter), and were grown for 15 days at 27 ± 2 °C. The conidia were harvested by scraping the surface of 14-15 days old culture gently with inoculation needle. The conidia were suspended in distilled water containing 0.1% Tween-80. The mixture was stirred by a magnetic shaker for 10 min and then 4 concentrations of spore's suspension were prepared from each of the fungal isolates (3 × 10 5 , 3 × 10 6 , 3 × 10 7 and 3 × 10 8 spores/ml).

Culture of entomopathogenic bacterium
One strain of Bacillus thuringiensis subsp. Kurstaki (Btk) was obtained from the Bio-insecticide Production Unit, Plant Protection Research Institute ARC, Giza, Egypt. T3 medium which was composed of (tryptone 3.0 g, tryptose 2.0 g, yeast extract 1.5 g, MnCl2 0.005 g, and NaH2PO4. H2O 8.9 g, adjusted PH at 6.8), was prepared and the final volume was made up to 1 L with distilled water. The medium was sterilized at 121 °C for 20 min, and inoculation was occurred with the standard inoculums. The inoculated flask was incubated on a shaker (142 rpm) at 28 °C for 72 h. (Attathom et al. 1995). Four concentrations (2 × 10 8 , 2 × 10 9 , 2 × 10 10 , and 2 × 10 11 spores/ml) were prepared by plate count method.

Bioassay tests Efficacy of entomopathogenic fungi on eggs of E. cautella
Fifty eggs of E. cautella (24:48 h. old) were placed each into a small glass jar (200 ml volume), every jar was treated with the 4 concentrations of each fungal concentration (3 × 10 5 , 3 × 10 6 , 3 × 10 7 and 3 × 10 8 spores/ ml) then filled with about 50 g Siwi date fruits and covered with cloths fixed with rubber bands. The reduction percentage of eggs hatchability was calculated after treatments. Then the calculated LC50 values of each fungus were combined to study their effects on E. cautella eggs. Then the percentage reduction in egg hatching was calculated.

Efficacy of entomopathogenic fungi and bacterium on larvae of E. cautella
B. bassiana, M. anisopliae and B. thuringiensis were sprayed against 30 individuals of 2nd E. cautella larvae that placed into a glass container (200 ml volume). The larval instar was chosen before penetrating the date fruit and feeding on it from inside. Four concentrations of each fungus (3.0 × 10 5 , 3.0 × 10 6 , 3.0 × 10 7 and 3.0 × 10 8 spores/ml) and 4 as well of the bacterium B. thuringiensis (2.0 × 10 8 , 2.0 × 10 9 , 2.0 × 10 10 and 2.0 × 10 11 spores/ ml) were tested. Each pathogen was applied by 2 methods (single and mixed) against the date fruit pest E. cautella in stored date fruit under laboratory conditions of 26 ± 2 °C and 65 ± 5% R.H. The experiment was replicated 4 times. The number of dead larvae in each jar was counted on specific dates after 3, 5, 7, and 14 days from the treatments and the percentages of mortality were recorded.

Combination of the three entomopathogens
The LC 50 value of the tested fungus and bacterium was calculated, to study their mixing effects.

Statistical analysis
Mortality rates of insects were corrected using the Abbott formula (Abbott 1925) compared to the control (untreated). LC 50 and LC 90 were calculated through the probit analysis as described by Finney (1971). Comparison between the mortality percentages using the LC 50 values of the three tested pathogens combinations were analyzed by Proc., ANOVA in SAS (SAS Institute 2006).

Virulence of entomopathogens
Infections' symptoms of E. cautella stages with the tested entomopathogens were illustrated in (Fig. 1).

B. bassiana and M. anisopliae on E. cautella eggs
The reduction percentages of E. cautella egg hatchability after separately treated at the concentrations (3.0 × 10 5 , 3.0 × 10 6 , 3.0 × 10 7 and 3.0 × 10 8 spores/ml) were ( (Fig. 2). The results showed that M. anisopliae was more effective than B. bassiana, and the percentage reduction in egg hatchability increased by increasing the concentrations. The LC 50 values were 6.0 × 10 7 spores/ml for B. bassiana and 2.0 × 10 7 spores/ml for M. anisopliae at 14 days of treatment (Table 3). As compared to control, the percentage reduction in the egg hatching for mixed fungal treatment was 66.6%.

B. bassiana and M. anisopliae on E. cautella larvae
Efficacy of 4 concentrations of the 2 fungi against E. cautella larvae at 26 ± 2 °C, 65 ± 5% RH, and different exposure periods were presented in Table 1. The results showed that the percentage of corrected mortality of the tested larvae increased by increasing the concentrations and or exposure time. Larval mortality was recorded (49, 62, 77, and 81%) for B. bassiana and (60, 66, 78, and 89%) for M. anisopliae after 14 days of exposure to the concentrations (3.0 × 10 5 , 3.0 × 10 6 , 3.0 × 10 7 and 3.0 × 10 8 spores/ml), respectively. The results showed that the corrected mortality % of E. cautella larvae increased by increasing the concentrations and or the exposure time as it was recorded (44, 52, 63, and 72%) at concentrations (2.0 × 10 8 , 2.0 × 10 9 , 2.0 × 10 10 and 2.0 × 10 11 spores/ml), respectively, after14 days of exposure. The LC 50 for B. bassiana and M. anisopliae were 0.5 × 10 6 spores/ml and 1.2 × 10 5 spores/ml, respectively, after 14 days of treatment and was 1.7 × 10 9 for B. thuringiensis after 5 days of treatments (Table 3). The results showed clearly that E. cautella larvae were more susceptible than the eggs. The same trend was recorded at the LC 90 level for the 2 tested insect stages.

Combined effect of the 3 entomopathogens on E. cautella larvae
The  El Shafei et al. Egyptian Journal of Biological Pest Control (2022)

Discussion
Results of the present study showed that EPF and bacterium (Bt) had a potential effect on E. cautella larvae.
Use of more than one pathogen to control the E. cautella larvae showed higher effects than using each one separately. The results are in agreement with those obtained by Alwaneen et al. (2020) who mentioned that B. bassiana isolates were screened against different developmental stages of E. cautella. The developmental stages were directly exposed to each fungus isolate at a concentration of 1 × 10 7 conidia/ml. It was observed that all the fungus isolates affected the hatchability of eggs, causing significant larval and pupal mortality. Bahmani et al. (2020) investigated the effects of B. thuringiensis kurstaki bacteria and B. bassiana pathogenic fungi on the population dynamics of Ephestia kuehniella L., the most dominant pest of date stored in Khuzestan Province. It was mentioned that regarding the highest potential of B. bassiana and Btk in reducing the stock pest population, it is very possible to exploit this interaction for biocontrol. In another consensual study, B. bassiana caused a reduction in E. cautella egg hatching reached (15.3%) and (85.22%) larval mortality at the highest concentration of the