Compatibility of entomopathogenic fungi and Azadirachta indica extract against the cotton pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) under controlled conditions

Compatibility of entomopathogenic fungi (EPFs) viz. Verticillium lecanii, Metarhizium anisopliae and Beauveria bassiana with Azadirachta indica extract (alone and in combinations) was evaluated against 2nd instar larvae of the cotton pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae) under controlled conditions. Results indicated that maximum mortality rate of P. gossypiella larvae was recorded at combination of the highest conidial concentrations of V. lecanii + M. anisopliae + B. bassiana along with A. indica extract, while minimum mortality rate was recorded at A. indica extract alone, 12 days post exposure interval. Maximum mycosis and sporulation from dead cadavers of P. gossypiella larvae were recorded at the highest concentration of B. bassiana, while the lowest mycosis and sporulation were recorded at the highest conidial concentrations of V. lecanii + M. anisopliae + B. bassiana along with A. indica extract. Maximum percent of pupation and adult emergence in P. gossypiella larvae was observed in the treatment, where A. indica extract was applied alone, while the minimum percent of pupation and adult emergence was observed at the treatment combination of the highest concentration of B. bassiana + V. lecanii + M. anisopliae along with A. indica extract was applied. EPFs proved their enhanced long-term protection potential for cotton crop against P. gossypiella. A. indica extract integrated with the EPFs can be a potential alternative to the chemicals and as an effective component of IPM program against P. gossypiella.

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.

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 2 nd 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 meshwindowed 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.

Preparation of conidial suspensions
EPF's conidial suspensions concentrations, 1 × 10 6 conidia ml −1 and 1 × 10 8 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 2 nd instar P. gossypiella larvae of uniform brood were treated by immersing them in to the conidial suspension (1 × 10 6 and 1 × 10 8 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).
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  (Table 1).
Maximum percent pupation and adult emergence in 2 nd 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 2 nd and 4 th 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.