Pathogenicity of native strains of Bacillus thuringiensis, Beauveria bassiana and Metarhizium rileyi as entomopathogens against the polyphagous borer, Conogethes punctiferalis (Guenée) (Crambidae: Lepidoptera)

Abstract

Background

The shoot and fruit borer, Conogethes punctiferalis (Guenée) (Crambidae: Lepidoptera), is a significant pest causing substantial economic losses across various crops. The need for alternative control methods has prompted the exploration of biological control using entomopathogenic fungi and bacteria. In this study, the pathogenicity of Beauveria bassiana, Metarhizium (Nomuraea) rileyi and Bacillus thuringiensis (Bt) against C. punctiferalis larvae and pupae was assessed through laboratory bioassays.

Results

Various concentrations of B. bassiana and M. rileyi spores, i.e. 1 × 10², 1 × 10⁴, 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ spores/ml, were tested alongside controls. Additionally, five strains of Bt (IIOR Bt-145, Bt-154, Bt-171, Bt-172 and Bt-127) were evaluated at concentrations of 0.5, 1.0, 1.5, 2.0 and 2.5 mg/ml. The LC₅₀ values for B. bassiana and M. rileyi were 7.9 × 10⁵ spores/ml and 8.7 × 10⁴ spores/ml, respectively, after 4 and 6 days of post-treatment using the spray method. In the larval dip method, the LC₅₀ values were 4.8 × 10³ spores/ml for B. bassiana and 2.0 × 10⁴ spores/ml for M. rileyi after 5 and 6 days of treatment, respectively. For pupae, the LC₅₀ values were 1.2 × 10⁶ spores/ml for B. bassiana and 4.3 × 10⁴ spores/ml for M. rileyi after 4 and 7 days of treatment, respectively. Similarly, the five strains of Bt were effective against C. punctiferalis. However, Bt-154 demonstrated the highest efficacy, with LC₅₀ values of 0.66 mg/ml in the spray method and 0.79 mg/ml in the larval dip method after 5 days of post-treatment.

Conclusion

The potential of entomopathogenic isolates as biocontrol agents against C. punctiferalis provided a promising alternative to synthetic insecticides in pest management. The efficacy of B. bassiana, M. rileyi and Bt strains suggests their suitability for integrated pest management strategies, potentially reducing reliance on chemical pesticides and minimizing the environmental impacts. Further field studies are warranted to validate the efficacy and practicality of these biocontrol agents in real-world agricultural settings.

Background

Conogethes punctiferalis Guenée (Crambidae: Lepidoptera), commonly known as the capsule borer or yellow peach moth, is a significant polyphagous pest with widespread distribution across tropical Asia, spanning from India through Southeastern Asia to Australia (Pena et al. 2002). Additionally, it has been introduced to areas beyond its native range, including Britain and Europe, where it is recognized as a detrimental pest. This species poses a substantial threat to agricultural and forest ecosystems, as it has the potential to cause severe damage to over 40 species of fruits, field crops and forest trees. Notable hosts include peach, apple, plum, chestnut, durian, citrus, mango, papaya, maize, sorghum, sunflower, castor and various pine species. In India alone, C. punctiferalis infests 36 crop plants from 23 families. Its impact extends globally, being identified as the most serious insect pest of papaya in Australia (Chay-Prove et al. 2000), Durio zibethinus in Thailand, fruit crops and maize in China (CPCI 2005), as well as over 20 fruit crops including Dimocarpus longan and Averrhoa carambola in Korea and Helianthus annuus and Macadamia ternifolia in New Zealand (CPCI 2005). Furthermore, C. punctiferalis poses a significant threat to high-value spice crops such as castor (Ricinus communis L.), turmeric (Curcuma longa L.) and ginger (Zingiber officinale Rosc.) in India (Duraimurugan and Lakshminarayana 2016).

The larval stage of this species represents the damaging phase, as it bore into stems, shoots, buds, fruits and seeds of various plants. Its cryptic behaviour during the initial stages of infestation poses a significant challenge for effective control measures. Boring activity can predispose fruits to secondary pathogens. Common symptoms include yellowing or browning of terminal shoots, fruit damage and the presence of bore holes (Molet 2015).

Microbial biopesticides, particularly entomopathogenic fungi (EPF) and entomopathogenic bacteria (EPB), hold considerable promise as alternatives to chemical pesticides due to their unique mode of action and ability to infect a wide range of sucking and chewing insect pests. They are deemed environmentally safe and represent novel tools in pest management (Vimala Devi et al. 2021), offering advantages such as low cost, high efficiency, safety for beneficial organisms and reduced residues in the environment (Lacey et al. 2001). The objective of the present study was to assess the potential efficacy of different EPF, namely B. bassiana and M. rileyi, and certain isolates of EPB (B. thuringiensis) against C. punctiferalis infesting castor under laboratory conditions.

Methods

Insect rearing

The insects utilized in these studies were sourced from a laboratory colony of C. punctiferalis, initially established from larvae gathered in October 2020 from castor fields at the Department of Entomology, ICAR-Indian Institute of Oilseeds Research. The culture of C. punctiferalis was maintained by collecting larvae at various stages from stray castor crop. Infested castor capsules were placed in glass jars containing additional castor capsules. The adults emerging from these larvae were released into an oviposition chamber for mating and oviposition. Newly hatched larvae were reared on fresh capsules within glass jars measuring 15 cm in diameter and 21 cm in height, covered with muslin cloth. Fresh capsules were provided as needed when the old ones dried out or were consumed by the larvae. Upon pupation, pupae were individually transferred to specimen tubes measuring 10.2 cm in length and 2.5 cm in diameter for adult emergence. Five pairs (5 males and 10 females) of freshly emerged adults were introduced into the oviposition chamber. This chamber was equipped with castor inflorescences and immature capsules held in a vial containing water. Similarly, castor inflorescences and immature capsules were replenished every other day and eggs were collected accordingly. The resulting homogenous larval populations in subsequent generations were utilized for conducting further experiments (Shivakumar et al. 2020).

Entomopathogens

B. thuringiensis (Bt) isolates Bt-127, Bt-145, Bt-154, Bt-171 and Bt-172 and the fungal isolates B. bassiana Bb-4513 and M. rileyi IIOR-SlMr maintained at Crop Protection Section, ICAR-IIOR were used.

Maintenance of B. thuringiensis culture

The five Bt isolates, namely IIOR Bt-127, Bt-145, Bt-154, Bt-171 and Bt-172, were cultured on nutrient agar slants. Following a 72-h incubation period, the inoculum from the pure Bt culture was introduced into sterilized Luria broth medium and placed in an incubating shaker set at 180 rpm and 30 °C for 17 h. Subsequently, Bt multiplication was achieved through solid-state fermentation, wherein the inoculum was transferred to sterilized solid wheat bran medium supplemented with molasses and allowed to incubate for 2–3 days.

Maintenance of B. bassiana and M. rileyi culture

The fungal isolates B. bassiana Bb-4513 and M. rileyi IIOR-SlMr were cultured on potato dextrose agar- and Sabouraud maltose yeast extract agar-specific medium, respectively. They were then incubated at 25 °C for 15 days.

Multiple concentration preparation of B. thuringiensis for bioassay

The fully grown Bt on wheat bran medium was filtered using distilled water, and the filtrate was then centrifuged at 10,000 rpm for 20 min. The supernatant was discarded, and the pellet was dried and powdered. This technical powder was utilized for larval bioassays at various concentrations of Bt technical powder, ranging from 0.5, 1.0, 1.5, 2.0 and 2.5 mg/ml, dissolved in water and 0.1% Tween 20 (Fite et al. 2019).

Multiple concentrations of B. bassiana and M. rileyi prepared for bioassay

For the fully grown fungal isolates, distilled water along with 0.1% Tween 20 was added and the mixture was filtered through muslin cloth. Pure conidial suspensions of B. bassiana and M. rileyi with concentrations of 1 × 10², 1 × 10⁴, 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ spores/ml were prepared using a hemocytometer (Fite et al. 2019).

Bioassay of B. thuringiensis isolates against C. punctiferalis larvae using larval dip method

Larvae were immersed in suspensions of Bt isolates IIOR Bt-145, Bt-154, Bt-127, Bt-171 and Bt-172 technical powder for 30 s, following which they were placed on filter paper and transferred to sterile insect rearing Petri dishes. Castor capsules or stem bits were provided daily as food. Various concentrations of Bt technical powder suspensions, specifically 0.5, 1.0, 1.5, 2.0 and 2.5 mg/ml, were employed for the insect bioassay. The control group was sprayed with distilled water and 0.1% Tween 20 (Senthil Kumar et al. 2016).

Bioassay of B. thuringiensis isolates against C. punctiferalis larvae using spray method

Larvae were placed on filter paper in a sterile insect rearing Petri dishes after spraying the filter paper with a volume of 3 ml suspension at multiple concentrations of IIOR Bt-145, Bt-154, Bt-171, Bt-172 and Bt-127 isolates technical powder, i.e. 0.5, 1.0, 1.5, 2.0 and 2.5 mg/ml diluted in distilled water and 0.1% Tween 20. The filter paper after inoculation was air dried before larvae were placed. Castor capsules or stem bits were provided daily as food. The filter paper of the control group was sprayed with 3 ml of distilled water and 0.1% Tween 20 (Chergui et al. 2020).

Bioassay of EPFs against C. punctiferalis larvae using larval dip method

Larvae were placed on filter paper after being immersed in the spore suspension for 30 s, following which they were transferred to sterile insect rearing Petri dishes. Castor capsules or stem bits were added daily as food. Conidial suspension at various concentrations of 1 × 10², 1 × 10⁴, 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ spores/ml was utilized for the insect bioassay. The control group was sprayed with distilled water and 0.1% Tween 20 (Senthil Kumar et al. 2016).

Bioassay of EPFs against C. punctiferalis larvae using spray method

Larvae were positioned on filter paper within a sterile Petri dish subsequent to the application of 3 ml of conidial suspension at various concentrations of 1 × 10², 1 × 10⁴, 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ spores/ml. The control group received a spray of 3 ml of distilled water and 0.1% Tween 20 (Chergui et al. 2020).

Bioassay of EPFs against C. punctiferalis pupae using the spray method

Pupae were placed onto filter paper within a Petri dish subsequent to spraying the filter paper with a volume of 3 ml of conidial suspension at various concentrations of 1 × 10², 1 × 10⁴, 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ spores/ml. The filter paper for the control group was sprayed with 3 ml of distilled water and 0.1% Tween 20 (Chergui et al. 2020).

Data analysis

Observations on the number of living and dead larvae in the treatments were recorded for all experiments to calculate the mortality percentage. Three replications per treatment, each containing 30 larvae, were utilized for the bioassay of both larvae and pupae. The data were subjected to one-way analysis of variance (ANOVA) using the statistical software SPSS Windows. Probit analysis for LC₅₀ calculation on specific days after treatment was also performed using the SPSS Windows software (Tesari et al. 2024).

Results

Bioassay of B. thuringiensis isolates against C. punctiferalis larvae

Following the bioassay with five isolates of Bt against C. punctiferalis larvae using different concentrations of technical powder (0.5, 1.0, 1.5, 2.0 and 2.5 mg/ml diluted in distilled water and 0.1% Tween 20), it became evident that Bt-154 exhibited significantly greater potential than the other tested Bt isolates. Bt-154 recorded lower LC₅₀ values of 0.79 mg/ml with the larval dip method and 0.66 mg/ml with the spray method of bioassay compared to the other isolates (Table 1).

Table 1 LC₅₀ values of Bacillus thuringiensis isolates tested against Conogethes punctiferalis larvae using larval dip and spray methods after 5 days of treatment

Larval dip method

Using larval dip method, at a concentration of 0.5 mg/ml, larval mortality ranged from 20.00 to 40.00%. This mortality increased to 20.00–60.00% at 1.0 mg/ml, 40.00–60.00% at 1.5 and 2.0 mg/ml and reached 80.00–100.00% at 2.5 mg/ml of technical powder of various Bt isolates after 5 days post-treatment, as detailed in Table 2. The results indicated that with an increase in concentration, the mortality percentage also increased. Larval mortality of 20.00% was observed in the control group of Bt-145, Bt-154 and Bt-171.

Table 2 Mortality percentages of Conogethes punctiferalis 5 days after treating with 5 concentrations of Bacillus thuringiensis isolates using larval dip and spray bioassay methods

Spray method

Using spray method, at a concentration of 0.5 mg/ml, larval mortality ranged from 23.33 to 43.33%. This mortality increased to 23.33–63.33% at 1.0 mg/ml and reached 43.33–63.33% at 1.5 mg/ml and further increased to 43.33–63.33% at 2.0 mg/ml and to 80.00% at 2.5 mg/ml of technical powder of various Bt isolates after 5 days post-treatment, as detailed in Table 2. The results indicated that with an increase in concentration, the mortality percentage also increased. Larval mortality of 6.60% was observed in the control group of Bt-172 and Bt-127 and 13.33% was observed in the control group of Bt-145 and Bt-154. Larval death induced by Bt was confirmed by the appearance of black discoloration over the entire body of the larvae, as depicted in Fig. 1.

Fig. 1

Effectiveness of entomopathogens against larvae of Conogethes punctiferalis

Bioassay of EPFs against C. punctiferalis larvae

Larval dip method

Following the larval dip method of bioassay with B. bassiana against C. punctiferalis larvae using different concentrations (1 × 10², 1 × 10⁴, 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ spores/ml), the mortality percentage increased with an increase in spore concentration and with an increase in days after treatment. At concentration of 1 × 10² spores/ml, the mortality ranged from 16.70 to 70.00% and 3–7 days after treatment, respectively, but with 1 × 10⁴ spores/ml, it ranged from 23.33 to 83.33% between the same period, respectively. At 1 × 10⁶ spores/ml, the mortality was between 30.00 and 90.00% from three to 7 days, while at 1 × 10⁷ spores/ml, it ranged from 46.70 to 96.70% at the same period, respectively, and it increased from 66.70 to 100.00% at the concentration of 1 × 10⁸ spores/ml between three to seventh day after treatment. No mortality was observed in the control group (Table 3).

Table 3 Mortality percentages of Conogethes punctiferalis larvae treated with 5 concentrations of entomopathogenic fungi using larval dip method

Following the larval dip method of bioassay with M. rileyi against C. punctiferalis larvae using different concentrations (1 × 10², 1 × 10⁴, 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ spores/ml), the mortality percentage increased with an increase in spore concentration and with an increase in days after treatment. At concentration of 1 × 10² spores/ml, the mortality ranged from 3.33 to 43.33% and 3–7 days, respectively, and at 1 × 10⁴ spores/ml, it ranged from 13.33 to 56.70% at the same period, respectively. After the period from 3 to 7 days post-treatment, the mortality ranged between 20.00 and 83.33% at concentration of 1 × 10⁶ spores/ml, respectively, while it was 36.70– 90.00% at 1 × 10⁷ spores/ml, respectively, and it increased to 60.00–100.00%, respectively, at 1 × 10⁸ spores/ml. No mortality was observed in the control group (Table 3).

Spray method

Following the spray method of bioassay with B. bassiana against C. punctiferalis larvae using different concentrations (1 × 10², 1 × 10⁴, 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ spores/ml), the mortality percentage increased with an increase in spore concentration and with an increase in days after treatment. At concentration of 1 × 10² spores/ml, the mortality ranged from 13.33 to 50.00% and 3–7 days, respectively, and with 1 × 10⁴ spores/ml, it ranged from 20.00 to 53.00% and 3–7 days, respectively. At 1 × 10⁶ spores/ml, the mortality was between 30.00 and 70.00%, at the same mortality period, respectively, while at 1 × 10⁷ spores/ml, it ranged from 36.70 to 97.00% and 3–7 days, respectively, and also, it increased from 43.33 to 100.00%, respectively, at concentration of 1 × 10⁸ spores/ml. No mortality was observed in the control group (Table 4).

Table 4 Mortality percentages of Conogethes punctiferalis larvae treated with 5 concentrations of entomopathogenic fungi using the spray method

Following the spray method of bioassay with M. rileyi against C. punctiferalis larvae using different concentrations (1 × 10², 1 × 10⁴, 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ spores/ml), the mortality percentage increased with an increase in spore concentration and with an increase in days after treatment. With 1 × 10² spores/ml, the mortality ranged from 3.33 to 30.00% after 3–7 days post-treatments, and with 1 × 10⁴ spores/ml, it ranged from 16.70 to 50.00%. At 1 × 10⁶ spores/ml, the mortality rate was between 26.70 and 63.00% after and 3–7 days post-treatments, respectively, while at 1 × 10⁷ spores/ml, it ranged from 40.00 to 77.00% it increased to 63.30–97.00% and 3–7 days, respectively, with 1 × 10⁸ spores/ml. No mortality was observed in the control group (Table 4).

Bioassay of EPFs against C. punctiferalis pupae using the spray method

Following the spray method of bioassay with B. bassiana against C. punctiferalis pupae using different concentrations (1 × 10², 1 × 10⁴, 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ spores/ml), at concentration of 1 × 10² spores/ml, the mortality ranged from 10.00 to 63.33% and 3–7 days, respectively, and with 1 × 10⁴ spores/ml, it ranged from 13.33 to 63.33% and 3–7 days, respectively. At 1 × 10⁶ spores/ml, the mortality was between 30.00 and 80.00% from 3 to 7 days, respectively, while at 1 × 10⁷ spores/ml, it ranged from 36.67 to 96.67% at the same period of treatment, respectively, and it increased to 53.33–100.00% with 1 × 10⁸ spores/ml between 3–7 days, respectively. No mortality was observed in the control group (Table 5).

Table 5 Mortality percentage of Conogethes punctiferalis pupae with 5 concentrations of entomopathogenic fungi using the spray method

Following the spray method of bioassay with M. rileyi against C. punctiferalis pupae using  different concentrations (1 × 10², 1 × 10⁴, 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ spores/ml), at concentration of 1 × 10² spores/ml, the mortality ranged from 0.00 to 13.33%, while at 1 × 10⁴ spores/ml, it was between from 6.67 to 46.67% for 3–7 days, respectively. At 1 × 10⁶ spores/ml, the mortality was between 16.67 and 60.00%, but at 1 × 10⁷ spores/ml, it ranged from 36.67 to 80.00% from 3 to 7 days, respectively, and it increased to 56.67 to 100.00% for the same period of treatment, respectively, at 1 × 10⁸ spores/ml. No mortality was observed in the control group (Table 5). After the death of larvae and pupae due to EPFs, the development of mycosis was observed, characterized by white sporulation due to B. bassiana infection and green sporulation due to M. rileyi infection, confirming that the death was caused by fungi only (Fig. 2).

Fig. 2

Effectiveness of entomopathogenic fungi against pupae of Conogethes punctiferalis

In the present study, the bioassay results underscore the efficacy of EPFs against C. punctiferalis larvae and pupae, as mortality escalates with high spore concentrations.

Additionally, the development of mycosis, characterized by distinctive sporulation patterns, provided further evidence of fungal infection as the cause of larval mortality. These findings highlight the potential of EPFs as a promising biocontrol agent for the management of C. punctiferalis, offering a sustainable and environmentally friendly alternative to chemical pesticides.

Concentration–mortality relationship of EPFs against larvae and pupae of C. punctiferalis

The LC₅₀ value of B. bassiana is 7.9 × 10⁵ spores/ml and the LC₅₀ value of M. rileyi was 8.7 × 10⁴ spores/ml at 4 and 6 days post-treatment using the spray method of bioassay. In the larval dip method, LC₅₀ value of B. bassiana was 4.8 × 10³ spores/ml, while it was 2.0 × 10⁴ spores/ml for M. rileyi at 5 and 6 days post-treatment. For pupae, the LC₅₀ values were 1.2 × 10⁶ and 4.3 × 10⁴ spores/ml for B. bassiana and M. rileyi, respectively, at 4 and 7 days post-treatments, respectively, as presented in Table 6.

Table 6 Effect of the tested EPF against Conogethes punctiferalis larval and pupal stages

Both bioassay methods were employed to assess the efficacy of Bt, B. bassiana and M. rileyi against C. punctiferalis larvae, while only the spray method was utilized for testing B. bassiana and M. rileyi against C. punctiferalis pupae. Following the bioassay with Bt, both methods demonstrated a maximum mortality of 80.00% with the highest concentration of 2.5 mg/ml after 5 days of treatment. However, among the five native isolates of Bt (IIOR Bt-127, Bt-145, Bt-154, Bt-171 and Bt-172), Bt-154 was found to be effective against C. punctiferalis larvae, recording lower LC₅₀ values of 0.79 mg/ml and 0.66 mg/ml with the larval dip and spray method of bioassay, respectively, after 5 days of treatment, than other isolates. With the EPF evaluated against larvae and pupae of C. punctiferalis, both B. bassiana and M. rileyi were found to be the most effective against the two stages.

Discussion

Entomopathogenic microorganisms, such as certain strains of bacteria and fungi, offer a promising alternative for the control of C. punctiferalis. These biocontrol agents specifically target and infect the pest insect, exerting their lethal effects while posing minimal risk to non-target organisms and the surrounding ecosystem. By harnessing the natural enemies of C. punctiferalis especially entomopathogenic bacteria and fungi, eco-friendly management practices can be implemented to effectively reduce pest populations while preserving environmental integrity and promoting sustainable agricultural practices.

B. thuringiensis

Efforts to develop biopesticides for controlling C. punctiferalis emphasize the need to screen new, potentially more potent isolates. Integrated pest management strategies could benefit from the incorporation of these biopesticides. However, it is worth noting that Bt has been found to have adverse effects on various biological aspects of larval, pupal and adult stages of many pests.

In the present study, we evaluated the efficacy of Bt across a range of concentrations, observing significant mortality rates among the tested isolates. This range facilitated the accurate estimation of the median lethal concentration (LC₅₀). Consistent with our findings, the previous research by Lalitha and Muralikrishna 2012 demonstrated high mortality rates in S. litura following Bt treatment.

The observed lower mortality of C. punctiferalis when exposed to Bt-171 and Bt-172 isolates in the present study suggested the potential development of resistance by C. punctiferalis larvae against Bt toxins. To achieve early mortality, high concentrations of Bt could be applied against C. punctiferalis larvae. These differences may stem from variations in insecticidal genes present or differences in the insect strain utilized, as noted by Lone et al. 2016. Additionally, Alsaedi et al. (2017) reported the susceptibility of Tuta obsoluta larval instars to Bt toxins.

The variation in LC₅₀ values among larval hosts exposed to different concentrations of Bt powder provides evidence for the disparity in mortality observed in the present study. These findings can be contextualized with the research of Lone et al. (2016), who demonstrated that the Bt isolate JK12 exhibited high toxicity against H. armigera, requiring a median lethal concentration of 0.184 mg/ml to cause 50.00% larval mortality.

Considerable variability exists in the effectiveness of Bt isolates against target insects, as noted by Yaradoni (1999). The degree of pathogenicity varies with the concentration of bacterial isolate, as well as the duration of exposure and the stage of metamorphosis of the silkworm (Savitri and Murali Mohan 2003). Knowles (1994) suggested that the variations in efficacy against different lepidopteran species may stem from differences in the number of cry genes and the absence of specific binding sites.

The disparity in LC₅₀ values among different Bt isolates suggested variations in their efficacy against the target pest species. Factors influencing this variation may include differences in the genetic makeup of the isolates, variations in the expression of insecticidal proteins and the specific mechanisms by which each isolate interacts with the target pest’s biology. For instance, Bt-154 showed a low LC₅₀ value indicated that this isolate’s efficiency was higher at the lowest concentrations compared to other isolates, implying potentially greater potency or a more effective mode of action against C. punctiferalis.

B. bassiana and M. rileyi

Both spray and larval dip methods demonstrated efficacy in inducing mortality among the target insects. These findings are consistent with the previous research conducted by Gonzalez-Cabrera et al. (2011) in bioassays targeting the coffee berry borer using fungal entomopathogens, wherein various application methods were employed. Specifically, high mortality among coffee berry borers treated with Bb using the dipping method was documented the effectiveness of different bioassay methods in inducing larval mortality (Gonzalez-Cabrera et al. 2011).

The larval dipping method offers advantages by providing direct exposure of spores to the insects, facilitating mortality. Moreover, this method reduces labour requirements, particularly beneficial when screening multiple isolates and proves useful in assessing the potency of different strains. Notably, our control experiment with entomopathogens did not result in any observed mortality, consistent with findings reported by Gonzalez-Cabrera et al. (2011). Furthermore, the implementation of biocontrol agents, such as EPF strains, has been shown to decrease pest incidence, as observed by Yun et al. (2017). The mortality rate observed in such cases was influenced by various factors including spore concentration; time elapsed after fungal treatment and temperature levels.

The present study elucidated that the screening of two EPF isolates revealed the virulence of B. bassiana and M. rileyi against larvae of C. punctiferalis. The majority of isolates induced larval mortality, indicating their capacity to effectively combat C. punctiferalis larvae. Specifically, B. bassiana and M. rileyi exhibited virulence under laboratory conditions. These findings are comparable to the research conducted by Douro et al. (2012), who identified isolates of B. bassiana and M. anisopliae that caused significant larval mortality in Heliothis armigera L.

Additionally, it was observed that the mycosis development of the EPF, M. rileyi was slower than B. bassiana. Furthermore, the mortality of larvae increased with higher spore concentrations, suggesting a concentration-dependent relationship in the efficacy of these EPF isolates against C. punctiferalis larvae. Similarly, Akmal et al. (2013) investigated the effectiveness of different entomopathogenic fungal strains against various aphid species under controlled laboratory conditions. They observed that the mortality of the aphids increased with higher spore concentrations.

In the present study, the efficacy of EPF in controlling insect pests has been well documented and added valuable insights into their potential application. The susceptibility of C. punctiferalis larvae to B. bassiana, determined through the spray method, aligns with previous findings by Swati et al. (2017), who reported significant pathogenicity of B. bassiana against H. armigera. Similarly, for M. rileyi indicate its substantial effectiveness, which is consistent with Senthil Kumar (2021) who demonstrated notable susceptibility of C. punctiferalis to Metarhizium.

In the larval dip method, B. bassiana and M. rileyi also showed significant pathogenicity. These results are corroborated by Fite et al. (2019) who reported high mortality rates in H. armigera larvae treated with these entomopathogenic fungi. Additionally, Ramanujam et al. (2020) provided evidence of the substantial impact of B. bassiana on S. frugiperda larvae, further supporting our findings.

It has been established that a concentration of 1 × 10⁸ spores/ml induces the highest mortality in C. punctiferalis larvae when inoculated with M. rileyi and B. bassiana. This result can be compared to the mortality observed in H. armigera larvae treated with B. bassiana-APPRC-9604 isolate at a concentration of 1 × 10⁸ conidia/ml, which was identified as a more virulent strain under laboratory conditions, reducing larval infection (Fite et al. 2019). Ana et al. (2018) also noted the highest susceptibility of S. frugiperda larvae to B. bassiana, resulting in 100.00% larval mortality at a concentration of 1 × 10⁸ conidia/ml. Kalvnadi et al. (2018) observed the virulence of B. bassiana and M. anisopliae isolates, respectively, causing a high larval mortality and affecting the biological parameters of H. armigera.

The present findings can be compared to those of Wraight et al. 2010, who conducted a study comparing the virulence of B. bassiana isolates against lepidopteran insect pests. Their research indicated that larvae of Heliothis zea and Spodoptera exigua were susceptible to fungal infection. Swathi et al. (2017) analysed the lowest LC₅₀ and observed the highest larval mortality (100%) against H. armigera when treated with B. bassiana.

Mycosis observed on the larvae in the present results, along with the outgrowth of white sporulation due to B. bassiana and green sporulation due to M. rileyi infestations on larval cadavers of C. punctiferalis, provided evidence that the mortality of larvae was attributed to the treated fungal isolates. These results can be compared to the findings of Lacey et al. (2015), who studied the role of fungal conidia, variations in concentrations of spore suspensions, larval species and larval instars in causing infection by entering the host body through the cuticle. The increase in mortality of larvae with the rise in concentration of spore suspension was compared to the study of Rogge et al. (2017), who reported that high conidial application rates led to a significant increase in mycosis, consequently reducing the incidence of wireworm, Agriotes obscurus (L.).

Conclusion

In conclusion, both Bt and EPF demonstrated remarkable potential as biocontrol agents against C. punctiferalis. Particularly, among the Bt isolates, IIOR Bt-154 exhibited superior virulence compared to others, under laboratory conditions, effectively reducing larval infestations. Additionally, both B. bassiana and M. rileyi were highly effective against both larvae and pupae of C. punctiferalis, showcasing their potential as efficient entomopathogenic fungi for integrated pest management strategies. These findings highlight the promise of utilizing these agents as biopesticides, offering a viable alternative to synthetic insecticides. However, to fully harness their potential, further efforts are needed to develop formulations for these virulent isolates, enhancing their shelf life and efficacy as entomopathogens. Ultimately, the future of integrated pest management for C. punctiferalis could be significantly enhanced through the strategic incorporation of these biopesticides into existing pest control strategies.