Evaluation of Metarhizium rileyi Farlow (Samson) impregnated with azadirachtin and indoxacarb against Helicoverpa armigera (Hubner)

Entomopathogenic fungi are the most versatile having a wide host range, capable of infecting insects at different developmental stages. In the present study, Metarhizium rileyi, at the concentrations of 102, 103, 104, 105, 106, 107 and 108 conidia/ml and sub-lethal concentrations of azadirachtin (1.02 and 1.53 ppm) and indoxacarb (0.72 ppm) were evaluated against the 1st, 2nd, 3rd, 4th and 5th larval instars of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) under laboratory conditions. M. rileyi applied at 106 conidia/ml caused a maximum mortality of 83.33 and 80.00% of 1st and 2nd larval instars of H. armigera, respectively. The maximum mortality of 3rd, 4th and 5th larval instars of H. armigera with 108 conidia/ml of M. rileyi was 83.33, 76.67 and 53.33%, respectively. When M. rileyi blended with azadirachtin at 1.02 ppm, the highest mortality rate of 86.21% at 106 conidia/ml against 2nd instar larvae was resulted. Similarly, M. rileyi applied at 108 conidia /ml mixed with azadirachtin (1.53 ppm) showed 89.66% mortality of 3rd instar larvae. The 2nd instar larvae treated with M. rileyi at 106 conidia/ml, mixed with indoxacarb (0.72 ppm), the corrected mortality rate was 82.14%. Concentration mortality response of 3rd instar larvae to M. rileyi blended with indoxacarb (0.72 ppm) was 85.71% at 108 conidia/ml. The median lethal concentration (LC50) values were 5.51 × 103, 1.86 × 104, 2.81 × 105 and 5.55 × 105 conidia/ml for 1st, 2nd, 3rd and 4th larval instars, respectively, after 7 days of treatment. M. rileyi when mixed with sub-lethal concentrations of azadirachtin (1.02 ppm) and indoxacarb (0.72 ppm) resulted LC50 values of 1.09 × 104 conidia/ml and 1.37 × 104 conidia/ml against 2nd instar larvae, respectively, after 24 hours. Similarly, M. rileyi mixed with sub-lethal concentrations of azadirachtin (1.53 ppm) and indoxacarb (0.72 ppm) resulted LC50 values of 3.12 × 108 and 3.06 × 105 conidia/ml against 3rd instar larvae, respectively, after 24 hours. The study revealed that the susceptibility of larvae decreased in case of large larval instars. M. rileyi can be utilized as one of the component of Integrated Pest Management Program for the eco-friendly management of H. armigera. As the application of M. rileyi @ 107 conidia/ml alone or in combination with azadirachtin (1.02 and 1.53 ppm) or indoxacarb (0.72 ppm) resulted to the highest mortality.

Page 2 of 7 Dev et al. Egyptian Journal of Biological Pest Control (2021) 31:142 than 47 families (Bird 2017). H. armigera has a high fecundity, high rate of fertility, high dispersal rate, long distance movement, overlapping generations per year under tropical and subtropical conditions, respectively, and resistance development against insecticides (Jones et al. 2019). For the management of this noctuid pest farmers mainly used synthetic insecticides, Excessive use of chemical insecticides to control the pest has led to development of pest resistance, pest resurgence, killing of natural enemies, environmental pollution besides being costly. Therefore, there is a need of development of alternative tools. Entomopathogenic fungi (EPF) are an alternative to chemical pesticides which is ecofriendly, safe to non-target organisms and prevent pesticides resistance (Leahy et al. 2014). EPF are the most versatile due to their wide host range, capable of infecting insects at different developmental stages and ability to penetrate through the host cuticle (Vega et al. 2012).
Metarhizium rileyi is a dimorphic hypomycete fungus and initially named as Botrytis rileyi (Farlow) and later on described as Spicaria rileyi (Farlow) Charles. Kish et al. (1974) re-described the fungus and kept in the genus, Nomuraea. According to Boucias et al. (2000), N. rileyi isolates were more closely related to Metarhizium anisopliae and M. flavoviride than to N. atypicola and N. anemonoides. Metarhizium spp. have been extensively exploited because it is ecofriendly and easy to mass produce (Greenfield et al. 2015). Metarhizium genus was originally comprised of four species, which were M. anisopliae, M. taii, M. pingshaense and M. guizhouense. N. rileyi isolates were closely related to M. anisopliae and M. flavoviride than to N. atypicola and N. anemonoides. Based on morphological and molecular characterization, N. rileyi has been changed to M. rileyi (Kepler et al. 2014). It is observed that sometimes farmers spray the crop with insecticides alone or in combination with EPF for the management of the pests. Therefore, it is necessitated to know the action of synthetic chemical insecticides in combination with the M. rileyi and determine their compatibility. Many authors have conducted the studies on the combination of pesticides with EPF (Kachhadiya et al. 2014). On the other hand, Ignoffo et al. (1975) reported that several chemical products applied in soybean crop inhibited growth as well as virulence of N. rileyi. The information on combined action of sub-lethal concentrations of synthetic chemical pesticides and M. rileyi is scanty. Therefore, the aim of present study was to evaluate the susceptibility of H. armigera larvae to M. rileyi incorporated with sub-lethal concentrations of azadirachtin and indoxacarb under laboratory conditions.

Rearing of insect culture
The culture of H. armigera was raised in in vitro (25 ± 0.5 °C, 70 ± 5% RH and 14L:10D photoperiod) conditions from caterpillars collected from the field on chickpea crop. The larvae were reared individually in rearing trays on chickpea sprouts. Larval food was changed daily or as per requirement until pupation. Pupae obtained were transferred to glass jars for adult emergence. Adults on emergence were shifted to rearing cages for mating and egg laying. Adults were provided with 30% honey solution (in cotton swabs) as food and strips of filter paper as substrate for egg lying. The insect was reared for 2 generations before using in experimentations.

Rearing of culture of M. rileyi and treatment of H. armigera
The nucleus culture of M. rileyi was obtained from National Bureau of Agricultural Insect Resources (NBAIR) Bangaluru and further multiplied on SDAY (Sabouraud dextrose agar + yeast extract medium). Newly inoculated slants were incubated at 25 ± 0.5 °C and 70 ± 5%RH. M. rileyi was evaluated against 1st, 2nd, 3rd, 4th and 5th larval instars of H. armigera. Harvesting of conidia was carried out from 15-days-old well sporulated culture in tubes by pouring 10 ml sterilized emulsified (0.5% Tween 80) distilled water in each tube. The concentration of conidia in the suspension was determined by a Neubauer Hemocytometer and further adjusted the conidial suspension of 10 8 or 10 7 conidia/ ml depending upon the harvested. Conidial suspension thus obtained was serially diluted in 1:9 ratio with sterilized emulsified distilled water to get test concentrations of 10 6 , 10 5 , 10 4 , 10 3 and 10 2 conidia/ml. For the combinations, different concentrations of azadirachtin and indoxacarb were fortified with the conidial suspension and larvae of H. armigera where treated by larval dip method for 10 s, and data were recorded after 24 h of treatment upto 7 days.

Data analysis
Mortality data were subjected to probit analysis as per Finney (1952). The mortality data falls in the range of 20-80% were subjected to probit analysis, and LC 50 / LC 90 values were calculated by IBM SPSS Statistics 20.

Effect of azadirachtin and indoxacarb on growth of M. rileyi
M. rileyi was tested against both azadirachtin and indoxacarb at tested concentrations and founded that they inhibited the growth of fungus over control (

Discussion
In the present study, high mortality rate of the early instars of H. armigera may be due to fragile and thin cuticle of the larvae which was easy for the germ tube of conidia to penetrate, germinate and caused mycelium growth. The present findings were in agreement with the findings of Manjula and Krishnamurthy (2005) who found mortality of 80-95% at 1st and 2nd larval instars of H. armigera at the concentration of 10 7 conidia/ml of M. rileyi. Similar to the present findings Gundannavar et al. (2008) recorded 100 and 97.50% mortality of the 1st instar larvae due to M. rileyi, at the concentration of 10 8 conidia/ml and 10 7 conidia/ml, respectively, whereas, a mortality of 95% at the concentration of 10 8 conidia/ ml of M. rileyi was recorded with the 2nd instar larvae.
In the present study, M. rileyi killed 83.33% of 3rd instar larvae of H. armigera at concentration of 10 8 conidia/ ml. These findings were in line with findings of Gundannavar et al. (2008) who reported 82.50% mortality at 10 8 conidia/ml. Similar, to present findings, Padanad and Krishnaraj (2009) reported that M. rileyi isolates tested against 3rd instar larvae of S. litura caused mortality in the range of 85-97%. M. rileyi @ 10 8 conidia/ml caused 76.67% mortality to the 4th instar larvae of H. armigera.  These findings were in accordance with the findings of Gundannavar et al. (2008) who recorded 75% mortality at same concentration. M. rileyi caused 53.33% mortality on the 5th instar H. armigera larvae, at concentration of 10 8 conidia/ml. The lowest mortality to the 5th instar larvae than early instar may be due to thick cuticle of the oldest instar larvae, which makes it difficult for the fungus to penetrate, germinate, and form mycelial growth and kill the larvae. Similar to present findings, Namasivayam and Arvind (2015) reported that the LC 50 values increased as the larvae grew older. As the instars advanced, a decrease in mortality was recorded. The present study was in agreement with the study of Patil et al. (2014) who noticed that early instars were highly susceptible with a mortality of 70.17% and mortality decreased significantly with the increase in age of the larvae. The present findings also corroborate the findings of Gundannavar et al. (2008) who reported 47.50% mortality at 10 8 conidia/ ml. Whereas, Mohamed et al. (1978) observed a high mortality (63%) at a concentration of 10 9 conidia/ml M. rileyi. In the present investigations, M. rileyi mixed with azadirachtin and indoxacarb separately enhanced the lethal effect of M. rileyi. The increase in the efficacy of the M. rileyi in the presence of azadirachtin and indoxacarb may be due to increased susceptibility of larvae. M. rileyi with indoxacarb (0.72 ppm) showed better performance than M. rileyi with azadiracthin (1.02 ppm) against 2nd instar larvae of H. armigera. The superiority of indoxacarb over azadirachtin may be due to more stress exhibited to the larvae. M. rileyi with azadirachtin (1.53 ppm) resulted to a slightly high mortality than M. rileyi mixed with indoxacarb (0.72 ppm) to the 3rd instar larvae of H. armigera might be due to interference of neem (azadirachtin) with insect development and formation of cuticle or the molting process (Rembold 1989). According to Zimmermann (1994), if new cuticle formation was affected in term of deposition, hardening and tanning it will reduce the barricading ability to fungus, thus the chance of mycosis might increase.

Conclusions
Susceptibility of larvae decreased with the increase in larval instars of H. armigera. M. rileyi impregnated with azadirachtin (1.02 and 1.53 ppm) and indoxacarb (0.72 ppm) inhibited the growth of M. rileyi but increased the lethal effect against H. armigera. Thus, it can be concluded that either M. rileyi at 10 7 conidia/ml alone or impregnated with azadirachtin (1.02 and 1.53 ppm) or indoxacarb (0.72 ppm) resulted almost equal mortality to the larvae of H. armigera. Hence, M. rileyi can be utilized as one of the components of IPM program for the ecofriendly management of H. armigera.