Background

Almost all field and forage, fruit and vegetable crops and forest and ornamental plantations are attacked by a wide range of sucking and chewing insect pests (Lehmann et al. 2020). Aphids (Hemiptera; Aphididae) and armyworms (Lepidoptera; Noctuidae) are among the destructive and economically important insect pests throughout the world (Singh and Singh 2020).

Among aphids, the peach-potato aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae) is a highly polyphagous pest species that infests and damages about 400 plant species from 40 families around the globe including Pakistan (Hlaoui et al. 2019). Moreover, M. persicae vectors about 100 plant viruses. Similarly, fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is one of the economically important and notorious lepidopterous pests. It is native to tropical and subtropical Americas but has recently been dispersed to African and Asian countries including Pakistan (Gilal et al. 2020). It infests more than 80 agricultural crops, particularly rice, maize, cotton, millet, sorghum and sugarcane crops (De Groote et al. 2020).

Farmers in Pakistan rely primarily on synthetic pesticides to combat aphid and armyworm infestations on their crops. However, extensive use of broad-spectrum persistent synthetic chemicals has led to many issues such as insecticides resistance (Zhang et al. 2021), secondary pest outbreaks (Gross and Rosenheim 2011), eradication of beneficial fauna (Bueno et al. 2017), environmental contamination and human health hazards (Gomes et al. 2020). In view of these emerging ecological consequences of synthetic insecticides, it is imperative to seek out relatively safer and environment-friendly pest control options such as entomopathogenic fungi (EPF).

A wide range of EPF, particularly belonging to Beauveria, Isaria, Lecanicillium and Metarhizium genera, have been evidenced as potential biological control agents exhibiting significant virulence and pathogenicity against various insect pests. M. persicae and S. frugiperda are naturally infected by many species of EPF, some of which are very effective biocontrol agents (Firake and Behere 2020). The occurrence of such EPF naturally in an environment or agro-ecosystem indicates their potential role as biotic factors regulating insect pest populations in the field (Meyling and Eilenberg 2007).

Moreover, as EPF have a great genetic variation among their different biogeographic strains, the virulence and pathogenicity of different isolates to target insect pests may vary considerably due to their differential enzymatic and molecular characteristics (Maistrou et al. 2020). The present laboratory study aimed to isolate, identify and assess the virulence and entomopathogenicity of indigenous strains of EPF against the aphid M. persicae and the fall armyworm S. frugiperda as model mandibulate and haustellate phytophagous pests of economic importance, respectively.

Methods

Rearing of S. frugiperda

Late instar larvae of fall armyworm (S. frugiperda) were collected from the maize crop (Zea mays L.; hybrid cultivar Pioneer-32B33) grown in the farm area of Agriculture College, University of Sargodha (32°07′54" N; 72°41′35" E). The collected larvae were reared individually in a solitary manner in glass Petri plates (90 mm diameter) under controlled conditions (25 ± 2 °C, 65 ± 5% RH and 14-h: 10-h photoperiod). Larvae were fed on chickpea-based artificial diet prepared according to protocol of Jin et al. (2020) with slight modifications. Diet was changed daily until pupation and the newly formed pupae were placed on moistened filter paper discs lined in glass Petri plates (90 mm diameter). Newly emerged adults were shifted to rearing cages (30 × 30 × 30 cm; Bugdorm-I, Taiwan) for mating and egg lying and were provided with 10% honey solution soaked in cotton swabs as food. Egg clusters were shifted to glass Petri plates (90 mm diameter) on artificial diet. The culture of S. frugiperda was reared in the laboratory up to F4 generation prior to their utilization in experimentation.

Rearing of M. persicae

Colonies of the peach-potato aphid (M. persicae) were randomly collected from the potato crop (Solanum tuberosum L.; white-skin cultivar Diamant) cultivated in the vicinity of Agriculture College, University of Sargodha (32°07′58" N; 72°41′32" E). This aphid population was reared on potted cabbage (Brassica oleracea L. var. botrytis) plants grown under controlled conditions (60 ± 10% RH and 27 ± 2 °C and 14-h: 10-h photoperiod). Old plants were replaced with new ones every week. The culture of M. persicae was reared in the laboratory for several generations before its utilization in the bioassays. Healthy and active insect individuals were used in all bioassays.

Isolation of EPF from naturally dead insects

Cadavers of naturally infected insects were collected from the leaf litter around agricultural field banks and from the sideway areas of an irrigation canal junction (32°08′0.5" N; 72°40′46" E). These sites were selected because of known insect pest activities with no application of insecticides and fungicides for the last few months. Insect cadavers were collected and placed in zip-lock polythene bags and were brought to the laboratory for isolation of EPF. Field-collected insect cadavers, potentially having a fungal infection, were surface-sterilized with 0.5% aqueous solution of sodium hypochlorite, followed by 3 rinsing with sterile distilled water, and then were dried by placing and rolling them on sterile filter paper sheet. Subsequently, these larvae were placed in glass Petri plates (90 mm diameter) lined with moistened sterile filter paper discs and were incubated at 25 ± 2 °C to stimulate the conidial germination (Herlinda et al. 2008).

Fungal growth on insect cadavers was monitored on daily basis. When a fungal growth was observed, the conidia and hyphae were transferred to glass Petri plates (90 mm diameter) lined with potato dextrose agar (PDA) medium with the help of a sterile inoculation needle for purification and identification. Petri plates were incubated at 25 ± 2 °C and were inspected daily for fungal growth. At the point when more than one type of growth was developed on the same plate, they were isolated by sub-culturing. For further purification, small inoculum of fungal mycelia was cut out with a sterile inoculation needle and was moved to new Petri plates lined with Sabouraud dextrose agar (SDA) medium.

Identification of entomopathogenic fungi

Identification of isolated fungal strains was based on cultural and morphological features and was done by observing the growth pattern and colony formation of the fungal cultures. Slides were prepared from each isolate to identify its morpho-taxonomic characters using an inverted trinocular microscope (XDS-3, Optika SRL, Italy) by inspecting conidial structure and morphology according to the available literature and identification keys (Mongkolsamrit et al. 2020). Two cultures were identified as EPF: Beauveria bassiana (Balsam) Vuillemin and Metarhizium anisopliae (Metschn.) Sorokin (Ascomycota, Hypocreales: Clavicipitaceae) and were selected for further determination of their entomo-virulence against the model insect pests i.e., M. persicae and S. frugiperda.

Conidial suspensions preparation

Isolated cultures of B. bassiana and M. anisopliae were further mass cultured on Sabouraud dextrose agar yeast extract (SDAY) medium lined in glass Petri plates (90 mm diameter) incubated at 25 ± 2 °C. Conidial suspensions of both fungal isolates were prepared by harvesting their 15-day-old cultures with the help of a sterile inoculation loop and suspending them in 10 ml sterile double-distilled water in sterile vials containing 0.1% Tween-80 as surfactant. The solution was gently mixed and filtered through a three-layer sterile muslin cloth to eliminate other mycelial mass. Conidial concentrations of both fungal filtrates were determined by improved Neubauer’s hemocytometer as described by Ibrahim et al. (2016). From the stock solution, serial dilutions were made by the concentrations of 1.0 × 109, 1.0 × 108, 1.0 × 107 and 1.0 × 106 conidia/ml and were stored in refrigerator at 4 ºC until their downstream utilization in pathogenicity bioassays.

Pathogenicity assays

Bioassay of isolated EPF strains against M. persicae

Leaf-dip bioassay method as described by Nazir et al. (2019) was followed for determining the virulence of promising isolates of B. bassiana and M. anisopliae against M. persicae. In brief, leaf discs (50 mm diameter) were prepared from freshly clipped cabbage (B. oleracea var. botrytis) leaves and were dipped in each conidial concentration for 15 s. These treated leaf discs were placed on sterile filter paper sheet to remove excessive solution and were then shifted in sterile glass Petri plates (60 mm diameter) lined with 1.0% agar solution. In the control treatment, leaf discs were dipped in sterile double-distilled water containing 0.1% Tween-80 solution. Ten late 3rd instar nymphs of M. persicae were released on the treated leaf discs using sterile camel hair brush, and Petri plates were incubated under controlled conditions (65 ± 5% RH and 25 ± 2 °C). Mortality of exposed aphid individuals was recorded on 3rd, 5th, 7th and 10th day post-exposure. Dead aphid nymphs were removed and placed immediately on moistened filter paper discs lined in glass Petri plates (60 mm diameter) and were inspected daily for the development of fungal mycelia in order to confirm their fungus infection-induced death (Additional file 1: Fig. S1).

Bioassay of EPF isolates against S. frugiperda

The virulence of B. bassiana and M. anisopliae isolates was determined against S. frugiperda by larval-dip bioassay method according to a previously described protocol (Ramanujam et al. 2020). In brief, freshly molted (0–6 h old) 3rd instar larvae of S. frugiperda were immersed in conidial concentrations for approximately 15 s. In control treatments, larvae were dipped in sterile double-distilled water having 0.1% Tween-80. Treated larvae were transferred in glass Petri plates (90 mm diameter) containing freshly cut leaves of cauliflower (B. oleracea var. botrytis) lined on 1.0% agar solution. Ten 3rd instar larvae of S. frugiperda were exposed in each Petri plate, and 5 replications were maintained for each treatment. Petri plates were incubated for 10 days under controlled conditions (27 ± 2 °C and 60 ± 5% RH). Leaves inside plates were changed every 2nd- or 3rd-day intervals. Larval mortality was recorded on 3rd, 5th, 7th and 10th day post-exposure. Dead larvae were removed and inspected for the fungal infection as described above (Additional file 1: Fig. S1).

Statistical analysis

All bioassays were conducted according to the completely randomized design (CRD) with 5 replications for each treatment. Using Statistix® Version 8.1 (Analytical Software, Tallahassee, FL), factorial analysis of variance (ANOVA) was run on the mortality data of insects taking conidial concentration and exposure time as factors. Treatment means were further compared by Tukey’s honestly significant difference (HSD) post hoc test at standard level of probability (α = 0.05). Probit regression analysis was performed to determine the median lethal concentration (LC50) and time (LT50) values using Polo Plus® software (LeOra Software, Parma, MO, USA, 2003).

Results

Virulence and pathogenicity of both indigenous isolates of B. bassiana and M. anisopliae were determined against laboratory-reared 3rd instar nymphs of M. persicae and 3rd instar larvae of S. frugiperda under laboratory conditions.

Mortality response of M. persicae to B. bassiana and M. anisopliae isolates

Factorial analysis of M. persicae bioassay revealed that there was a significant effect of both fungal concentration (F3, 64 = 76.36; P ≥ 0.001 for B. bassiana and F3, 64 = 31.53; P ≥ 0.001 for M. anisopliae) and time (F3, 64 = 119.87; P ≥ 0.01 for B. bassiana and F3, 64 = 71.65; P ≥ 0.01 for M. anisopliae) factors and their interactions (F9, 64 = 5.14; P ≥ 0.001 for B. bassiana and F9, 64 = 3.56; P ≥ 0.001 for M. anisopliae) on the mortality of M. persicae nymphs (Table 1).

Table 1 Mean percent mortality of late 3rd instar nymphs of Myzus persicae exposed to different concentrations of indigenous isolates of entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae under laboratory conditions
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Mean percent mortality of M. persicae nymphs was conidial concentration and time dependent as it increased along with the increase of conidial concentrations and exposure time. In case of B. bassiana isolate, aphid nymphal mortality increased significantly from day 3 to day 10 at all concentrations (R2 = 0.97–0.99). Maximum nymphal mortality (94%) was exhibited by the highest concentration (1.0 × 109 conidia/ml) of B. bassiana recorded on 10th day of bioassay, while minimum aphid mortality (8–31%) was observed for the lowest concentrations (1.0 × 106 and 1.0 × 107 conidia/ml) on 3rd and 5th day of bioassay (Fig. 1). Similar trend of gradual and significant increase in the mortality of M. persicae nymphs was recorded for M. anisopliae isolate. Nymphal mortality increased significantly from day 3 to day 10 for all conidial concentrations (R2 = 0.86–0.96). Maximum percent mortality (65%) was found at the highest conidial concentration (1.0 × 109 conidia/ml) on 10th day of bioassay, while the minimum aphid mortality values (6 – 24%) were recorded for low concentrations (1.0 × 106 and 1.0 × 107 conidia/ml) at 3rd and 5th day of bioassay (Fig. 2).

Fig. 1
figure 1

Percent corrected mortality (mean ± S.E.) of late 3rd instar nymphs of peach-potato aphid Myzus persicae bioassayed against different concentrations of indigenously isolated strain of entomopathogenic fungus Beauveria bassiana under laboratory conditions. Concentrations C1–C4 correspond to 1.0 × 106–1.0 × 109 conidia/ml. Small letters at bar tops indicate significant difference among the concentrations, while capital letters indicate overall significant difference among the mortality at different time intervals (factorial ANOVA followed by Tukey's HSD test at α = 0.05)

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Fig. 2
figure 2

Percent corrected mortality of late 3rd instar nymphs of peach-potato aphid Myzus persicae bioassayed against different concentrations of indigenously isolated strain of entomopathogenic fungus Metarhizium anisopliae under laboratory conditions. Concentrations C1–C4 correspond to 1.0 × 106–1.0 × 109 conidia/ml. Small letters at bar tops indicate significant difference among the concentrations, while capital letters indicate overall significant difference among the mortality at different time intervals (factorial ANOVA followed by Tukey's HSD test at α = 0.05)

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Probit regression analysis of data regarding percent corrected mortality of M. persicae nymphs revealed that B. bassiana was most virulent and fast-acting against 3rd instar nymphs of M. persicae than M. anisopliae isolate. Median lethal time (LT50) values were 4.57 and 6.48 days for 1.0 × 109 conidia/ml and were 6.86 and 7.88 days for 1.0 × 108 conidia/ml of B. bassiana and M. anisopliae, respectively (Table 2). Similarly, medial lethal concentration (LC50) values of 1.67 × 107 and 7.75 × 106 conidia/ml at 7th day and 1.12 × 108 and 8.70 × 107 conidia/ml at 10th day were recorded for B. bassiana and M. anisopliae, respectively (Table 3).

Table 2 Median lethal time (LT50) values for the indigenous strains of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae bioassayed against late 3rd instar nymphs of peach-potato aphid Myzus persicae under laboratory conditions
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Table 3 Median lethal concentration (LC50) values for the indigenous strains of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae bioassayed against late 3rd instar nymphs of peach-potato aphid Myzus persicae under laboratory conditions
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Pathogenicity of B. bassiana and M. anisopliae isolates against S. frugiperda larvae

Factorial analysis revealed a significant effect of fungal concentrations (F3, 64 = 31.63; P ≥ 0.001 for B. bassiana and F3, 64 = 43.40; P ≥ 0.001 for M. anisopliae) and time (F3, 64 = 300.63; P ≥ 0.01 for B. bassiana and F3, 64 = 721.93; P ≥ 0.01 for M. anisopliae) factors and their interactions (F9, 64 = 12.88; P ≥ 0.001 for B. bassiana and F9, 64 = 7.44; P ≥ 0.001 for M. anisopliae) on the larval mortality of S. frugiperda (Table 4). For both fungal isolates, mean percent mortality of S. frugiperda larvae appeared in a time- and concentration-dependent manner as it increased along with the increase of exposure time and conidial concentration. For B. bassiana isolate, larval mortality increased significantly from day 3 to day 10 at all concentrations (R2 = 0.82–0.93). Maximum percent mortality (76%) was observed by the highest concentration (1.0 × 109 conidia/ml) recorded on 10th day of bioassay, while minimum mortality (2–4%) was observed for the lowest concentrations (1.0 × 106 and 1.0 × 107 conidia/ml) on 3rd and 5th day of bioassay (Fig. 3). For M. anisopliae isolate, larval mortality increased as well significantly from day 3 to day 10 at all concentrations (R2 = 0.91–0.94). Maximum percent mortality (88%) was caused by the highest concentration (1.0 × 109 conidia/ml) recorded at 10th day of bioassay, while the minimum mortality values (0–6%) were observed for the lowest concentrations (1.0 × 106 and 1.0 × 107 conidia/ml) at 3rd day of bioassay (Fig. 4).

Table 4 Mean percent mortality of 3rd instar larvae of fall armyworm Spodoptera frugiperda exposed to different concentrations of indigenous isolates of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae under laboratory conditions
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Fig. 3
figure 3

Percent corrected mortality of 3rd instar larvae of fall armyworm Spodoptera frugiperda bioassayed against different concentrations of indigenously isolated strain of entomopathogenic fungus Beauveria bassiana under laboratory conditions. Concentrations C1–C4 correspond to 1.0 × 106–1.0 × 109 conidia/ml. Small letters at bar tops indicate significant difference among the concentrations, while capital letters indicate overall significant difference among the mortality at different time intervals (factorial ANOVA followed by Tukey's HSD test at α = 0.05)

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Fig. 4
figure 4

Percent corrected mortality of 3rd instar larvae of fall armyworm Spodoptera frugiperda bioassayed against different concentrations of indigenously isolated strain of entomopathogenic fungus Metarhizium anisopliae under laboratory conditions. Concentrations C1–C4 correspond to 1.0 × 106–1.0 × 109 conidia/ml. Small letters at bar tops indicate significant difference among the concentrations, while capital letters indicate overall significant difference among the mortality at different time intervals (factorial ANOVA followed by Tukey's HSD test at α = 0.05)

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Probit analysis data revealed that M. anisopliae was most virulent and fast-acting against 3rd instar larvae of S. frugiperda than B. bassiana isolate. Median lethal time (LT50) values were 7.75 and 8.71 days for 1.0 × 109 conidia/ml of B. bassiana and M. anisopliae, respectively, while these were 7.03 and 7.93 days for 1.0 × 108 conidia/ml of B. bassiana and M. anisopliae, respectively (Table 5). Similarly, LC50 values of 2.84 × 107 and 8.84 × 105 conidia/ml were recorded at 10th day for B. bassiana and M. anisopliae, respectively (Table 6).

Table 5 Median lethal time (LT50) values for the indigenous strains of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae bioassayed against 3rd instar larvae of fall armyworm Spodoptera frugiperda under laboratory conditions
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Table 6 Median lethal concentration (LC50) values for the indigenous strains of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae bioassayed against 3rd instar larvae of fall armyworm Spodoptera frugiperda under laboratory conditions
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Discussion

Contemporary issues of environmental contamination and health hazards being manifested by the extensive and recurrent use of highly persistent and hazardous synthetic insecticides necessitate looking for relatively safer and environment-friendly pest control options such as EPF which have been effective against a wide number of insect pest species (Litwin et al. 2020). However, these fungi exhibit considerable genetic variations among their biogeographic strains and the virulence and pathogenicity of different isolates to target insect pests may vary according to their differential enzymatic and molecular characteristics (Maistrou et al. 2020).

This laboratory work isolated, identified and assessed the virulence and pathogenicity of 2 promising indigenous strains of B. bassiana and M. anisopliae against M. persicae and S. frugiperda as a model mandibulate and haustellate phytophagous pests of economic importance. B. bassiana and M. anisopliae are ubiquitously found soil-born fungi capable of parasitizing a wide range of insect and mite pests (McGuire and Northfield 2020).

Bioassay results of aphids revealed that the indigenous isolate of B. bassiana was more pathogenic against 3rd instar nymphs exhibiting significantly a high mortality and minimum LT50 and LC50 values than those of M. anisopliae. These results are in line with the study of Bugti et al. (2018) in which four hemipteran pests including M. persicae were exposed to different conidial concentrations (1.0 × 102 to 6.75 × 105 conidia/mm2) of a B. bassiana strain (Bb-202) and demonstrated that B. bassiana showed the highest pathogenicity to M. persicae and caused maximum mortality (100%) with LC50 and LT50 values of 6.7 × 104 conidia/ml and 5.2 to 8.24 days, respectively. Earlier, Kim et al. (2013) demonstrated the filtrates of B. bassiana were most pathogenic against M. persicae among 47 cultural filtrates of Isaria, Lecanicillium, Beauveria and Cordyceps spp. Similar results were revealed by Nazir et al. (2019) evaluating 3 strains of B. bassiana and Lecanicillium lecanii against M. persicae adults. Also it was found that Bb-72 and Bb-252 strains of B. bassiana caused the highest mortality rate (95 and 91%, respectively) of M. persicae with minimum LC50 values (3.09 × 104 and 1.29 × 104 conidia/ml, respectively) and also showed mutual compatibility of these B. bassiana strains.

On the other hand, bioassays with armyworms revealed that the isolate of M. anisopliae was relatively more virulent against 3rd instar larvae of S. frugiperda and showed maximum mortality with minimum LT50 and LC50 values than those of B. bassiana. These results are in accordance with those of Cruz-Avalos et al. (2019) showed that among various strains of B. bassiana, M. anisopliae and Nomuraea rileyi isolated from soil samples and from naturally infected S. frugiperda larval cadavers, the isolates of M. anisopliae, particularly isolate Ma41, showed the highest larval mortality (100%) with LC50 value of 2.8 × 105 conidia/ml. A similar laboratory bioassay revealed 100% mortality of S. frugiperda larvae and eggs by a virulent strain of M. anisopliae with LT50 value of 2 to 3 days at 1.0 × 108 conidia/ml concentration (Lezama-Gutiérrez et al. 1996).

Many previous studies have demonstrated that B. bassiana and M. anisopliae are not only virulent and pathogenic against M. persicae and S. frugiperda, respectively, under laboratory bioassays (Nazir et al. 2019), but also have been effective under the field conditions and appeared promising options for sustainable management of S. frugiperda and other lepidopterous insect pests (Mwamburi, 2021) and against sucking insect pests including M. persicae (Dannon et al. 2020). In cage and field experiments, aqueous conidial suspensions of B. bassiana isolates (CG-864 and PL-63) were demonstrated to reduce the M. persicae population and infestation by 60 to 80% than the control (Filho et al. 2011).

However, the present findings are not in line with those of Montecalvo and Navasero (2021) who demonstrated that the virulence of B. bassiana and M. anisopliae varied according to different life stages of S. frugiperda. In this study, B. bassiana appeared more virulent to 1st than 6th larval instars with LC50 values of 0.06 × 108 to 9.43 × 108 conidia/ml, respectively, but LT50 values were 4.6 to 7.5 days, respectively. However, interestingly M. anisopliae isolate was more pathogenic to 3rd instar S. frugiperda larvae than B. bassiana although their difference was statistically non-significant. Moreover, the virulence of indigenous isolates may vary according to target insect pests. For instance, Gebremariam et al. (2021) showed that the indigenous Ethiopian isolates of B. bassiana were more pathogenic and lethal to G. mellonella than M. anisopliae isolates from the same soil samples.

Moreover, some studies have revealed the potential role of these EPF in phytopathogen antagonism, endophytism, rhizosphere colonization and in triggering the plant growth hormones (Ramos et al. 2020). Similarly, these insect parasitic fungi are also compatible with other pest control tactics including conventional and differential chemistry synthetic insecticides and along with other non-chemical control strategies (Quintela et al. 2013). For instance, both B. bassiana and M. anisopliae fungi have been shown compatibility with chlorpyrifos and spinosad (Rivero-Borja et al. 2018) and with pheromone traps (Akutse et al. 2020) against S. frugiperda.

Conclusions

It is concluded that the indigenous isolate of M. anisopliae was more virulent to S. frugiperda larvae than B. bassiana isolate, while the later fungal isolate appeared to be more lethal to M. persicae nymphs than the former one, exhibiting significant mortality and minimum LT50 and LC50 values. These results corroborate the effectiveness of different strains and isolates of B. bassiana and M. anisopliae against M. persicae and S. frugiperda, respectively, and advocate the significance of considering indigenous isolates of microbial biocontrol agents against native and exotic insect pests. However, field evaluations of these indigenously isolated promising fungal strains against these target insect pests and on their natural enemies constitute the future perspectives of this work.