Background

The cotton pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), is one of the major economic cotton pests that causes considerable damages to cotton in Pakistan (Jaleel et al. 2014) and many other countries (Parmar and Patel 2016). Different approaches such as chemical insecticides and growing resistant cultivar (transgenic Bt cotton containing Cry1Ac toxin) have been used to manage the pest control (Heuberger et al. 2014), but they have not given optimal control levels of the pest (Mohamed et al. 2016). Plant extracts such as Nicotiana tabacium and Azadirachta indica have widely been used to control insect pests. A. indica (Neem) has been used for years in Indo-Pak against several insect pests and is still used for stored grain pest (Rajendran and Sriranjini 2008). Due to its broad host range, inexpensive production and no harmful impact on environment (Mathew 2016) makes it a safer alternative method to control some insect pests.

The entomopathogenic fungi (EPFs) are among the most effective and environmental friendly biological control agents that invade their host insect through the cuticle and play a key role in the regulation of insect pest population in natural ecosystem (Niu et al. 2019). EPFs can be used against a wide range of insect pests and their nonspecific actions and antagonistic natures give them broad host range ability (Ong and Vandermeer 2014). More than 700 species of fungi belonging to 90 genera among Beauveria bassiana, Metarhizium anisopliae, Verticillium lecanii, Purpureocillium lilacinum, and Isaria fumosorosea are the widely used ones as biological control agent against many agricultural pests (Khan et al. 2012; Rizwan et al. 2019). The addition of plant extracts which act as both adjuvant (Nursal and Ilyas 2019) and bio-pesticide (Dougoud et al. 2019) can heighten the coverage of leaf and persistence of EPFs (Świergiel et al. 2016) resulting in enhanced performance of EPFs and plant extracts in combination for the suppression of some insect pests such as P. gossypiella (Vashisth et al. 2019).

This study aimed to check the effect of EPFs; B. bassiana, M. anisopliae and V. lecanii and the plant extract (Azadirachta indica) on some biological aspects of P. gossypiella under laboratory conditions.

Materials and methods

P. gossypiella culture

Different growth stages of P. gossypiella larvae were collected from cotton fields where their population did not expose to any insecticidal applications. All the stages of P. gossypiella were placed separately in labeled plastic vials and transferred to the laboratory. Larvae were maintained by feeding them on green bolls at 27 ± 0.5 °C until pupation. Larval discrepancy on the basis of their sex was performed, following the method of Dharajothi et al. (2010) for moth copulation (Jothi et al. 2016). Moths were released in insect rearing cages measuring 28 cm height and 24 cm diameter for egg laying, at the rate of 20 pairs per cage, and were fed upon 1 ml multivitamin and protein mixed with 100 ml honey solution (20%) along with fresh terminal buds and leaves of cotton brought from unsprayed cotton plot, inserted in a small conical flask contains water to keep them fresh egg lying space for adults (Muralimohan et al. 2009). Water-soaked cotton twigs were placed at the bottom of the flask to maintain the moisture level of the tissues and were replaced every 2nd day. These twigs were transferred into translucent plastic containers (5 cm height and 4 cm diameter) sealed with muslin cloth and rubber band for egg hatching (Parker 2005).

The larvae were maintained in plastic trays on green bolls at 26 ± 2 °C, 65 ± 5% R.H. and 11 h light–13 h dark photoperiod until pupation. The pupae were placed in plastic vials (4 × 5 cm diameter × height) with a mesh-windowed lid and a disk of filter paper at the bottom, retained until adult emergence (Muralimohan et al. 2009). Glass wares were rinsed with distilled water followed by sterilization and were used for the preparation and storing of diet.

Entomopathogenic fungi

Two commercial formulations of 3 EPFs viz. V. lecanii, M. anisopliae and B. bassiana were procured from AgriLife SOM Phytopharma (India) Limited® (www.agrilife.in) in the form of talc powder. Formulations, at 2 different concentrations (1 × 106 and 1 × 108 CFU/ml), were tested against P. gossypiella. Hemocytometer and potato dextrose agar (PDA) were used to determine the conidial concentrations and germination in conidial suspension, respectively. Measurement of conidial germination was computed by randomly counting 200 conidia in each plate at 25 ± 2 °C, 18 h after incubation (Atta et al. 2020).

Preparation of conidial suspensions

EPF’s conidial suspensions concentrations, 1 × 106 conidia ml−1 and 1 × 108 conidia ml−1 alone and in combination with A. indica extract were prepared by dissolving in distilled water and in 5% extract as basic solution while using hemocytometer.

Preparation of Azadirachta indica plant extract

Plant extract of A. indica was prepared by adopting the methodology of Ali et al. (2017). Fresh collected leaves of A. indica were sufficiently washed by distilled water and dried in shadow, followed by electric grinding to get fine powder. Fine A. indica powder (50 g) was dissolved in distilled water (500 ml) in a 2.5 liter sized conical flask by heating the solution at 60 °C and shaking the flask continuously with a magnetic stirrer for 6 h. Solution was filtered, using Whatman no. 1 after sieving with muslin cloth to remove any solid particles. Rotary evaporator was used to evaporate the solution in vacuumed conditions in hot air oven at to bring the dry plant extract to a constant volume 50 ml. The solution thus obtained was considered as 100% A. indica extract, stored at 4 °C for further investigations.

Bioassay

Thirty fresh molted 2nd instar P. gossypiella larvae of uniform brood were treated by immersing them in to the conidial suspension (1 × 106 and 1 × 108 ml−1) concentrations, i.e., B. bassiana, V. lecanii, M. anisopliae and A. indica extract (5%) alone and in combination for 10 s (Derbalah et al. 2014). After the treatment, larvae were placed into sterile Petri dishes (9-cm diameter) for air drying for 10 min. The treated larvae were maintained in labeled plastic trays with artificial diet for further investigations, i.e., mortality, sporulation, mycosis, pupation and adult emergence under laboratory conditions. Mortality rate was calculated at 4, 8 and 12 days of time intervals after which sporulation and mycosis were computed. Mortality data was calculated by Abbott’s formula (Abbott 1925).

$$ \mathrm{Corrected}\ \mathrm{mortality}\%=\left(1-\frac{n\ \mathrm{in}\ T\ \mathrm{after}\ \mathrm{treatment}}{n\ \mathrm{in}\ {\mathrm{C}}_{\mathrm{o}}\ \mathrm{after}\ \mathrm{treatment}}\ \right)\times 100 $$

where n = insect population, T = treated, Co = control

Sporulation and mycosis

Dead cadavers of P. gossypiella stiffs were collected from treatments where EPFs were applied (alone and in combination), for sporulation and mycosis and were transferred to plastic vials from sterile Petri dishes for refrigeration at 4 °C. Solution of sodium hypochlorite (0.05%) was used for surface sterilization of the collected cadavers for 2–3 min, followed by 2–3 washings, using distilled water (Leland and Gore 2016). The cadavers were then placed in the Petri dishes for a week with PDA for incubation at 75 ± 5% R.H. and 25 ± 1 °C. Microscope was used for the observation and identification of external growth of the fungi on the treated cadavers. A drop of Tween-80 was added and stirred for 10 min with distilled water (20 ml) to mix with the cadavers, selected from each replication, which were already mycosed for the determination of sporulation. Hemocytometer coupled with microscope was used to determine the total number of conidia ml−1 (Rizwan et al. 2019).

Assessment of pupation and adult emergence of P. gossypiella

All the remained larvae after treatments were evaluated further reared in plastic trays (5.5 cm × 6 cm, depth and diameter) with artificial untreated diet (Muralimohan et al. 2009) to allow them to continue their development until pupal stage to calculate percent pupation. Pupae were placed individually in a Petri dish (8-cm diameter) until adults emerged to calculate percent adult emergence.

Statistical analysis

The data was analyzed with the Statistix® (Version 8.1) statistical package, using analysis of variance (ANOVA) in CRD to determine the effects of individual and interacted application of variables. Tukey’s HSD test for mean separation was used to compare mean values at P < 0.05 (Sokal and Rohlf 1995).

Results and discussion

Mortality rates of P. gossypiella

Thirty 2nd instar larvae of P. gossypiella, treated with different EPFs concentrations and A. indica (AI) (alone and/or in combination) showed significant effects at different exposure intervals. At 4 and 8 day intervals, all 3 EPFs combined with AI extract (Ba2 + Vl2 + Ma2 + AI) showed the highest mortality of 48.67 and 57.33%, while lowest the mortality rate, recorded in Bb1 (22.27 and 28.93%), respectively. Similarly at 8 day interval, all the 3 EPFs combined with AI extract (Ba2 + Vl2 + Ma2 + AI) showed the highest mortality rate (74.67%), whereas the lowest one (37.33%) was recorded in Vl1. Mortality rates of all treatments was compared to the control, where 0.00, 1.33 and 2.67% rates were recorded at 4, 8 and 12 day intervals, respectively (Table 1).

Table 1 Percent mortality (mean ± SE, n = 5) of 2nd instar Pectinophora gossypiella larvae due to Beauveria bassiana, Verticillium lecanii, Metarhizium anisopliae concentrations and Azadirachta indica extract (alone and in combination) at different exposure intervals. Means sharing with same lower case letters are not significantly different from each other at 0.05 level of significance, i.e., P < 0.05
Full size table

Mycosis and sporulation from dead cadavers of P. gossypiella

Effects of the lowest and the highest concentrations of EPFs, alone and in combination, on percent mycosis and sporulation (conidia ml−1) from dead cadavers of treated 2nd instar P. gossypiella larvae were recorded highly significant (P < 0.01) (percent mycosis: F6,34 = 46.7; sporulation: F6,34 = 48.7). Maximum percent mycosis and sporulation (94.20 ± 1.10% and 157.20 ± 1.67 conidia ml−1, respectively) was recorded at the dead cadavers of B. bassiana (Bb2)-treated P. gossypiella at the highest concentration (1 × 108 conidia ml−1), while the minimum percent mycosis and sporulation (37.20 ± 1.31% and 103.40 ± 1.05 conidia ml−1, respectively) was recorded at the dead cadavers of P. gossypiella treated with combination of A. indica + the highest concentration (1 × 108 conidia ml−1) of B. bassiana, V. lecanii and M. anisopliae (Bb2 + Vl2 + Ma2 + AI) (Table 2). These results indicated that AI demonstrated inhibitory effects on the mycosis and sporulation.

Table 2 Percent mycosis and sporulation (conidia ml−1) (mean ± SE, n = 5) from dead cadavers of 2nd instar Pectinophora gossypiella larvae due to the highest concentrations of Beauveria bassiana, Verticillium lecanii, Metarhizium anisopliae (1 × 108 conidia ml−1) (alone and in combination). Means sharing with same lower case letters are not significantly different from each other at 0.05 level of significance, i.e., P < 0.05
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Pupation and adult emergence from treated second larval instar of P. gossypiella

Effects of both lowest and the highest concentrations of EPFs, alone and in combination with A. indica extract, while that of the highest concentration of EPF (1 × 108 conidia ml−1) on percent pupation and progeny of the 2nd instar P. gossypiella larvae, were highly significant (P < 0.01) (percent pupation: F11,59 = 37.0; percent adult emergence: F11,59 = 22.8). Maximum percent pupation and adult emergence (90.67 ± 1.28% and 84.00 ± 2.47%, respectively) from P. gossypiella were recorded in control, while minimum percent pupation and adult emergence were zero recorded in P. gossypiella treated with a combination of the highest concentration of B. bassiana + V. lecanii + M. anisopliae (1 × 108 conidia ml−1) and A. indica (Bb2 + Vl2 + Ma2 + AI). Alone A. indica extract-treated P. gossypiella demonstrated 87.33 ± 1.73% pupation and 64.67 ± 3.08% adult emergence (Table 3).

Table 3 Percent pupation and percent adult emergence (mean ± SE, n = 5) of 2nd instar Pectinophora gossypiella larvae due to Beauveria bassiana, Verticillium lecanii, Metarhizium anisopliae concentrations and Azadirachta indica extract (alone and in combination). Means sharing with same lower case letters are not significantly different from each other at 0.05 level of significance, i.e., P < 0.05
Full size table

The EPFs conidial suspension concentrations alone, or combined with, A. indica extract showed promising potentials against P. gossypiella larvae by causing significantly high mortality rates after treatment within 4, 8 and 12 day intervals. B. bassiana, V. lecanii and M. anisopliae concentrations and A. indica extract (alone and in combination) at different exposure intervals showed the highest mortality rates. Similarly, at 8 day intervals, all the 3 EPFs combined with AI extract (Ma2 + Vl2 + Ma2 + AI) showed the highest mortality rate (74.67%). In the present investigations, P. gossypiella pupation as well as adult emergence were also significantly affected. B. bassiana, V. lecanii and M. anisopliae concentrations and A. indica extract (alone and in combination) at different exposure intervals caused high mortality rates among the treated 2nd instar P. gossypiella larvae due to their effects on specific hydrolytic enzyme such as proteinase, chitinase and lipase that affect cuticle (Kurtti and Keyhani 2008).

Introduction of an exogenous biological agent into an environment, with the aim towards its permanent establishment to control the pests present therein has been an effective technique over the long term (Kenis et al. 2017). Maximum percent mycosis and sporulation was recoded from the dead cadavers of B. bassiana-treated P. gossypiella at 1 × 108 conidia ml−1 concentration, while minimum percent mycosis and sporulation was recoded from the dead cadavers of combination of the highest concentrations of B. bassiana + V. lecanii + M. anisopliae. The results are in line with the findings of Riasat et al. (2011) who reported that the maximum mycosis (86.47%) and sporulation (153.22 conidia ml−1) were observed in treatments where the lowest concentration of B. bassiana (2.23 × 107 conidia Kg−1) alone was applied against adults of Rhizopertha dominica (F.) (Coleoptera: Bostrichidae). They also witness our results showing the antagonistic behavior of combined EPFs. They reported low rates of mycosis and sporulation in the treatments, where high concentrations of diatomaceous earth were mixed with B. bassiana. Similar results were also documented by Tefera and Pringle (2003), who found the highest mycosis and sporulation in cadavers of Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) treated with B. bassiana alone. The results of present finding are in line with the findings of Ashraf et al. (2017) applied 1.4 × 104 ml−1 conidial concentration of M. anisopliae individually against stored grain insect pests and reported that percent mycosis and sporulation was high.

Maximum percent pupation and adult emergence in 2nd instar larvae of P. gossypiella was observed in treatment where AI extract was applied alone, while minimum percent pupation and adult emergence was observed in treatment where combination of the highest concentrations of B. bassiana + V. lecanii + M. anisopliae along with AI extract was applied. These results are in agreement with Sufyan et al. (2019), who documented that both pupation and adult emergence of 2nd and 4th larval instars of C. partellus were maximum at low concentration of entomopathogens (alone and in combination), while minimum pupation and adult emergence were recorded at high concentrations of entomopathogens (alone and in combination).

Conclusion

This study concluded that the integration of EPFs and A. indica extract can prove a successful alternative to traditional chemicals and may become effective component of IPM program against P. gossypiella and some other insect pests. Further studies under field conditions are required.