Insecticidal activity and possible modes of action of secondary metabolites of some fungal strains and wild plants as natural pesticides against Spodoptera frugiperda

Background The fall armyworm (FAW), Spodoptera frugiperda , is an invasive and destructive pest to certain strategic crops, especially maize in Egypt. This research was conducted mainly to obtain secondary metabolites from some wild plants and fungal strains, use them in controlling FAW, and investigate their mode of action. Results The ethyl acetate extracts of Cladosporium cladosporioides and Verticillium lecanii , as well as the alkaloid extracts of Ricinus communis and Nicotiana glauca , were extracted to obtain their secondary metabolites. The sec‑ ondary metabolite contents were identified by gas chromatography mass spectrometry and NMR. The toxicity of all extracts against the 3rd‑instar larvae of FAW was evaluated. The possible mode of action of the extracts was stud‑ ied via their effects on larval enzyme activities and larval tissue. The toxicity results illustrated that, the extract of C. cladosporioides was more effective with LC 50 229 ppm, followed by the extract of V. lecanii with LC 50 341 ppm and N. glauca with LC 50 404 ppm, while the least effective extract was R. communis with LC 50 1110 ppm after 72 h of treat‑ ment. While, the results of larval enzyme activities showed that C. cladosporioides , V. lecanii, and R. communis extracts led to significant activity of AST, ALT, ACP, and ALP enzymes, but GST and AchE were inhibited in treated larvae compared with control. While N. glauca alkaloid extract caused significant inhibition of AST, ALT, ACP, AchE, and GST enzymes, ALP was activated in the treated larvae compared with control. The results of larval tissue slides indicated that the most affected tissues were the cuticle layer and the membrane lining of the midgut, in addition to the fatty bodies.

subsequently spread throughout the continent and across Asia.However, fall armyworm had a wide range of plants hosts and could feed on over 350 plant species.Damage of FAW has been estimated to cause up to 13 billion US dollars per annum in crop losses throughout sub-Saharan Africa.According to CIMMYT Annual Report [5], FAW had devastated almost 1.5 million hectares of cultivated corn crops in six African countries.In Egypt, FAW was firstly recorded in maize field in a village in Kom Ombo city of Aswan governorate, Upper Egypt 2019 season according to the Agricultural Pesticide Committee (APC) and Ministry of Agriculture and Land Reclamation.As the north coast of Africa are environmentally suitable for FAW infestations besides its wide range of host plants, destructive damage would be expected, and it is contingent to harm other strategic crops such as cotton, soybean, and rice.Therefore, the population of FAW S. frugiperda should be under control.
So, many interested in the plant protection field in Egypt started to study several methods to suppress the FAW S. frugiperda population and reduce its damages.Among of these methods, using entomopathogenic fungi and plant extracts.Many previous studies reported that the mode of action of most entomopathogenic fungi refer to their production from bioactive secondary metabolites or enzymes and toxic proteins [6,7].In this study, authors used secondary metabolites of entomopathogenic fungi Cladosporium sp. and Verticillium sp. as well as alkaloid extract of wild plants castor bean, Ricinus communis and tobacco, Nicotiana glauca.
The fungus of Cladosporium sp.belongs to Ascomycota division.It was used as an entomopathogenic fungus to control several of insects in many previous studies.Shaker et al. [8] and Elbanhawy et al. [9] evaluated the efficiency of secondary metabolites which were extracted from Cladosporium cladosporioides against cotton aphids, Aphis gossypii.They found high toxicity effect where LC 50 values were 24 and 36 ppm against nymphs and adults of aphids, respectively.Also, Habashy et al. [10] mentioned that spores suspension of C. cladosporioides caused high mortality percentage of spotted spider mites, Tetranychus urticae.Abdullah [11] isolated Cladosporium sp., Verticillium sp., Epicoccum sp., Alternaria sp., Fusarium sp., Penicillium sp., and Aspergillus sp.during his M.S. thesis from aphid and whitefly.The toxicity of isolated fungi was tested against aphids and whitefly.He found that Cladosporium sp., Verticillium sp., and Epicoccum sp. were more toxic fungi compared with other tested fungi.Also, Bahar et al. [12] isolated Cladosporium sp. from eggs of Helicoverpa armigera (Lepidoptera: Noctuidae) and tested its pathogenicity against eggs and larvae of H. armigera, cotton aphids, Aphis gossypii and whitefly, Bemisia tabaci.Verticillium lecanii is an opportunistic Ascomycete's fungus and widely distributed.V. lecanii (Zimm.) is commonly known as the "white holo.," and it causes mycosis of numerous insects belonging to the orders Lepidoptera, Coleoptera, and Homoptera [13].Lecanicillium lecanii, L. longisporum, L. attenuatum, L. nodulosum, and L. muscarium are among the new taxonomic entities divided from the species V. lecanii.There are number of important species from the Verticillium spp.group, which are used as biocontrol agents to control insect pests and diseases in agriculture [14].There are 15 productions of bioinsecticides belonged to Verticillium spp.commercialized worldwide [15,16].
Several previous studies reported that the crud extract of secondary metabolites of castor plant R. communis found to has insecticidal activity against pests such as sugarcane aphid, Melanaphis sacchari [20], acaricidal and insecticidal activities against Haemaphysalis bispinosa Neumann adult and hematophagous fly Hippobosca maculata Leach [21], malaria vector Anopheles gambiae [22] and in controlling the termites which damage the wood of Mangifera indica and Pinus longifolia [23].
Tobacco plant (Nicotiana glauca) is a flowering plant in the tobacco genus Nicotiana of family Solanaceae.The alkaloid fraction of N. glauca was analysis by UPLC/ MS and GC/MS, anabasine was found to be the major constituent (60%).In addition to, five compounds were identified as nicotine, nornicotine, ammodendrine, chlorogenic acid, and rutin [24].The acute toxicity of N. glauca was due to the highly percentage of anabasine compound [25].It was found that N. glauca plant had antioxidant activity as a result of its content of flavonoids and phenolic compounds, which recorded their highest values in the leaves and flowers [26].As a result of the presence of a high percentage of alkaloids in N. glauca, great importance was given to its use as antimicrobial agents [27] and insecticides to control aphids [28].The aim of this study is to extract the secondary metabolites from entomopathogenic fungi, C. cladosporioides and V. lecanii, as well as from wild plants, R. communis and N. glauca.In addition to evaluating the influence of their crud extracts on larval mortality, enzyme activity, and larval tissue of the fall armyworm, S. frugiperda.

Insect rearing procedure
The colony of fall armyworm was established initially from larvae collected during the summer season of 2022 from corn field located in Dakahliah governorate.Larvae were reared individually to avoid cannibalism and were fed on fresh leaves of castor oil plants Ricinus communis L. replaced every 2 days, depending on how long they remain green and fresh.Larvae were kept in climatic control chamber (24 ± 1°C, 70% RH, 14L: 10D photoperiod) till pupation.Pupae were collected and placed in PVC container filled with sand under same condition until adult emergence.Following emerging, moths were collected and held in 1 L. glass mason jars, 20-40 moth per jar were kept together to encourage mating and were fed on honey bee solution from a cotton wick hanged from the jar top.Jars were kept in a growth chamber under same previous conditions with pieces of zigzag shaped papers to provide dark arena for eggs oviposition.Once mated adult started oviposition, egg mass were collected and held in plastic containers 200 ml in climatic control chamber until hatching.In order to obtain larvae for the experiment, a number of five neonate larvae were kept in plastic container 100 ml and were provided with small pieces of corn cob.Once larvae reached third instar, we isolate and count the proper number of larvae for the experiment then they were subjected to treatments.

Fungal strains
Fungal strains Cladosporium cladosporioides and Verticillium lecanii were kindly obtained from the Microbiology Department, Faculty of Agriculture, Mansoura University, Egypt.Both fungal strains were cultured on potato dextrose agar medium (PDA) made from potato infusion 200 ml, dextrose 20 g, and agar 15 g per liter, then incubated at 25°C. Figure 1 shows the colonies growth on potato dextrose agar medium and spore shapes under light microscope of both fungal strains.

Extraction of fungal metabolites from their broth cultures
One hundred ml of potato dextrose broth medium (PDB) was prepared in a 250-ml conical flask and sterilized in an autoclave.One disk of solid fungal culture was used to inoculate 100 ml of sterilized PDB medium.The inoculated medium was incubated to 7 days at 25 °C.After incubation period, one litter of sterilized PDB medium was inoculated by 100 ml broth culture in 2 litter conical flask and incubated to 21 days at 25 °C.After incubation period, the broth culture was filtered to remove the mycelium and obtain to the filtrate by Whatman no1 filter paper, Sigma-Aldrich.The volume of filtrate was measured then the equal volume from ethyl acetate was added.The mixture was poured in separating funnel and moved well for homogenate then left to 2 h.The upper layer was taken then ethyl acetate was evaporated by rotary evaporator system to obtain the crud extract of fungal metabolites.This method was conducted as described by Abdullah [29].

Gas chromatography-mass spectrometry (GC-MS) analysis
Trace GC-TSQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG-5MS (30 m × 0.25 mm × 0.25 µm film thickness) was used to perform the chemical composition of fungal strains extracts.The temperature of the column oven was initially maintained at 50 °C then raised by 5 °C/min to 250 °C and maintained for two minutes then raised to final temperature 300 °C by 30 °C/min, where it was held for two minutes.The temperatures of injector and MS transfer line were maintained at 270, 260 °C, respectively.The carrier gas was Helium at a constant flow rate of 1 ml/min.Electron impact (EI) mass spectra were collected at 70 eV ionization voltages over the range of m/z 50-650 in full scan mode.The ion source temperature was set at 200 °C.By comparing the components' retention times and mass spectra to those in the WILEY 09 and NIST 14 mass spectral databases, the components were identified [29].

Plant material
Ricinus communis L "Euphorbiaceae" seeds were collected from plants in the villages of Shoha and Salamoun, beside to Mansoura city (31.065745°N-31.453642°E),

Extraction of secondary metabolites from plants 2.6.1 Processing of Ricinus communis L. (Seeds)
The dried seeds of R. communis L. (1 kg) were grinded and extracted with methanol (2L × 5).The methanol extract was concentrated by rotary evaporator to obtained 300 ml oil extract.A certain amount of oil was exposed to PTLC (Preparative Thin Layer Chromatography, silica gel Merck GF 254 precoated plates 20 × 20 cm on aluminum sheets) with eluent system CHCl 3 : MeOH (6:1) to isolated major compound (1), (25 mg, R f 0.44), which gave positive result (orange color) with Dragendroff 's reagent.

Processing of Nicotiana glauca
The dried powder aerial parts of N. glauca (1 kg) extracted with methanol (1.8L × 5).The methanol extract was concentrated by rotary evaporator till dryness (83 gm).The methanol extract was dissolved in small amount of methanol and then added distilled water acidified by 2N HCl till pH 3. The acidified mixture was extracted by separating funnel with chloroform three times.The acidified aqueous extract was basified using ammonia solution

Bioassay experiment
Spray bioassay method was used to evaluate the toxicity of four crud extracts as natural pesticides against S. frugiperda under laboratory conditions.Five serial concentrations of each crud extract were prepared (500, 1000, 1500, 2000, 2500 ppm).Ten third-instar larvae of S. frugiperda were transferred to each jar (10D * 25H cm).Pieces of castor leaves were used to feed larvae.Two milliliter of each concentrate were sprayed on larvae and castor leaves in each jar.Two milliliter of water were used to treat control group.The number of live larvae was recorded every day.Mortality percentages were calculated and corrected by Abbot's formula [31].LC 50 , confidence limits and slope values were calculated as described by Finney method [32].Also, toxicity index was calculated as described by Sun [33].

Estimation of the changes in insect enzyme activities
Fourth-instar larvae of S. frugiperda were used to investigate the effect of tested secondary metabolites on insect enzymes activities.The same method of bioassay experiment was repeated but LC 50 of each crude extract was used to spraying the larvae.Also, water was used to spray control group.After four days, the live larvae were weighted and frozen in Eppendorf tube until analysis.All enzyme activities were estimated colorimetrically by UV-visible spectrophotometer, model V1200, China.The estimated insect enzymes were aspartate transaminase (AST), alanine transaminase (ALT) according to Reitman and Frankel [34] at 520 nm, acid phosphatase  (ACP), alkaline phosphatase (ALP) according to Powell and Smith [35] at 510 nm, acetyl choline esterase (AchE) according to Simpson et al. [36] at 515 nm, and glutathione S-transferase (GST) according to Pan et al. [37] at 540 nm.

Determination of histological changes in insect tissue
Also the fourth-instar larvae of S. frugiperda were treated by LC 50 of each extract using spray technique.Two live larvae of treated and untreated larvae were taken after five days of treatment and saved in 10% formalin solution.Tissue processing, sectioning, and staining were conducted according to Gaaboub et al. [38] in Faculty of Medicine, Mansoura University, Egypt.

Toxicity effect of fungal and wild plants extracts against S. frugiperda
Table 1 shows the toxicity effect of ethyl acetate extracts of two fungal strains (C.cladosporioides & V. lecanii) and alkaloid fraction of N. glauca as well as methanol extract of R. communis) against S. frugiperda.
The more effective extract after 48 h of treatment was the alkaloid extract of N. glauca (LC 50 = 788 ppm) followed by the ethyl acetate extract of C. cladosporioides (LC 50 = 886 ppm), while there were different results after 72 h of treatment where the extract of C. cladosporioides was more effective extract (LC 50 = 229 ppm) followed by the extract of V. lecanii (LC 50 = 341 ppm) and N. glauca (LC 50 = 404 ppm).Also, there were different values of toxicity line's slope between all tested extracts.This illustrate that could be there were different mode of actions of tested extracts against S. frugiperda.In addition, toxicity index showed that S. frugiperda was more susceptible to C. cladosporioides extract than other tested extracts.Figure 2 shows some abnormal symptoms of treated S. frugiperda larvae as necrosis and analysis of larvae body also the larva could not get rid of some parts of the cuticle layer, which led to the larva not growing normally.

The changes in insect enzymes activity
In this study, the changes of five insect enzymes activities were estimated in the treated larvae of S. frugiperda.The results in Table 2 show that C. cladosporioides, V. lecanii, and R. communis extracts led to significant activity of AST, ALT, ACP, and ALP enzymes, but GST and AchE were inhibited in treated larvae compared with control, while N. glauca alkaloid extract caused significant inhibition of AST, ALT, ACP, AchE, and GST enzymes, but ALP was activated in the treated larvae compared with control.

Histological changes of treated larvae
Figures 3, 4, 5, 6, and 7 show the histological changes of treated 4th-instar larvae by C. cladosporioides, V. lecanii, N. glauca, and R. communis extracts as well as control treatment.The description of histological changes is under each cross section's photo.The most affected tissues were the cuticle layer and the membrane lining of the midgut, in addition to the fatty bodies.
Tables 3 and 4 show the identified compounds in the ethyl acetate extract of C. cladosporioides and V. lecanii broth cultures by GC-MS analysis.Thirteen compounds were identified in C. cladosporioides extract.The major compounds were n-hexadecanoic acid (Palmitic acid), hexadecanoic acid, methyl ester, 9, 12-Octadecadienoic acid (Z, Z)-(Linoleic acid) and 9,12-octadecadienoic acid, methyl ester, (E,E)-where their peak area percentages were 31.63%,14.74%, 17.97%, and 13.14%, respectively.On the other hand twelve compounds were identified in V. lecanii extract.Three compounds had the more percentages of peak area compared with other compounds.These compounds were tetramic acid (13.22%), 7-Isopropyl-1,4a-dimethyldecahydronaphthalene # (Selinane) (16.09%) and methyl ester of ricinoleic acid (14.9%).On the other hand, two wild plants (R. communis and N. glauca) were selected according to their alkaloids, which have a wide range of uses in many fields, especially as insecticides.Alkaloids are among the most important compounds that have a toxic effect on insects [59].The analysis of R. communis extract indicated that ricinine (Compound 1) was the major constituent in the extract [39].Ricinine alkaloid was used as a natural pesticide against Tetranychus urticae and two predatory phytoseiid mites [40].As well, ricinine can be used as an insecticide against leaf-cutting ants [60].The extracts of R. communis were used by [20] as botanical insecticides to control the sugarcane aphid, Melanaphis sacchari.
In addition, the anabasine (Compound 2) was found to be the major constituent of the alkaloid fraction of N. glauca [24].The anabasine alkaloid has been widely used as a pesticide.Its insecticidal effect is due to interaction with nicotinic acetylcholine receptors [61,62].Alkaloid fraction of N. glauca was analyzed by GC/MS technique.Compound (2) was identified as the major constituent in the fraction (Rt = 17.34, 65.52%) by comparing their mass spectra with those of their analogous reported by NIST library (Fig. 11).The GC/MS analysis indicated that the molecular ion peak at m/z 162 as [M] +.  [24,41].Figure 12 shows the chemical structure of anabasine (compound 2).

Conclusion
In conclusion, natural pesticides would have a promising role in terms of controlling the FAW where they avoid environmental damage, reduce synthetic pesticides, and overall reduce control cost.Thus according to this study, it was recommended that, alkaloid extracts of tested wild plants and ethyl acetate extracts of fungal strains be used as natural pesticides to control the fall armyworm, S. frugiperda.

Fig. 1
Fig. 1 The colonies growth of C. cladosporioides and V. lecanii on potato dextrose agar medium and their spore shapes under a light microscope

Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7
Fig. 3 Cross section through untreated 4th-instar larvae of S. frugiperda, stained using hematoxyline, Eosin (H, E X200), showing normal fibrous and cellular layer of cuticle (CT) also normal basement and peritrophic membranes in the midgut (MG) as well as normal cells of fat bodies (FB)

Table 1
Toxicity of secondary metabolites of tested fungal strains and wild plants against S. frugiperda Fig. 2 Abnormal symptoms of S. frugiperda larvae after treatment by crude extracts of fungal strains and wild plants under laboratory conditions

Table 2
Changes in the enzyme activities in the treated larvae of S. frugiperda caused by the extracts of fungal strains and plants a,b,c,d,e The same letter in the same column means it is not significant at P 0.05.LSD The least significant difference 33% till pH 11 then extracted with chloroform (Alkaloid fraction 1.85 gm).

Table 3
GC-MS analysis of ethyl acetate extract of the liquid culture of C. cladosporioides

Table 4
GC-MS analysis of ethyl acetate extract of the liquid culture of V. lecanii R.t.: Retention time (min)