Effects of Various Salts on The Efficacy of Bacillus thuringiensis against the Larval Instar of Fall Armyworm, Spodoptera frugiperda

This research aims to study the efficacy of some chemical additives along with commercial compounds for B. thuringiensis (Agrien) to increase its ability to control the Fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae). Hence


INTRODUCTION
Fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae), is a significant pest with a very broad host range of plants, where it has been observed on more than 353 plant crops, such as maize, sweet corn, sorghum and rice (Casmuz et al., 2010 andMontezano et al., 2018). In addition, S. frugiperda can infest and damage vegetable crops including capsicum, chilli, tomato, pumpkins, cucumber, beans, eggplant and other vegetables (Acharya et al., 2020). The first outbreak of Fall Armyworm (FAW) in Africa was reported in West Africa in late 2016 and Sisay et al. 2019). In Egypt, FAW is considered one of the most serious pests, and according to the Agricultural Pesticide Committee (APC), Ministry of Agriculture the FAW was recorded on maize fields in a village of Kom-Ombo city, Aswan Governorate, Upper Egypt in May 2019 (Dahi et al., 2020). Subsequently, it invaded Qena, Sohag, Luxor and Assuit Governorates causing damage to maize fields (Hend et al., 2022). In general, the fall armyworm is a major limiting factor affecting crop and vegetable production, not only in Egypt but also in many other countries. The current use of synthetic insecticides is not effective against fall armyworms; this leads to the use of high doses with repeated applications. The extensive use of insecticides has led to the development of insect resistance, with subsequent pest population outbreaks, a negative impact on the environment, and a great threat to human health (Bakr and Abd El-Bar 2017;Sisay et al., 2019). The best alternatives to the use of chemical pesticides are biopesticides, which are based on microorganisms and are beneficial for agriculture and less harmful to the environment and health (Oliveira et al., 2018). Among the group of bacteria used for biocontrol means, Bacillus thuringiensis has been successfully used for many years as both a bioinsecticide (Fang et al., 2009;Raymond et al., 2010;Oliveira et al., 2018 andGC et al., 2020).
Therefore, the main objective of the current study is to assess the impact of some chemical salts additives on B. thuringiensis (Agrien) to increase its ability to control the fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae).

Field and Semi-Field Experiments:
In order to achieve the objective of this study one Kirat (175 m 2 ) was planted with cabbage (Brassica oleracea) seedlings at Qaha research station, Qalubya Governorate in October 2022. To assess the efficacy of Agrien as a Bt. Compound and some chemical additives (inorganic salts) against the fall armyworm larvae were applied as flow: Agrien was sprayed at the recommended rate of 250 gm/ fed and serial dilutions of the selected samples were incorporated with certain concentrations of additive and then were used to treat the cabbage leaves before feeding to the larvae at three concentrations; 0.1%, 0.25% and 0.5% with the following salts Zinc sulfide (ZnS), Potassium chloride (KCl), Calcium oxide (CaO), Sodium bicarbonate (NaHCO3) and Potassium Sulfate (K2SO4). Mixtures were prepared by adding 1.25 gm from Agrien to each concentration of salts. The treatments were sprayed on cabbage plants using a solo motor under field dilution rate (250 L water / fed.).

Rearing of Fall Armyworm; Spodoptera frugiperda:
A stock culture of Spodoptera frugiperda larvae was provided from the cotton leaf worm Department, Plant Protection Research Institute; Agricultural Research Centre (ARC), Giza, Egypt. The S. frugiperda larvae were maintained under controlled conditions; 65 ± 5% relative humidity (RH) and 27±1 °C temperature; (Dahi et al., 2020). The larvae were reared on cabbage leaves as natural food until entered the pupae stage. The pupae were observed daily until the adult moths emerged. Moths were transferred to a plastic container (20 lit. capacity with 27 cm height and 24 cm diameter), covered with muslin cloth. The plastic containers were supplied with a cotton plug soaked in 10% honey solution as a sugar source for moths feeding. Another piece of muslin cloth was hung inside the cage for oviposition (Sharanabasappa et al., 2018;Haq et al., 2022). Eggs of S. frugiperda were collected from insect colonies and kept in an incubator at 65 % R. H and. 25 °C for hatching. Newly emerging larvae were transferred into Petri dishes and were supplied with discs of cabbage leaves to perform the experiments. Bioinsecticide: Agrein 6.5% WP. Bt. compound produced by the agricultural genetic Engineering Research Institute, ARC, Giza, Egypt. It contains Bacillus thuringiensis aegypti that distributes a different profile with various combinations of genes from groups; cry1, cry2, cry8, and cry9.

Bioassay with B. thuringiensis and Chemical Additives:
Bacillus thuringiensis formula (Agrien), as well as three concentrations for the five inorganic salts mentioned above mixed with Agrien, and control, were assayed against the 2 nd instar larvae of S. frugiperda. The 2 nd instar larvae were selected for the present study. Such cabbage leaves were randomized collected from each treatment after application with the materials under study and transferred to the laboratory and provided to the starved 2nd instar of S. frugiperda to feed on. The treated leaves were replaced with untreated ones after 24 hours. The assay was replicated five times for each concentration; twenty-five 2 nd instar larvae were used in each treatment. The mortality was recorded 3, 5, and 7 days after feeding and the corrected mortality percentage was calculated.

Biochemical Studies:
Biochemical studies were conducted in the Department of Insect Physiological, Plant Protection Research Institute, Agricultural Research Centre to explain the effect of tested Bt formulation alone and its combination with chemical additives on some larvae enzymes.: Protease, Amylase, Acetylcholinesterase ( AChE), and Acid phosphatase (ACP).
Enzyme activities in second-instar larvae were determined. The larvae were fed on either cabbage leaves that were first treated with Agrine or cabbage leaves treated with Agrine combined with 0.25% of Zinc Sulfide (ZnS), Potassium Chloride (KCl), Calcium Oxide (CaO), Sodium Bicarbonate (NaHCO3), or Potassium Sulfate (K2SO4).

Enzymes Determination: Preparation of Larval Enzymes Solution:
The samples of larvae used in enzyme assays were obtained from those subjected to the experimental biopesticide. The larval enzyme solution was prepared according to the method described by Ishaaya et al. (1971). The enzyme solutions were obtained by homogenizing 10 second-instar larvae, representing ca. 2 g. larval weight, in 20 ml distilled water, using a chilled glass Teflon grinder. The homogenate was centrifuged at 8000 r.p.m. for 15 min at 5°C, the deposits were discarded and the supernatants were kept in a deep freezer till use.

Determination of Enzymes Activities:
The determinations of Amylase activity were based on the digestion of starch respectively, by spectrophotometric methods Ishaaya et al. (1971). The determination of Protease activity: The proteolytic activity was determined by the casein digestion method described by Ishaaya et al. (1971).The determination of Acid phosphatase activity: Acid phosphatase activity was measured according to the method of Laufer and Schin (1971).The determination of Acetylcholinesterase (AChE) activity: was measured according to the method described by Simpson et al. (1964).

Statistical Analyses:
Data were statistically analyzed. Mortality data were corrected according to Abbott`s formula (Abbott, 1925). For testing the homogeneity of the control agent Chisquare analysis "χ2" method was used (Snedcor and Cochran 1982). Data analysis has been employed in this study SPSS Ver. 23 was used to compute ANOVA (P<0.05).

The Efficiency of Certain Control Treatments against the 2 nd Instar Larvae of Spodoptera frugiperda:
The efficiency of mixing Bacillus thurigensis (Agrien) with five inorganic salts in three different concentrations and (Agrien) alone were tested against 2 nd instar larvae of Spodoptera frugiperda in a laboratory to explore their potential to control the pest. Data represented in Table (1) revealed that the Bt+KCl 0.5% recorded the highest reduction percentage 60.0% flowed by Bt (Agrien) with 54.67%. On the contrary, the Bt + K2SO4 0.1% treatment recorded the lowest reduction percentage13.3%. According to the chi-square test; there are significant differences between the 16 treatments whereas χ 2 = 12.59 and χ 2 = 15.51 sig. at 0.05. These treatments could be divided into three groups based on their reduction percentage, 1 st group (a) contains on Bt+KCl (0.5%), Agrien, Bt.+KCl (0.25%), Bt.+ZnS (0.5%), Bt.+ ZnS (0.25%) and Bt.+ CaO (0.5%) respectively. The second group (b) contains Bt + NaHCO3 (0.25%), Bt+KCl (0.1%), Bt + CaO (0.25%), Bt + CaO (0.25%), Bt + ZnS (0.1%), Bt + K2SO4 (0.5%), Bt + NaHCO3 (0.1%) and Bt + CaO (0.1%); while the 3 rd group contains on two treatments only Bt + K2SO4 (0.25%) and Bt + K2SO4 (0.1%). Salama, et al. (1989) and Hefez et al. (1998) said that inorganic salts such as CaO, CaCO3, ZnSo4 and K2CO3 could potentiate the activity of Bt. formulation (Agerin) against the corn borers, Chilo agamemnon and Ostrinia nubilalis. In addition, (Girgis 2007) found that the addition of K2Co3 and CaCO3 increased the effectiveness of some biopesticides against Phthorimaea operculella under greenhouse and laboratory conditions. Zhang, et al. (2013) pointed out that many inorganic salts can increase the activity of Bacillus thuringiensis against P. xylostella larvae at different levels when combined with them, as he found calcium salts (calcium hydroxide, calcium chloride, calcium carbonate and calcium sulfate) have the preference increasing the activity of B. thuringiensis, followed by some other salts such as Sodium carbonate, potassium hydroxide and sodium acetate. Priyanka, et al. (2021) indicated that treatments of B. thuringiensis in addition to inorganic salts were significantly effective against fall armyworm larvae compared with B. thuringiensis alone.

Effect of the Tested Compounds on The Activities of Some Enzymes: Effect on Acetylcholinesterase Enzymes (AchE):
Acetylcholinesterase (AchE) is considered a key enzyme in the nervous system of insects and is responsible for the hydrolysis of the neurotransmitter acetylcholine for ending the neurotransmission process. Thus, if this hydrolysis does not take place as a result of degradation or inhibition in the expression of AchE, build-up of acetylcholine occurs and leads to repeated firing of neurons and ultimately death of the insect (McCaffery, 1999;Gunning and Moores, 2001).
Data in Table (2) pointed that the activity of acetylcholinesterase increased significantly in the larvae of Spodoptera frugiperda under four treatments; Bt+ZnS 0.25%, Bt+KCl 0.25%, Bt+CaO 0.25%, and Bt+K2SO4 0.25% compared to the control. On the other hand, there were no significant differences between the control and the other two treatments (Agrien and Bt+NaHCO3 0.25%). The activity of (AChE) was significantly higher in Bt+ K2SO4 0.25% treatment as compared to the control and Agrien.
The activity of (AchE) correlated inversely and significantly with the total corrected mortality percentage (% CM) whereas r= 0.648 (Fig. 1). It is noticed that (% CM) was recorded at 17.33% when the concentration of (AchE) was 174.33 ug AchBr/min/g.b.wt and (% CM) was 54.67 when (AchE) recorded at 92.67 ug AchBr/min/g.b.wt. Gao (1992) said that increased expression levels of (AChE) in response to pesticide exposure showed insect tolerance and increased (AChE) activity, which seems to be a dominant mechanism conferring resistance in lepidopteran pests. Pesticides mostly, exert toxicity by inhibiting enzyme (AChE) (Bolton and Lim 1991;Muthusamy et al. 2011) which degrades acetylcholine (AChE), an essential neurotransmitter in the central nervous system (CNS) of insects, rodents, and humans (Jones 2005). Table 2: Effect of certain treatments on some enzyme activities of fall armyworm, Spodoptera frugiperda (J.E. Smith).
Means followed by different letters are significantly different according to Duncan's multiple range comparisons (DMRTs). Means followed by the same letter are not significantly different.

Effect on Acid Phosphatase (ACP) Enzymes:
Three of the tested compounds significantly inhibited the activity of Acid phosphatase enzyme (ACP) compared with the control, Agrien gave the lowest decrease (430.67 mU/g.b.wt) followed by Bt+KCl 0.25% and Bt+K2SO4 0.25% (675.67 and 754.33 mU/g.b.wt), respectively as compared with the control (810.3 mU/g.b.wt). On the contrary, the Bt+NaHCO3 0.25% increased phosphatase activity (1056.00 mU /g.b.wt). Figure (2) showed that the corrected mortality percentage correlated reversely and significantly with the activity of acid phosphatase enzyme where r=0.529. The activity of phosphatase enzymes indicates the efficiency of digestion and absorption of nutrients in the stomach and their transfer to fat bodies. The decrease in the activity of this enzyme group can indicate a lack of digestive function and decreased metabolism due to a reduction in releasing phosphate groups for energy manufacture (SenthilNathan 2006;Younes et al., 2011;Selin-Rani et al., 2016). Kamel et al. (2010) revealed that the ACPE activities decreased significantly in larvae of cotton leafworm Spodoptera littoralis after 48 hours of Agrien treatment compared to the control. Changes in ACPE activities after treatment with Bt indicate that changing the physiological balance of the midgut might affect these enzymes.

Effect on Protease Enzymes:
All treatments resulted in the inhibition of protease enzyme activity compared to the control. There were significant differences between the control and all other treatments. On the other hand, there were no significant differences between Agrien, Bt+ZnS 0.25% and Bt+KCl 0.25% (Table2). This result inagreement with Hassan et al (2015) who found that addition of Sodium bicarbonate (NaHCO3), and calcium carbonate (CaCO3) to Bacillus thuringiensis commercial formula induced a significant increase in the proteolytic activity in the second instar larvae of Spodoptera littoralis (Boisd.) compared to the control.
Protease enzyme activities showed inverse non-significant correlation with the total corrected mortality percentage (% CM) whereas r=0.40 (Fig. 3). Salama, et al. (1989) said that inorganic salts demonstrated a considerable potentiation of the endotoxin action against the greasy cutworm Agrotis ypsilon. The impact of these salts has on the proteolytic enzymes like protease found in the midgut of insects may be connected to the salt's mood of action. Additionally, the efficacy of Bt was significantly increased by calcium salts such calcium carbonate and calcium oxide.

Effect on Amylase Enzymes:
Data illustrated in Table (2) revealed the Agrien treatment resulted in the highest decrease in the activity of the Amylase enzyme (60.97 µg glucose /g.b.wt.) compared to the control (109.17 µg glucose /g.b.wt.) followed by Bt+ZnS 0.25%, and Bt+KCl 0.25% (80.03 and 96.67 µg glucose /g.b.wt.), respectively. Also, there were significant differences between the control and the three treatments mentioned above. Conversely, the activity of the Amylase enzyme increased under two treatments, Bt+NaHCO3 0.25% (153.33 µg glucose /g.b.wt.) and Bt+K2SO4 0.25% and (115.67 µg glucose /g.b.wt.) compared with the control (109.17 µg glucose /g.b.wt.). Figure (4) shows the simple correlation coefficients between the activity of the Amylase enzyme and corrected mortality percentage and the relations between them were inverse. Moreover, the corrected mortality percentage has a positive significant correlation coefficient, r=0.604 with Amylase enzyme activity where the high reduction percentage was 54.67% when the amylase activity was low (60.97 µg glucose /g.b.wt.) under Agrien treatment while Bt+ KCl 0.25% at 54.67% and 46.7%, respectively while the low one was recorded under Bt+K2SO4 0.25% treatment at 17.33% when the amylase activity was high (153.33 µg glucose /g.b.wt.). Hammati et al. (2022) found that both proteases and amylases were most active in the larval midgut extract under alkaline conditions of pH 11 and 10, respectively. Yezdani et al. (2010) said that several chemicals compounds; (NaCl, KCl, MgCl2 and CaCl2) had variance effects on the enzyme activity in the midgut of Glyphodes pyloalis Walker (Lepidoptera: Pyralidae). Whereas, the highest amylase activity was recorded in the case of KCl at 20 mmol/L. Ca 2+ ions increase the activity of α-amylase in G. pyloalis since αamylase is a metalloproteinase and require calcium for maximal activity (De Sales et al., 2008).

CONCLUSION:
The chemical additives with Bacillus thuringiensis (Agrien) caused a disturbance in the activities of enzymes such as Amylase, Protease, Acid Phosphatase (ACP), and Acetylcholinesterase (AchE) which play essential roles in the insect body. This indicates that many physiological functions, in the insect body, have been disrupted. Disturbance of the enzyme activities can be considered sub-lethal effects of the tested compounds but could finally lead to death.