SILVER NANOPARTICICLES FROM ENTOMOPATHOGENIC FUNGI AGAINST THE SPINY BOLLWORM, EARIAS INSULANA IN MAIZE CROP

Article information Article history: Received:18/3/2023 Accepted:2/5/2023 Available:30/6/2023


INTRODUCTION
The spiny bollworm, Earias insulana (Lepidoptera: Noctuidae) is considered one of the most important corn pests in the world. It causes severe damage, resulting in a significant loss in both quality and quantity of corn yield. Various conventional chemical insecticides are available which offer some protection against these pests, but they have created many problems (resistance, secondary pest outbreaks, environmental pollution, etc….) (Dhakal and Singh, 2019). The death or harm to microorganisms caused by pesticides affects the fertility rate of the soil. Nanotechnology has become one of the most promising novel approaches for pest control in recent years, it also employs nanoparticles (NPs) having one or more dimensions in the order of 100 nm or less (Auffan et al., 2009).
The trials for evaluating nanotechnology in controlling insects are based on their size-dependent qualities high surface-to-volume ratio and increasing chemical reactivity and penetration in the living cells (Medina et al., 2007), A wide variety of nanoparticles materials are used against some insects in the laboratory. Goswami et al., (2010), Abdel-Raheem et al., (2019a, 2020a, Saad and Abdel-Raheem, 2020), studied that the applications of different nanoparticles such as silver nanoparticles (AgNPs), aluminum oxide nanoparticles, (Al2O3) zinc oxide, (ZnO) and titanium dioxide nanoparticles (TiO2), in pest control of rice weevil, Sitophilus oryzae, and the Red Palm Weevil, Rhynchophorus ferrugineus Olivier (Coleoptera: Curculionidae). Also, Stadler et al., (2010) Successfully applied alumina against stored grain pest Callosobruchus maculatus. Debnath and Seth (2011) demonstrated the application of SNP could significantly increase the mortality effect of NPs by increasing the time after application. Vinutha et al., (2013), Abdel-Raheem (2019b), Abdel-Raheem et al., (2009, 2020b recorded the potential of nanoparticles against Helicoverpa armigera. Entomopathogenic fungi (EPF) have been studied as biological control agents. Two isolates of various microorganisms were isolated from Cassida vittata, Metarhizium anisopliae and Beauveria bassiana (Saleh et al., 2016 andAl-Keridis 2017). The EPF are infecting the host by contact and penetrating through the insect cuticle. The host can be infected by direct treatment, transmission of inoculum from treated insects, cadavers to untreated insects, or by a new generation of spores. Eggs, Larvae and adults were contaminated by B. bassiana and M. anisopliae, reaching 50-100% mortality, (Zaki and Abdel-Raheem 2010, Sabry et al., 2011. Fungi, bacteria, algae, and plant extracts are known to synthesize silver nanoparticles (Ag NPs) (Sabbour and Abdel-Raheem 2016, Nisha et al. 2017, Dimetry et al. 2019, and Sabbour et al. 2020. Fungi such as Verticillium species are known to produce Ag NPs (Zonorodiam, et al., 2016). This study aimed to evaluate the bioefficacy of Nanoparticles of EPF, B. bassiana, M. anisopliae and V. lecanii as fungal spores and silver Nanoparticles on different life stages of E. insulana (larvae and Pupae) under laboratory conditions.

Commercial Compound
A compound product called Bio Catch containing V. lecanii, was used in the experiment. The concentration of EPF used was (1x106 spores/ ml). Spores were harvested from 14-day-old cultures by rinsing with sterilized water and adding 0.5 % Tween 80. The suspensions were filtered to remove mycelium clumps, and spore concentrations were determined using a Hemocytometer (0.1mm x 0.0025mm2). The concentrations were (1 x 10 6 spores /ml) from each EPF. The grown fungal cultures were centrifuged at 12000 rpm fungal for 30 min at 25 ˚C and the supernatant was used for the synthesis of Ag NPs.

Insect rearing
The laboratory strain of E. insulana was obtained from laboratory of Pests & Plant Protection Department, National Research Centre, Cairo, Egypt. Where reared for several generations away from any contamination with insecticides on an artificial diet.

Bioassay
Larvae and pupae of E. insulana were treated by the B. bassiana, M. anisopliae and Bio Catch (V. lecanii). Each treatment group consisted of 100 larvae and pupae divided into 5 groups, with 20 individuals in each group. The control group was treated with sterilized water. The mortality rates of E. insulana were recorded daily, and kept at 24 ±2 ºC and 65 ±5 % R.H.

Biosynthesis of Silver Nanoparticles
Silver nanoparticles were synthesized by using 50 ml aqueous solution of 1 mM Ag No3 treated with 50 ml of fungi culture (these particles prepared for all fungal isolates and commercial products) supernatant in a 250 ml conical flask and the PH was adjusted to 8.5. The whole mixture was incubated at 40 ºC at 200 rpm for 6 days under dark conditions. The control was maintained without adding the culture supernatant to the solution of Ag No3.

Bioassay studies
E. insulana was placed in sterile Petri dishes having food and sterile filter paper. The silver nanoparticle solution was sprinkled over the filter paper. Which was allowed to air dry aseptically. The Petri dishes were then incubated at 24±2ºC for 3 days, and the mortality rate was recorded two days after the treatment and % mortality. The experiment was replicated three times.

Data Analysis
Mortality data were recorded and mortality percentages were calculated for nymphs and adults. The corrected percent mortality by use of Abbott's formula, 1925. Student's t-test or one-way ANOVA was used to compare the effects of the experimental and control treatments. Statistical analyses were performed by the Stat View for Power PC software, version 4.5 (Abacus Concepts, Inc., Berkeley, CA, USA).

RESULTS & DISCUSSION
Data of the treated nymphs of E. insulana with B. bassiana, M. anisopliae and Bio Catch (V. lecanii) as fungal spores and their Silver NPs particles were presented in Table ( 1). It appears that both treatments, fungal spores and bio-synthesized Ag NPs, have a positive impact on the EPF (Entomopathogenic Fungi) being studied, as compared to the control group.Six days post-treatment, up-to (70%) mortality rate of E. insulana was recorded in the treated larvae. The percent mortality rates attained 60, 50, and 47% by infection with fungal spores from B. bassiana, M. anisopliae, and V. lecanii, respectively. Furthermore, the percentage mortality rates were 70, 60, and 58% by infection with biosynthesized Ag NPs from B. bassiana, M. anisopliae and V. lecanii respectively, at the same time. B. bassiana recorded the highest mortality (70%) in the larvae of E. insulana, when treated with nanoparticles or with fungal spores after 6 days will the lowest was (47%) when treated with V. lecanii fungal spores. The control group, which did not undergo any treatment, maintained a constant value of 5.0. This suggests that there was no significant impact on the EPF's activity in the absence of treatment.  Will, at the infection with biosynthesized Ag NPs from B. bassiana, M. anisopliae and V. lecanii the mortality rates recorded 65, 60, and 50%, respectively. B. bassiana was the highest % mortality (65%) in the pupae of E. insulana, when treated with the nanoparticles or with the fungal spores (57%) after 6 days, while the lowest (50%) was when treated with V. lecanii biosynthesized.
The control group, as before, shows a constant value of 5.0, indicating no significant change or treatment effect. The standard error values (S.E) provided in the last row of the table indicate the variability or margin of error associated with the mean values. The S.E for the fungal spores treatment is 1.31, while for the bio-synthesized Ag NPs treatment, it is 1.51. These values represent the range within which the true mean values are expected to fall. The implementation of nanotechnology in agriculture has resulted in the development of efficient strategies for pest insect control. Abdel-Raheem et al. (2009, 2019, 2020a & b) demonstrated that the mortality rate of larvae significantly increased when exposed to M. anisopliae spores compared to the control group. Similarly, Saleh et al. (2016) and Abdel-Raheem (2019b) suggested that variations in pathogenicity rates among different fungal species may be attributed to the presence of certain polar compounds with antimicrobial activity, ranging between 1000 and 1500 Dalton, extracted from fungi. Additionally, Moustafa et al. (2019) discovered that newly hatched larvae of Earias insulana exhibited high susceptibility to the toxic effects of M. anisopliae. Entomopathogenic fungi have been shown to effectively induce mortality in P. gossypiella at different stages, as reported by Niu et al. (2019), Rizwan et al. (2019), and Omar et al. (2021).

CONCLUSIONS
The results proved that the use of the Ag NPs synthesized through the application of spore suspension and biosynthesized silver nanoparticles showed efficacy against E. insulana larvae and pupae stages. B. bassiana had the highest potential as well as it was more effective than M. anisopliae and V. lecanii.

ACKNOWLEDGMENT
This research has been financed by National Research Centre (Project no. E 120-706).