Occurrence and Distribution of Entomopathogenic Fungi in Cultivated Soil and Its Efficacy Against Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) Under Laboratory Conditions

1. Introduction

The tomato fruit worm, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), is a highly destructive and cosmopolitan pest (Kriticos et al. 2015, Dinesh et al. 2017, Chen et al. 2018, Fathipour et al. 2020). Its larval stage is highly voracious and infests over 181 host plant species belonging to more than 47 families (Bird 2017, Mishra and Omkar 2021). This pest successfully adapts to various agro-ecosystems due to its polyphagous nature, facultative diapause, high reproductive capacity, and migratory potential (Chaitanya et al. 2014, Jones et al. 2019, Katsikis et al. 2020, Riaz et al. 2021). In Pakistan, from the late 20th century to the early 21st century, this pest was identified as a significant factor in yield reduction and threatening major field crops (Karim 2000).

Tomatoes, Lycopersicon esculentum (Mill.) are among the valuable crops targeted by H. armigera. In Pakistan, tomatoes are the second most produced crop in the horticultural sector (Chohan and Ahmad 2008). The initial instars of H. armigera feed on green shoots and foliage, while subsequent stages target buds, inflorescences, and fruiting bodies, resulting in serious damage to post-harvest produce (Wang and Li 1984, Czepak et al. 2013, Pratissoli et al. 2015). This noctuid pest is estimated to cause around 53% fruit loss in tomatoes (Saljoqi et al. 2022). Farmers in Pakistan and around the world mainly rely on chemical applications to control this pest (Shaheen 2008, Fite et al. 2019). However, the excessive use of broad-spectrum insecticides has raised serious concerns about their harmful effects on human health, the environment, non-target species, disturbs ecological balance and leads to pest resurgence and the development of pest resistance (David 2008, Shah et al. 2013, Sun et al. 2019, Riaz et al. 2021). According to the Arthropod Pesticide Resistance Database (APRD) (http://www.pesticideresistance.org/DB/), this pest has become resistant to over 40 active ingredients. This situation has increased interest in finding more eco-friendly alternatives to reduce the harmful impacts of insect pests and support sustainable farming (Sezen and Demirbag 2006).

One such approach is biological control with insect-invading pathogens (Ai et al. 2018). Entomopathogenic fungi (EPF) are eco-friendly microbial agents naturally found in various terrestrial habitats (Sanchez-Peña 2000, Sevim et al. 2010, Mahankuda and Bhatt 2019); widely recognized worldwide in integrated pest management due to their broader host range, ability to cause lethal mycosis in insect pests through cuticle penetration, and capability to control different developmental stages more effectively than other strategies (Vega et al., 2012; Maina et al., 2018). Moreover, EPF strains promote long-term environmental sustainability by germinating on insect cadavers, thereby enhancing their inoculum density and disseminating their network throughout the ecosystem (Moore et al. 2012).

Metarhizium spp. are frequently used or tested as eco-friendly microbiological agents because they are easy to produce on a large scale (Greenfield et al. 2015). Metarhizium rileyi (Farl.), formerly known as Nomuraea rileyi (Kepler et al. 2014), is a potential EPF that can cause lethal epizootics in various insects, including noctuid pests (Jaronski 2014, Ramos et al. 2024). Various studies have reported the potential of M. rileyi for developing a microbial pesticide against lepidopterous pests (Vega-Aquino et al. 2010, Hatting et al. 2012). M. rileyi is widely used in control strategies for H. armigera (Yuan and Yong 2010, Inglie et al. 2017) due to its primary features, including environmental friendliness and host specificity (Sinha et al. 2016). Aspergillus is another important genus within Ascomycota, encompassing more than 250 species (Kotta-Loizou 2021). Some of these species have been identified as entomopathogenic, such as A. niger (Kaur et al. 2016), A. parasiticus (Nnakumusana 1985, Khan et al. 2025), and A. flavus (Askar et al. 2024). Aspergillus spp. employ a unique infection method by initiating lipid peroxidation, followed by a series of events that ultimately compromise the host’s immunity. They release toxic compounds that cause gut tissue leakage and induce septicemia, leading to the death of the host insect (Karthi et al. 2024).

Mensah et al. (2015) reported the effective control of H. armigera through the use of local Aspergillus sp. (BC 639), which also had a minimal impact on beneficial organisms. Similarly, Sebayang et al. (2021) explored native EPF species as microbial agents against H. armigera and isolated A. flavus and other EPF species from infected H. armigera larvae. These species have been utilized as microbial agents against other agricultural pests, including Oligonychus coffeae (Mazid et al. 2015), Dysdercus koenigii (Kumari et al. 2019), Spodoptera frugiperda (Idrees et al. 2021), and Spodoptera litura (Kaur et al. 2016, Kaur et al. 2025). However, in Pakistan, the potential of these entomopathogens viz., M. rileyi, A. niger, A. parasiticus, and A. flavus has never been investigated against the noctuid pest, H. armigera.

Moreover, understanding the composition and distribution of indigenous species is vital for preserving fungal strains in soil, which is necessary for effectively managing local pest populations in agro-ecosystems (Meyling and Eilenberg 2006, Hussein et al. 2010). Additionally, laboratory evaluations are crucial to determine effective concentration levels, ensuring the success and safety of future field applications against this pest. The study’s objectives were to explore indigenous EPF strains in cultivated soils of KP province and evaluate their potential against the local pest, H. armigera, using the topical inoculation method under laboratory conditions. The main parameters examined included the comparative distribution of fungal isolates in plain areas versus foothills and between agronomic versus horticultural crops, along with first mortality, total mortality (%), pupal recovery (%), adult emergence (%), and the determination of LC₅₀ and LT₅₀.

2. Materials and Methods

2.1. Study Area

Isolation of entomopathogenic fungi and pathogenicity bioassays were performed in the Biocontrol and Plant Pathology laboratories of Nuclear Institute for Food Agriculture (NIFA), Peshawar. The morphological identification of isolated EPF species was done in the Vector Biology Laboratory of the Institute of Zoological Sciences, University of Peshawar.

2.2. Collection of Soil Samples

Soil samples were collected from four agronomic (rice, maize, sugarcane, mustard) and four horticultural crops (tomato, potato, guava, peach) from distinct geographical terrains in Khyber Pakhtunkhwa province (Plains: Peshawar and Charsadda and Foothills: Abbottabad and Haripur) during 2023-24. The details regarding the geographic coordinates of surveyed regions are given in Figure 1.

From each locality, five soil samples (~200 g each) were collected from a depth of 5-10 cm at five random points by using a hand shovel with three replicates per point and stored in sterile zip-lock bags (28×20 cm) at 4 °C in a refrigerator until used (Mantzoukas et al. 2022).. The samples from each site were mixed to get a unit sample and sieved through a 16 mm mesh to remove roots, stones, litter, etc. Soil samples were then spread on rectangular trays and kept open to remove excessive moisture to avoid nematode infestation as suggested by Quesada-Moraga et al. (2007).

2.3. Isolation of Entomopathogenic Fungi

Galleria bait method was used for isolating the entomopathogenic fungi from collected soil samples (Zimmerman 1986). The wax moth larvae, Galleria mellonella (Lepidoptera: Pyralidae), were originally collected from infested bee combs acquired from local beekeepers. The G. mellonella culture was sustained on a suitable diet, specifically bee wax, and kept in rectangular trays within insect rearing cages (60 x 60 x 60 cm) under controlled conditions i.e., 26 ± 2℃ temp., 65±5% R.H. and continuous darkness (Sevim et al. 2010). The dry soil samples were first moistened to field capacity (Quesada-Moraga et al. 2007), and then, after thorough mixing, from each unit sample, five sub-samples were taken into autoclaved glass vials. At the top of the soil, about 1 cm space was left and vial lids were provided with needle holes for proper aeration. Five healthy 3rd instar larvae were added to each vial using sterile forceps and incubated at 26 ± 2℃ for ten days. The vials were inverted daily to ensure regular larval movement through the soil. After incubation, the soil was examined, and the bait larvae were removed. The larval cadavers were first surface sterilized in 3% sodium hypochlorite (disinfectant) for 3 min, then washed with sterile distilled water for 3 min as suggested by Sookar et al. (2008) and individually incubated at 26 ± 2℃ on wet filter paper in sterile glass petri plates for three days to enhance pathogen sporulation.

The cadavers with symptoms of fungal infection were transferred to Sabouraud Dextrose Yeast Agar (SDAY) plates comprising 10 g dextrose, 2.5 g peptone, 2.5 g yeast extract and 20 g agar in 1 litre of distilled water (supplemented with antibiotics 100 mg/l to inhibit bacterial growth) (Sevim et al. 2010) inside a laminar flow cabinet in Plant Pathology Laboratory, NIFA. They were kept at 26 ± 2℃ temperature, 60±5% relative humidity in complete darkness till the growth of fungal mycelium. The culture obtained from an infected bait larvae was considered an isolate. The name code for isolates were based according to crop: Maize (M), Sugarcane (S), Mustard (Mu), Rice (R), Potato (P), Tomato (T), Guava (G) and Peach (P); replication (R1-R3) and sample number within crop (S1-S5) and thus samples were labeled as MR1S1, RR2S5, etc.

2.4. Identification of EPF Isolates

The fungal isolates were identified based on morphological traits, such as colony appearance, spore size and shape, etc., using a taxonomic key in Vector Biology Laboratory of the Institute of Zoological Sciences, University of Peshawar (Humber 2005). Pure culture plates of each fungal isolate were then submitted to the First Fungal Culture Bank of Lahore, Pakistan (FCBP) for confirmation. The identified fungal cultures were deposited in the Biocontrol laboratory of Nuclear Institute for Food Agriculture (NIFA), Peshawar, and Vector Biology Laboratory of the Institute of Zoological Sciences, University of Peshawar.

2.5. Insect Rearing

H. armigera larvae were collected from the NIFA field area and initially reared on a natural diet (peas) in plastic cups. Pupae were collected and transferred to glass vials for adult emergence. The emerged adults were coupled, kept in 2.5 L plastic jars covered with muslin cloth as a substrate for oviposition, and provided with a 10% sugar solution. The muslin cloth with eggs was changed daily and shifted to plastic boxes, with an artificial diet for hatchlings, comprising of chickpea powder (79.6%), scorbic acid (1%), methyl para hydroxyl benzoate (0.8%), ascorbic acid (0.3%), streptomycin (0.3%), yeast (12.7%), agar (0.1%) and vitamin B12 (0.0001%) by weight in distilled water (1000 ml). After hatching, the third instars of the F1 generation were used in bioassays. All developmental stages of H. armigera were kept under controlled conditions of 26 ± 2℃ temperature, 65±5% relative humidity, and L14: D10 hr photoperiod.

2.6. Pathogenicity of Entomopathogenic Fungi on H. armigera

Bioassays were performed in the Biocontrol laboratory of NIFA to evaluate the pathogenicity of isolated EPF species against H. armigera. Conidial suspensions were prepared from 2-3 weeks old pure EPF cultures in 10 ml of distilled water with Tween-80 (0.1%) in sterile 15 ml tubes and quantified using a Hemocytometer (Sorathiya et al. 2023). The desired concentrations of each fungal strain, i.e., 1×10⁸, 1×10⁷, and1×10⁶ conidia/ml, were adjusted through serial dilution. Three replicates were used per treatment and each replicate contained nine 3rd instar of H. armigera, which were topically inoculated using a micropipette with a volume of 10 microliter of conidial suspensions. In a control treatment, the larvae were treated similarly, using only distilled water. The bioassay was repeated twice and during the experimental period, the larvae were provided with artificial diet and adults were fed with 10% sugar solution.

2.7. Parameters to Be Studied

2.7.1. Percentage Diversity of Soil-Dwelling EPF

To calculate the percentage diversity of a particular identified EPF strain (x), the following formula was used:

% Diversity of ‘x’ = ${\displaystyle \frac{\mathrm{T}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{c}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{t} \mathrm{o}\mathrm{f} \mathrm{I}\mathrm{d}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{f}\mathrm{i}\mathrm{e}\mathrm{d} \mathrm{E}\mathrm{P}\mathrm{F}}{\mathrm{N}\mathrm{o}.\mathrm{o}\mathrm{f} \mathrm{I}\mathrm{d}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{f}\mathrm{i}\mathrm{e}\mathrm{d} \mathrm{E}\mathrm{P}\mathrm{F} \mathrm{s}\mathrm{p}\mathrm{e}\mathrm{c}\mathrm{i}\mathrm{e}\mathrm{s} \left(\mathrm{x}\right)}}$ × 100

2.7.2. Concentration-Response Bioassay

Concentration-response tests were conducted for different parameters. In each treatment, as noted above, nine healthy third instars were placed individually in petri plates and topically inoculated with conidial suspension and in the control group, treated with distilled water. The first larval mortality data for each treatment’s replication were recorded for 5 days following the inoculation of EPFs. The larvae showing no indication of movement were considered dead. The total mortality and median lethal concentration (LC₅₀) for each fungal treatment were determined at different post-inoculation intervals: total mortality after 7 and 14 days, LC₅₀ after 3, 7, and 14 days. The effect of different concentrations of EPFs on the pupal period and adult emergence of H. armigera was also assessed. The median lethal time (LT₅₀) was also estimated for each fungal concentration.

2.8. Research Design

A Completely Randomized Design (CRD) was followed, with 3x replicates. The aspect used was the conidial concentration treatments (T) with the following levels for each fungal species: 1×10^6, 1×10⁷, and 1×10⁸ conidia/ml. The bioassay was conducted twice, comprising a total of thirteen treatments, including a control group.

2.9. Statistical Analysis

The bioassay data (percent mortality, pupal recovery and adult emergence) were analyzed using one-way ANOVA with Statistix version 8.1 software to determine means and different parameter values for tested fungal treatments. The means of significant treatments were compared with Tukey’s HSD (Honestly Significant Difference) test at a significance level of α = 0.05. To estimate LC₅₀ and LT₅₀ and their associated P values, degrees of freedom, chi-square and slopes of tested EPF species, probit analysis was performed on bioassay data using SPSS v16.

3. Results

3.1. Isolation and Identification of EPF Isolates

A total of 209 fungal isolates were recovered from baited wax moth larvae. Based on morphological traits identified using a taxonomic key (Humber 2005) and confirmed by the Fungal Culture Bank of Pakistan (FCBP), fungal isolates were classified into four different EPF species: Nomuraea rileyi, Aspergillus parasiticus, Aspergillus flavus, and Aspergillus niger. Their morphological details are presented in Figure 2.

3.2. Comparative Distribution of Fungal Isolates in Different Agro-Ecological Zones of KP Province

The comparative distribution of EPF isolates was determined in cultivated soil from diverse agro-ecological zones, including plains and foothills of KP Province. Of the total 209 isolates, 133 were obtained from cultivated soils in the plain area districts, i.e., Peshawar and Charsadda, while the remaining 76 isolates came from the foothills districts, i.e., Haripur and Abbottabad. Our results revealed that the comparative distribution of all isolated EPF species was higher in the soil of the foothills than in the plain areas (Figure 3). Overall, fungal species composition was uniform throughout, with the highest percentage distribution of Aspergillus niger i.e., 48% in the foothills and 37% in the plain regions, while Nomuraea rileyi had the lowest i.e., 15% in the foothills and 11% in the plain areas.

3.3. Comparative Distribution of EPF Isolates in Different Cropping Systems

The percent distribution of fungal isolates was compared across different cropping systems viz., agronomic and horticultural crops of Plains and Foothills in KP Province. Of 209 isolates, the highest number (119) was found in various horticultural crops. The percent distribution of nearly all fungal isolates in horticultural crops was relatively higher than in different agronomic crops (Table 1). Overall, the highest percent distribution was recorded for A. niger i.e., 43% in horticultural crops and 39% in agronomic crops, while A. parasiticus had the lowest percent distribution with 6% in horticultural and 5% in agronomic crops. (Table 1).

3.4. Concentration-Response Bioassays

1. First larval mortality of H. armigera exposed to different EPF concentrations 

First mortality is the duration in which different EPF concentrations caused the first larval death to be recorded over 5 days. Results showed that at relatively high concentration levels (1 × 10⁸, 1 × 10⁷ conidia/ml), the first larval mortality in all fungal treatments occurred in a minimum time i.e., within 2 days. Nomuraea rileyi was found to be the most effective, causing the first mortality in the minimum time (Table 2).

2. Total Percent Mortality of H. armigera exposed to different EPF concentrations at three post-inoculation intervals 

The total mortality for each treatment was recorded at two post-inoculation intervals (PII): 7, and 14 days against the third instar of H. armigera. The recorded data were analyzed using ANOVA to assess the differences among treatments and the group means were compared with Tukey’s HSD (Honestly Significant Difference) test at a significance level of α = 0.05. Our findings revealed that at 7d PII, N. rileyi exhibited the highest mortality percentage in the third instar at 1×10⁸ conidia/ml followed by A. niger at the same conidial concentration, while the lowest mortality was recorded in control group (F=13.1, P=0.000). At 14d PII, the highest mortality against the third instar was recorded with higher concentrations of tested EPF and no mortality occurred in the control treatment (F=10.1, P=0.000) (Table 3). Among the tested EPF species, N. rileyi demonstrated the highest total mortality with 1×10⁸ conidia/ml at different post-inoculation intervals.

3. Effect of different EPF concentrations on Pupal Recovery (%) and Adult Emergence (%) of H. armigera 

The statistical analysis revealed that different conidial concentrations of N. rileyi significantly reduced the percent pupal recovery and adult emergence of previously treated H. armigera larvae (Table 4). The highest percent pupal recovery was recorded in the control group, which significantly decreased with increasing concentration levels and reached its lowest at higher concentrations of 1×10⁸, 1×10⁷ conidia/ml of N. rileyi, and 1×10⁸ conidia/ml of other tested species (F=13.1, P=0.000). Similarly, the lowest percent emergence was observed in higher concentrations of the tested EPF species. While the highest percent adult emergence was recorded in the control group (F=3.01, P=0.00) (Table 4).

4. LC₅₀ and LT₅₀ Values of the Tested EPF Species Against H. armigera 

Using SPSS v16 and a probit analysis on mortality data, the LC₅₀ and LT₅₀ values were calculated for the EPF species, setting P<0.05 as the significance level. We measured LC₅₀ values at three post-inoculation intervals: 3, 7, and 14 days, against the third instar of H. armigera. Our findings showed that the LC₅₀ values of the tested EPF species were time-dependent and decreased significantly as the post-inoculation interval increased. At 3d, the lowest LC₅₀ was recorded for N. rileyi (1.36×10⁸ spores/ml), followed by A. niger, A. flavus (1.15×10⁹ spores/ml) and Aspergillus parasiticus (1.66×10⁹ spores/ml) (Table 5). At 7d, N. rileyi was the most virulent, with the lowest LC₅₀ (2.40×10⁶ spores/ml), followed by A. parasiticus (3.14×10⁶ spores/ml) and A. niger (8.06×10⁶ spores/ml), while A. flavus was the least effective, showing the highest LC₅₀ (3.18×10⁷ spores/ml) (Table 6). At 14d, the lowest LC₅₀ was recorded for A. flavus (4.00×10⁵ spores/ml) followed by N. rileyi (7.19×10⁵ spores/ml), A. parasiticus (8.05×10⁵ spores/ml) and A. niger (1.88×10⁶ spores/ml) (Table 7).

Median Lethal Time (LT₅₀) 

Median lethal time (LT₅₀) was also estimated for each fungal concentration, i.e., 1×10⁶, 1×10⁷, and 1×10⁸ conidia/ml. At 1×10⁶ conidia/ml, the lowest LT₅₀ was observed in A. parasiticus (8.77 days), followed by A. flavus (9.62 days), M. rileyi (9.72 days), and A. niger (12.9 days) (Figure 4 a-c). At 1×10⁷ conidia/ml, M. rileyi showed the lowest LT₅₀, followed by A. flavus and A. parasiticus, with A. niger having the highest LT₅₀ (10.6 days) (Figure 4 b). Similarly, at 1×10⁸ conidia/ml, M. rileyi was the most virulent with the lowest LT₅₀ (5.45 days), followed by A. niger (8.25 days) and A. parasiticus (8.49 days), while the highest LT₅₀ was recorded for A. flavus (8.55 days) (Figure 4 c).

4. Discussion

Many studies have reported the isolation and identification of fungal entomopathogens in different habitats (Sookar et al. 2008, Tkaczuk et al. 2014, Gorczyca et al. 2018). However, in Pakistan, due to limited knowledge about the occurrence and distribution of native fungal species, no microbial pesticide has been registered or made commercially available to farmers for local pest control (Iqbal et al. 2021). This study confirms the presence of potential EPF in cultivated soils. Our results revealed that the distribution percentage of fungal isolates was higher in the soil of the foothills compared to the plains, indicating a significant presence in the soils of the higher altitude districts (Haripur and Abbottabad). These findings align with those of Wakil et al. (2013), who evaluated the occurrence of fungal entomopathogens in different regions of Punjab province and reported the highest distribution of fungal isolates in soil from higher altitudes (above 600 m). While Liu et al. (2021) identified ultraviolet exposure as a major factor negatively affecting the occurrence and persistence of EPF species in soil, which contradicts our findings, as foothill soils, being at higher altitudes, are more exposed to ultraviolet radiation than those in plain areas. The difference may be due to variations in the organic content and soil texture that significantly influence the occurrence of EPF in soil habitats (Uzman et al. 2019).

The comparative distribution of fungal isolates was higher in different horticultural crops than in agronomic fields. Our results are in partial agreement with Qayyum et al. (2021), who reported higher fungal distribution frequency in fruits (52.50%) and vegetables (42.50%) than in field crops (37.50%). Similarly, Sookar et al. (2008) also observed that the occurrence and distribution of EPF were significantly influenced by habitat type. They recovered a larger number of Metarhizium anisopliae isolates from the vegetable soil than from other plantations. Notably, in our results, Aspergillus was the most prevalent genus, with Aspergillus niger found as the most abundant EPF species among the fungal isolates, followed by A. flavus. Our results are supported by Qayyum et al. (2021), who reported A. niger as the most abundant fungal strain with a distribution frequency of 27.50% followed by A. flavus with 22.50% occurrence in sampled soil. This is because various ecological and environmental factors influence the distribution and abundance of EPF in the soil habitat (Tkaczuk et al. 2014). Similarly, Wakil et al. (2013) recovered Aspergillus as the most common genus from soil samples.

The virulence of isolated EPF species, identified as N. rileyi, A. parasiticus, A. niger, and A. flavus was evaluated in bioassays against the third instar of H. armigera. The larvae were found susceptible to all tested fungal entomopathogens, resulting in significant mortality, especially at their highest concentration 1×10⁸ conidia/ml. Our results showed a dose-dependent mortality response, with N. rileyi as the most effective treatment, causing the highest percentage mortality, followed by Aspergillus species. These findings are partly consistent with those of Hazarika et al. (2016), who reported concentration-dependent mortality with N. rileyi and identified it as a promising biocontrol agent, causing 86% mortality in H. armigera at a concentration of 1 × 10⁹ spores/mL. The difference in the maximum mortality recorded in the present study and Hazarika et al. (2016) is attributed to variations in strains and the concentrations assessed in both studies. Our results are highly consistent with those of Dev et al. (2021), who found M. rileyi to be highly virulent against H. armigera, with the highest mortality observed in the third instar at 1×10⁸ conidia/ml. M. rileyi causes host-preferential epizootics in susceptible larvae mainly by blocking respiratory structures, leading to host death (Sabbour and Abdel-Rahman 2013). Similarly, Ramos et al. (2024) recorded an effective mortality rate (70 to 98.7%) in Spodoptera frugiperda larvae when treated with 24 different isolates of M. rileyi in bioassays.

Several studies have reported the entomopathogenic potential of Aspergillus species against various insect pests, including lepidopterans (Fitriana et al. 2021), with limited research focused on H. armigera. Mensah et al. (2015) assessed the efficacy of Aspergillus sp. (BC 639) against Helicoverpa spp. and predatory insects both in vitro and in cotton fields. They reported effective pest control using Aspergillus spp. with minimal effects on beneficial organisms, maintaining crop yield. Similarly, Kaur et al. (2016) reported a 63% larval mortality in Spodoptera litura using ethyl acetate extract of A. niger compared to 3% in the control group.

The pupal recovery and adult emergence of treated H. armigera larvae were significantly reduced with increasing conidial concentration. The lowest percent pupal recovery and percent emergence were observed at different concentrations of N. rileyi, followed by Aspergillus species. These findings partially agree with those of Mantzoukas (2019), who reported concentration-dependent pupation and emergence when exposing H. armigera larvae to different concentrations of Metarhizium robertsii, with the lowest pupation and emergence observed at the highest concentration (1 × 10⁷ conidia/ml). However, Shanthakumar et al. (2010) found no significant effect of EPF concentration on pupation and recorded 90% pupal recovery in S. litura at both lower and higher concentrations of N. rileyi (10⁴ and 10⁷ conidia/ml). Similarly, Hatting et al. (2012) observed accelerated pupation in H. armigera after treating the larvae with N. rileyi using both topical and oral inoculation methods. The observed discrepancies in pupal recovery results may be attributed to the genetic makeup of fungal strains isolated from different geographical regions (Bidochka et al. 2000, Couceiro et al. 2022). Additionally, variations in methodology, including inoculation techniques and the susceptibility of targeted pests to fungal infections, also explain the contrasting outcomes.

Probit analysis revealed that the estimated LC₅₀ values of the tested EPF species were time-dependent and significantly decreased as the PII increased. N. rileyi was found to be most virulent, exhibiting the lowest LC₅₀ at 7d PII, followed by A. parasiticus, A. niger and A. flavus. These results are in accordance with those of Liu et al. (2019), who reported an LC₅₀ of 6.24×10⁶ conidia/ml for M. rileyi against third-instar S. litura. Similarly, Ramos et al. (2024) documented the LC₅₀ range (2.04×10⁵ to 1.05×10⁶ conidia/mL) of 5 different N. rileyi isolates against S. frugiperda larvae. However, LC₅₀ results for N. rileyi are in partial agreement with those of Dev et al. (2021), who determined the LC₅₀ of 2.81×10⁵ spores/ml for M. rileyi against H. armigera third instar. This partial variation in the findings may be attributed to the genotypic variation, related to their adaptation to different native environments (Bidochka et al. 2000, Couceiro et al. 2022).

LT₅₀ values of almost all fungal treatments (except A. parasiticus) were dose-dependent and significantly decreased with increasing conidial concentrations. These findings are supported by Yang et al. (2024), who also observed concentration-dependent LT₅₀ values of M. rileyi against S. frugiperda larvae. Contrary to these results, Ramos et al. (2024) recorded LT₅₀ values for 1×10⁸ conidia/ml concentration of five M. rileyi isolates ranging from 7.04 days to 9.46 days against the second instar of S. frugiperda. The observed discrepancies in the LT₅₀ results may be attributed to experimental conditions and targeted lepidopterous species.

Based on our findings, the distribution of entomopathogenic fungi in the different agro-ecological zones and cropping systems was not uniform throughout the soil habitat. EPF species viz., N. rileyi, A. parasiticus, A. flavus and A. niger, isolated from cultivated soil in Khyber Pakhtunkhwa province of Pakistan, effectively managed the local pest, H. armigera in laboratory conditions. Among all the fungal species tested, N. rileyi showed the most promising control, followed by Aspergillus species. N. rileyi caused deadly mycosis in H. armigera and showed a detrimental impact on its biological parameters as well. Hence, it is potentially suitable for the eco-friendly management of this pest and would be a sustainable alternative to synthetic insecticides. Naturally occurring EPF causes deadly mycosis in insect pests, with the potential to infect all their life stages. Their incorporation into IPM strategies will ensure the production of healthier organic crops by addressing the insecticide residue and resistance issues. Further, field trials using the extracts of isolated EPF species against H. armigera are needed.