The fungal pathogens act primarily by cuticular degradation, followed by the development of fungal hyphae and spores on the insect body. The cuticular degradation may be due to the secretion of hydrolytic enzymes such as proteases, lipases, and chitinases which play an important role in the infection process (Banu et al. 2010).
Among the sole and combinational treatments of EPF, the combination of L. lecanii with B. bassiana was found superior over the sole treatments (Fig. 1). When these biopesticides used in sole, L. lecanii was found significantly superior to B. bassiana at the tested doses (4 and 6 g/l). These bioagents were found effective in reducing the mealy bug population by causing mortality at the range of 42.71% to more than 90% at 15 DAT. The maximum mortality in the combination of both B. bassiana and L. lecanii may be attributed to potentiation but with limited synergetic action of both the entomopathogens. When used in sole, the L. lecanii recorded 83.96% mortality at the highest concentration and was almost 2 times significant than B. bassiana that recorded only 56.67% mortality at the same concentration. Of the two EPF, L. lecanii showed a high mortality on all stages of mealy bugs, which is in agreement with the results of Russell and Paterson (2006). The highest efficacy on mealy bugs could be because of production of mycotoxins, viz., helvolic acid, bassianolide, cytochalasin C and D, destruxins, and faster mycelial growth to penetrate into the body of the host which caused septicemia to the mealy bug, leading to early death as compared to other entomopathogens like Metarhizium anisopliae (Metschinkoff) Sorokin and B. bassiana (Banu et al. 2010).
The treatments with B. bassiana recorded lower nymphal mortality (around 20–30%) on grape mealy bug than to L. lecanii. Similarly, Amala et al. (2014) reported that B. bassiana was less effective against grape mealy bug than L. lecanii with LC50 and LT50 values of 1.5 × 106 and 3.04 days, respectively. Jeyarani et al. (2011) also reported that B. bassiana could cause 50% mortality of cotton mealy bug, Planococcus solenopsis Tinsley (Hemiptera: Pseudococcidae) at 3.6 × 107 spore/ml concentration and is in conformity with present findings, wherein the present trial the spore concentration used was 2 × 108 cfu/ml. Kanitkar et al. (2020) revealed that application of Brigade-BL (B. bassiana) at 5.0 ml/l with 2 sprayings resulted in reduction of mealy bug colonies up to 67.82 and 75.68% in foundation and fruiting pruning, respectively. Similarly, it has not resulted in any abnormality and phytotoxic effects like leaf chlorosis, tip burning, necrosis, epinasty, and rusting on grapevines and were found compatible with Buprofezin 25 SC chemical insecticide.
Treatment with insecticide, imidacloprid at 0.25 ml/l significantly affected the mealy bug population by recording mortality of more than 90%. The systemic nature of imidacloprid in tuber dip bioassay, resulted in effective suppression of mealy bugs whereas, the topical application of contact insecticides many times fail to cause mortality in mealy bugs because of the heavy waxy coatings on the body surface. Reports of Meyerdirk et al. (2004) also suggested some biological characteristics of the pest like secretion of wax, waxy ovisac, and the ability to oviposit on foreign substrates which led to difficulty in control of mealy bugs. Perhaps, this difficulty could be quite evident especially in contact insecticides compared to systemic insecticides.
The use of a wetting agent is very important prerequisite to dissolve waxy secretions in mealy bugs for easy deposition and penetration of fungal bodies. Though wetting agent itself caused negligible mortality of mealy bugs, its addition along with EPF is an essential pre-requisite, as the mode of entry of these EPFs is through the cuticle. In addition to the wetting agents, several other additives have also proved promising in recording increased mortality of mealy bug. A similar kind of effort made by Chavan and Kadam (2010) recorded a maximum of 82.50% mortality of M. hirsutus nymphs in formulations containing V. lecanii + glycerol 8% + Tween 80 at 1% and Arachid oil 0.5%.
The early stages of mealy bugs are more susceptible to fungal biopesticides in general and L. lecanii in specific. In the present investigations, the first and second instar nymphs of M. hirsutus were selected and probably are more susceptible to EPF like L. lecanii than the later instars. In addition, EPFs had latent infection periods ranging from 5 to 10 days, which results in the escape of infection in late-stage nymphs. Ten days after EPF treatment, the highest mortality of 65.10 and 58.99% of the first and second nymphal instars of M. hirsutus was recorded, respectively, whereas 45.96 and 30.66% mortality in third instar nymphs and adult mealy bugs, respectively (Makadia et al. 2009).
In addition to these factors, the time required for infection, moribund, and mortality is important and depends on the stage of the pest. So, the present study was found to be in conformity with the finding of Halder et al. (2013), where the % mortality of nymphs increased with the increase in time where, 6 days after the spraying, V. lecanii (at 2 × 109 cfu/g) caused the mortality of 67.11%, followed by B. bassiana (1 × 108 cfu/g) and M. anisopliae (1 × 108 cfu/g) with 62.85 and 56.52% mortality, respectively. Among the 3 tested fungal pathogens, L. lecanii at 1 × 108 spores /ml was proved to be an efficient entomopathogen with increased efficiency as the infection time prolonged. The mean fecundity of adult females treated with L. lecanii was lower (54.20 eggs/female) with a significant reduction in the hatchability of the eggs (25.20). This is reflected clearly in the present studies, indicating greater post pathogenicity and speed of kill in L. lecanii (63.34% in 9 days) than B. bassiana (35.84% in 9 days). Further, the virulence of L. lecanii was maintained even in combination with B. bassiana. L. lecanii recorded 26.00 to 82.28% mortality from first to fourth nymphal instars of mealy bug, M. hirsutus infesting custard apple (Makadia et al. 2009).
Among the botanical oils/formulations, significantly high cumulative nymphal mortality was noticed in neem oil at 15 ml/l (81.36%) and was statistically on par with neem oil + pongamia oil at 10 + 10 ml/l (79.65%), neem oil at 10 ml/l (78.09%), and IIHR neem soap at 10 g/l (77.67%). In general, the formulations made of or extracted from neem proved effective in managing the mealy bug population. Neem oil acts in many ways like insect growth regulator, feeding deterrent, and larvicidal activity. So, multiple modes of action of neem-based botanical oils qualify it as a unique insecticidal product and proved superior compared to pongamia oil. At 15 ml/l dose, neem oil was found significantly superior to all the other botanicals tested.
Verghese (1997) reported 5 and 2.5% neem seed kernel extracts as highly effective in causing mortality of early instar M. hirsutus nymphs after 24 and 48 h, after treatment under laboratory condition, while commercial product, Econeem® (Azadirachtin) 1500 ppm was effective on late instar mealy bugs after 24 and 48 h. of treatment. On the contrary, Banu et al. (2010) reported a moderate mortality in nymphs and adults (42.22 and 55.56%, respectively) of cotton mealy bug, P. solenopsis with NSKE at 5%, while neem oil + Nirma® powder caused 46.67 and 57.78% mortality of nymphs and adults, respectively, after 48 h of treatment. This might be due to thick waxy coating and high body mass of late instar nymphs and adults. However, early instar nymphs were more susceptible to neem oil at high concentrations (15 ml/l) or in combination with pongamia oil at 10 + 10 ml/l. The results are in close agreement with Kumar et al. (1989) on the coffee green scale, Coccus viridis (Green) (Hemiptera: Coccidae), and Manjunath et al. (1992) on mealy bug, M. hirsutus.
More than 70% mortality was exhibited by commercial neem products are supported by Shekhar and Sathyaprasad (1998) who reported appreciable mortality of nymphs (95 to 98%) with Rakshak® and Amruthaguard® (0.5%), while their efficacy reduced on adults (around 51%) of M. hirsutus under laboratory conditions.
The time taken for causing more than 50% mortality of second or third instars was shortest (3 days) in case of neem oil at 15 ml/l (60.00% mortality), indicating quicker penetration and action. The results are corroborated with findings of Halder et al. (2013) who reported LT50 for neem oil 5% as 93.71 h, while it is blending with a fungal pathogen like L. lecanii, B. bassiana, and M. anisopliae at 1:1 ratio exhibited LT50 of 87.67, 88.34, and 90.82 h, respectively. So, in the present findings, the superiority of neem oil as a sole treatment is quite evident compared to its blends.
The treatments with pongamia oil at 15 ml/l and 10 ml/l were recorded moderate nymphal mortality of 36.63 and 35.11%, respectively. On the contrary, Verghese and Tandon (1987) reported that pongamia oil (2%) was very effective in killing crawlers of M. hirsutus, which could be because of high concentrations compared to present studies.
Fish oil rosin soap (FORS) at 10 ml/l recorded moderate nymphal mortality (63.38%) and was on par with FORS at 15 ml/l (60.74%), followed by FORS at 5 ml/l (44.37%). All the combinations of treatments with FORS gave moderate to high mortality and it could be due to enhancement of botanical penetration into insect body by dissolving the waxy filament layer. Overall, FORS concentrations performed inferior than neem oil at similar concentrations.