Field evaluation of two entomopathogenic fungi; Beauveria bassiana and Metarhizium anisopliae as a biocontrol agent against the spiny bollworm, Earias insulana Boisduval (Lepidoptera: Noctuidae) on cotton plants

Background: The Earias spp. are devastating pests which reduce cotton yield up to 40% as seed cotton. Efficacy of Metarhizium anisopliae and Beauveria bassiana as bio-insecticides were investigated against the spiny bollworm, Earias insulana Boisduval (Lepidoptera: Noctuidae), under field conditions in Egypt throughout two successive seasons 2018 and 2019. Results: Results showed E. insulana population reduction during both seasons after bio-insecticidal applications. M. anisopliae strains (S1, S2) treatments showed significant reductions in the mean numbers of infested bolls with E. insulana after 14 days from the 1st spray. Reduction percentages of E. insulana infestation reached to 77.74 and 76.51% respectively, after application of M. anisopliae strains (S1, S2) treatments; then, the infestation reduction percentages increased to 88.48 and 85.41% after the 2nd spray by the same fungal strains; then, the infestation reduction percentages increased to 90.16 and 90.84% after 3rd spray of the same fungal strains in season 2018. In 2019 season, the infestation reduction percentages of E. insulana was 85.48 and 80.75%, after the 1st spray of M. anisopliae strains (S1, S2), respectively, which increased to 92.40 and 89.87%, after 14 days from the 2nd spray by the two fungal strains respectively, and then increased to 94.12 and 93.73%, after 14 days from the 3rd spray of M. anisopliae (S1 and S2) respectively. In season 2018, the infestation reduction percentage of E. insulana by B. bassiana strains (S1, S2) recorded 73.09 and 71.89%, respectively and 81.04 and 82.89% respectively, in season 2019, after 14 days of the 1st spray. While after the 2nd spray of the two tested B. bassiana strains, the infestation reduction percentage of E. insulana was 85.41 and 85.41% respectively, in season 2018, whereas it was 89.16 and 89.16% respectively, in season 2019. Then after 14 days of the 3rd spray by the same fungus strains, the reduction percentage of E. insulana increased to 86.56 and 85.35%, respectively, in season 2018, and 90.83 and 90.83% in season 2019. Conclusions: Tested strains of B. bassiana and M. anisopliae fungi proved their potential for decreasing E. insulana infestation percentages through 2–3 spray treatments under field conditions.


Background
Cotton (Gossypium barbadense) (Malvaceae) is the most important industrial crop worldwide (Johnson et al., 2014). In Egypt, cotton plants like most of the field crops are attacked by many lepidopterous pest species (Gaaboub et al., 2012). Insect pests are the most limiting factors that decrease cotton production and cause severe damages to the crop yield (El-Heneidy et al., 2015). The spiny bollworm, Earias insulana Boisduval (Lepidoptera: Noctuidae), the mid-late season pest, usually threats cotton plants in Egypt (El Hamaky et al., (1990). The damage of fruits (green bolls) is frequently more destruction than the other parts of the cotton plant. E. insulana and the pink bollworm, Pectinophora gossypiella (Saund.) (Lepidoptera: Gelechiidae), are the most destructive insect pests of cotton in Egypt (Amin and Gergis, 2006). Biopesticides based on bacteria, viruses, entomopathogenic fungi, and nematodes are often considerable scope as a plant protection agent against several insects. Many works extensively investigated the field bio-efficacy of the entomopathogenic fungi (EPF) such as Beauveria bassiana (Balsamo) Vuillemin (Deuteromycotina: Hyphomycetes) (Wraight et al., 2010).
The present study aimed to evaluate the potential of EPF, as bio-insecticides against the spiny bollworm, E. insulana, under field conditions. The present study aimed to evaluate the potential of EPF, as bio-insecticides against the spiny bollworm, E. insulana under field conditions.

Preparation of specific media
The isolates were cultured on Sabouraud dextrose agar medium g/l containing 40 g glucose, 20 g peptone, 20 g agar, 2 gm yeast extract, and 1000 ml of distilled water in flasks. These flasks were autoclaved at 21°C for 15 min.

Preparation of tested bioagent
The two sources were prepared as solutions as follows: Source 1 (S1): Fungal cultures were grown on Sabouraud dextrose agar medium (Dextrose 40gm, agar 20gr, peptone 10g)/1000 ml distilled water and incubated at 25±2°C in darkness for 14 days. Conidial suspensions were prepared by scraping cultures with a sterile objective glass and transferred to 10 ml of sterile water containing 0.05% Tween 80 in a laminar flow chamber. The mixture was stirred for 10 min the hyphal debris was removed by filtering the mixture through fine mesh sieve. The conidial concentration of final suspension was determined by direct count 1×10 8 using hemocytometer with final concentration of 1×10 8 spore/ml. Source 2 (S2): To prepare stock solution (1×10 8 ), spore/ml from the commercial bioagent was weighted, 5gram of powder, and dissolved in 900 ml sterile water.

Field application
Field study was conducted at Talaat-El Agamy experimental farm, henno, Kafr El-Sheikh, Egypt. Studies covered two successive cotton growing seasons 2018 and 2019. Cotton variety Giza 96 was cultivated on April 7, 2018, and on April 13, 2019 seasons. The experimental design was complete randomized block, the whole cultivated area (350 m 2 ) was divided into equally 5 plots each was divided to 3 replicates, each plot was treated by one of the tested 4 bioinsecticides strain individually with a concentration of 1×10 8 spore/ml)/l water. The last plot was left untreated as control. Cotton seeds were sown at 20-cm distance between hills. Two sources of the 2 EPF were applied against E. insulana on infested cotton green bolls when infestation reached 3% (the economic threshold). Spraying of the tested fungi took place on cotton plants 3 times on July, 8th, 22nd and 5th August, respectively through the 2 seasons. Fungal applications were carried out at 4 pm using a knapsack motor sprayer (20-l capacity). To evaluate the effect of the 4 EPF strains against E. insulana, samples of 25 bolls/plot were picked randomly before and 1 week after applications. Sampling continued weekly until harvest. The collected bolls were carefully dissected, and the numbers of larvae, infested bolls, and reduction percentages were recorded. The reduction percentages in the field experiment were calculated.

Statistical analysis
Data of E. insulana infestation after 7 and 14 days of treatments were statistically analyzed by one-way analysis of variance (ANOVA) (P < 0.05 %) Duncan's multiple range test of means (Duncan, 1955

Results
Effect of the tested entomopathogenic fungi on E. insulana under field conditions In the control plot, larval population increased gradually throughout all the inspection weeks in the 2 studied growing seasons (Tables 1 and 2). Data in Table 1 showed that applications of M. anisopliae (S1, S2) treatments significantly reduced the mean numbers of infested bolls with E. insulana after 7 days post the 1st spray with the reduction percentages of 60.69 and 51.74%, respectively, while 77.74 and 76.51% after 14 days from the 1st spray the reduction percentages increased to 88.37 and 61.89%, 7 days from the 2nd spray. The reduction % by M. anisopliae (S1, S2) applications attained 88.48 and 85.63%, respectively, after 14 days from the 2nd spray. Afterward, the percentages of infestation reduction reached 92.5 and 90.16% after 7 days from the 3rd spray: 90.13 and 90.84% after 14 days from the 3rd spray in season 2018.
The reduction percentage of E. insulana in case of B. bassiana (S1, S2) in season 2018 recorded 54.61 and 49.24%, respectively, after 7 days from the 1st spray, and 73.09 and 71.89%, respectively, after 14 days, while it was 85.09 and 83.57%, respectively, after 7 days from the 2nd spray, and 85.41 and 85.41%, respectively, after 14 days from the 2nd spray; then, the reduction percentage increased to 89.00 and 88.12%, respectively, after 7 days from the 3rd spray, and 86.56 and 85.35%, respectively, after 14 days from the 3rd spray.
Results in Table 2 clarified that application of M. anisopliae (S1, S2) treatments showed significant reductions in the mean numbers of infested bolls with E. insulana than the control after 7 days from the 1st spray in season 2019 and these percentages reached to 79.54 and 72.72%, respectively, and then increased to 85.48 and 80.75%, respectively, after 14 days from the 1st spray, whereas, the reduction percentages were 91.02 and 87.59%, respectively, after 7 days from the 2nd spray, and they were 92.40 and 89.87%, respectively, after 14 days from the 2nd spray of M. anisopliae (S1, S2) treatments in season 2019. Afterward, the reduction percentages of E. insulana Infestation increased to 95.42 and 95.12%, respectively, after 7 days from M. anisopliae (S1, S2) spray, and reached to 94.12 and 93.73%, 14 days from the 3rd spray. Also, Table 2 shows the reduction percentages of E. insulana by the 1st application of B. bassiana (S1, S2) (70.61 and 74.69%, respectively), after 7 days and (81.04 and 82.89%, respectively) after 14 days of treatments. While for the 2nd spray of B. bassiana (S1, S2) treatments, the reduction percentages of E. insulana were 86.48 and 88.42%, respectively, after 7 days whereas they were 89.16 and 89.16%, respectively, after 14 days from treatments. Then, the reduction percentages of E. insulana increased to 92.93 and 92.78%, respectively, after 7 days from the 3 rd spray of B. bassiana (S1, S2) whereas they were 90.83 and 90.83%, respectively, after14 days from the 3rd spray by the same strains, during season 2019.
Data in Table 3 shows that there were significant differences between means of infestations after 7 and 14 days treatments when the p-value is <0.05).

Discussion
Obtained results showed that both M. anisopliae and B. bassiana strains were elicited effects toward E. insulana under field experiment. M. anisopliae (S1, S2) showed the best effect toward E. insulana infestation reduction percentage after 3 sprays of each treatments, under field conditions followed by B. bassiana S1, S2. In this respect, Abd-ElAzeem et al. (2019) investigated the biological activities of spores and metabolites of some fungi against the newly hatched larvae of E. insulana. Results showed that the fungi M. anisopliae had more effectiveness to the newly hatched larvae. Also, spore suspensions of the all fungal isolates had the highest larval mortality than fungal metabolites. Moustafa et al. (2019) found that toxicity of M. anisopliae was high in case of E. insulana treatment. Dar et al. (2020) reported that M. anisopliae and B. bassiana either isolates or (W.P.) with Economy Micron ULVA (15 L./Fed.) were the most effective applications in reduction percentage of boll infestation with E. insulana followed by Bacillus thuringiensis, whereas, Hegab and Zaki (2012) found that B. bassiana gave a low larval mortality against E. insulana.

Conclusion
It could be concluded that B. bassiana and M. anisopliae proved to be efficient EPF against E. insulana. These fungi induced significant reductions for the mean numbers of infested bolls with the pest. B. bassiana and M. anisopliae are commonly used as pathogens against many insect species. Generally, the tested strains can be suggested as promising fungi to be used in biological control program of E. insulana in the field application.