Pathogenicity of entomopathogenic fungi against the aphid and the whitefly species on crops grown under greenhouse conditions in India

The aphids, Myzus persicae (Sulzer) and Aphis gossypii (Glover) (Hemiptera: Aphididae) and the whiteflies, Trialeurodes vaporariorum (Westwood) and Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) are the most damaging pests of greenhouse crops, which cause vector-borne viral diseases and its damage includes chlorosis, necrosis, and fruit abortion. This review article addresses the protected cultivation of vegetable crops (cucumber, capsicum, tomato, and gerbera), important insect pests of greenhouse crops in India along with its management by entomopathogenic fungi (EPF) and increase in the virulence by different genetically modified techniques. Due to excessive and indiscriminate use of insecticides over the years, these insect pests became resistant to these insecticides. So, there is a need for the effectual substitutes to manage these pests. Biological control is a foundation of integrated pest management (IPM) that plays a key role in the repression of arthropod pests. Among different IPM program, the uses of different microbial formulations are ecofriendly and safe for life and proven a boon for the farmers and entrepreneurs. EPF are the most effective in reducing aphid and whitefly populations on vegetable crops recommending its organic production under greenhouse conditions and could be a part of IPM. In the future, these insect pests will become more resistant to entomopathogens. Some novel techniques such as genetic engineering of fungal formulations will be required to increase the efficiency of various entomopathogens as these techniques are well adopted by countries like the USA, China, and European countries but its use in India and needs to be improved in the near future.


Background
In the changing scenario of increasing human population, decreasing cultivable land and water resources, there is a need to produce more food from available land and water. One technology being used in this direction is growing crops under protected cultivation, which has shown enormous potential during the last few decades and is well adopted for vegetable and ornamental crops. Protected cultivation of vegetable crops hold good for manifold increase in productivity compared to open-field conditions (Singh 2013;Pekkeriet et al. 2015, andChoudhary 2016). In the recent past, there has been a tremendous increase in area under protected cultivation (around 20 million hectares area all over the world). In India, the area under protected cultivation is around 46,000 hectares with productivity of 327 thousand tons, which is mainly confined to Andhra Pradesh, Maharashtra, Karnataka, Himachal Pradesh, Punjab, Haryana, and Rajasthan Senthilkumar et al. 2018). The farmers are growing vegetables, flowers, and other high-value crops under the protected environment and earn high profit. Among the vegetables, capsicum, chili, cucumber, tomato, and brinjal are the main crops grown under protected environment. The productions of these crops are greatly influenced by the insect pests (Vashishth 2009 andSood et al. 2012a). The whitefly, Trialeurodes vaporariorum (Westwood) and Bemisia tabaci (Gennadius); red spider mite, Tetranychus ludeni (Zacher) and T. urticae (Koch); serpentine leaf miner, Liriomyza trifoli (Burgess); Polyphagotarsonemus latus (Banks); thrips, Franklinella ocidentalis (Pergande); Scirtothrips dorsalis (Hood); tobacco caterpillar, Spodoptera litura (Fabricius) and aphid, Aphis gossypii (Glover) and Myzus persicae (Sulzer) are economic pests infest crops under protected cultivation (Sood et al. 2018;Singh and Joshi 2020). Among these, the aphids, M. persicae and A. gossypii (Hemiptera: Aphididae) and the whitefly, T. vaporariorum and B. tabaci (Hemiptera: Aleyrodidae) are the major insect pests, which cause vector borne viral diseases and their damage includes chlorosis, necrosis, wilting, stunting, flower and fruit abortion, leaf distortion, and defoliation (Sayed et al. 2019). Due to the availability of favorable environmental conditions under protected cultivation, the management of these pests is quite challenging. It is mainly done by chemical pesticides but due to excessive and indiscriminate use of these pesticides, insect pests become resistant against them. Additionally, inauspicious effects of chemical insecticide residues on the crop (Van Lenteren 2000), killing of non-target organisms (Pilkington et al. 2010), and development of resistance to pesticides (Sood and Sood 2005;Pappas et al. 2013) are linked with its excessive use.
Various parasitoids, predators, pathogens, and botanicals are being exploited for the management of these pests (Ali et al. 2017 andUllah et al. 2019). Entomopathogenic fungi (EPF) have been identified as potential control agents against aphids and the whitefly species under protected conditions (Saito and Sugiyama 2005). Beauveria bassiana (Balsamo) Vuillemin, Lecanicillium lecanii (Zimmerman), Metarhizium anisopliae (Metsch.), and Paecilomyces fumosoroseus (Wize) are the key tools for the management of various agricultural insect pests, including whiteflies, mealy bugs, aphids and thrips in outdoor and greenhouse crops (Wraight et al. 2000;Daniel and Wyss 2010;Shah and Shukla 2014).
This review will elucidate the fundamental information on the crops grown under protected conditions; discuss major insect pests of crops grown under protected cultivation in India along with its management with the use of different commercial and indigenous fungal formulations and genetically engineered entomopathogens.

Protected cultivation of vegetable crops in India
The concept of protected cultivation came into existence in seventeenth century when natives of Netherlands and England used artificial structures (similar to polyhouse structures) to produce the crop under adequate climatic conditions (Janick et al. 2007). In today's world, the Netherlands is forefront in the production of largecapacity polyhouses to enhance the crop yield and quality (Muijzenberg and Erwin 1980). The adoption of new agricultural techniques is foremost in the agriculture leading countries to produce the crop with high quality and revenue from low-cost investment. The uses of various agricultural approaches put India out of frontier (Navale et al. 2003 andKanzaria et al. 2017). The protected cultivation includes growing crops under polyhouse, greenhouse, net house which are made up of materials like acrylic, polycarbonate, polyethylene which help to protect the crops from environmental factors. Nowadays, India is becoming popular for protected cultivation among the farmers and entrepreneurship because of its profitability and sustainability. Protected cultivation is the most important way to produce a large variety of horticultural crops, including tomato, capsicum, cucumber, and other vegetable crops . Under protected conditions, various factors like temperature, humidity, light, soil, and water are controlled. Additionally, the economics of capsicum under protected cultivation was studied by Sreedhara et al. (2013) and they revealed that India contributes onefourth of world production of capsicum with an average annual production of 0.9 million tons from an area of 0.885 million hectares with a productivity of 1266 kg per hectare and out of total production; Andhra Pradesh stands first with production of 748.5 thousand tons followed by Karnataka from an area of 76 thousand hectares with a productivity of 131 thousand tons. Weintraub (2007)  Abundances of aphid and whitefly species on crops grown under protected cultivation in India and temperate regions of the world. More than 1420 species of whiteflies associated with agricultural crops throughout the world. Among different species of whiteflies, B. tabaci being one of the most widely distributed pests found in tropical and subtropical regions of the world, where it infests over 600 different cultivated and wild plant species from 63 plant families (Oliveira et al. 2001). In India, B. tabaci was first recorded on cotton at Pusa (Bihar) in 1905 (Misra and Lamba 1929). Seventeen plant whitefly species belonging to widely separate families were reported in the Kalyani area of West Bengal. In Andhra Pradesh, B. tabaci was found to survive on 14 off-season hosts (Verma et al. 1989). Later, (Arneja 2000) from Punjab reported B. tabaci from 16 different host plants comprising field crops, vegetables, and weeds. Konar during (1997) also observed the three species of Aleyrodidae associated with Citrus reticulate (mandarin) orchards in the Darjeeling district of West Bengal. Whereas, in Lakshadweep (Dubey et al. 2004) reported the occurrence of 12 species of whiteflies representing 11 genera. From Himachal Pradesh, (Bhalla and Pawar 1977) recorded 10 species of whiteflies belonging to 6 genera. Bemisia tabaci was recorded on crops such as Capsicum annum, Cucumis sativus, Lycopersican esculentum, Gerbera jamesonii, and Solanum melongena in different regions of India (Reddy and Kumar 2006;Sood and Sharma 2010;Ibrahim et al. 2011;Kumar et al. 2017;and Padhi et al. 2017;Khanzada et al. 2018) (Table 1). In India, the incidence of T. vaporariorum was recorded first at Thummantty in the Nilgiri hills of Tamil Nadu and Himachal Pradesh on crops such as potato, capsicum, tomato, and cucumber (David 1971;Sharma et al. 2006;Sood et al. 2006;Vashisth 2009;Sood et al. 2012b;Sood and David 2012). So far, It has been reported on 120 host plants belonging to 38 plant families, mainly Solanaceae (15 species), Compositae (17 species), Labiatae (8 species), Acanthaceae (7 species), Leguminosae and Onagraceae (6 species each), and Malvaceae and Verbenaceae (5 species each) from different parts of India (Mohan et al. 1988;Krishnan andDavid 1999, andBakshi et al. 2003).
The wide range of aleyrodid fauna, B. tabaci and T. vaporariorum has drawn the maximum attention due to their wider host range and the losses incurred. Bemisia tabaci is distributed in the warmer regions Byrne and Bellows (1991) whereas T. vaporariorum is a serious pest in temperate region under glasshouses and field crops with summer region (Hill 1987). It is a serious pest in temperate regions under protected cultivation situations and in field crops where the summers are warm enough. It is considered as a "New World" species having distribution throughout Europe, parts of Africa, Asia, Australasia, North America, and South America (Hill 1987). Moreover, the infestation of greenhouse whitefly occurs in 249 host plants from 84 families of different crops and ornamental plants in temperate regions from six continents Russell (1977). The whitefly breeds were found throughout the year and it completes 13 generations in a year under protected environment in India (Sood et al. 2014). Adults of T. vaporariorum are tiny (1 mm long) snowy whiteflies with a covering of white waxy powder on wings. They are mostly found on the under surface of the leaves (Hill 1987). One hundred and fourteen virus species are transmitted by whiteflies (Aleyrodidae). Whiteflies, B. tabaci, and T. vaporariorum transmits 111 and 3 species, respectively. Of the whitefly-transmitted virus species, 90% belongs to the Begomovirus genus, 6% belongs to the Crinivirus genus, and the remaining 4% belongs to the Closterovirus, Ipomovirus, and Carlavirus genera (Jones 2003). The host plants were found to have a pronounced effect on the selection, feeding, oviposition, and development of whitefly. Overall, the yield loss due to various insect pests ranged from 30-40% among different vegetable crops in India (Sharma et al. 2017). However, loss due to whitefly under protected conditions varies among different vegetables. In capsicum, whitefly, B. tabaci caused 13.60% yield loss (Singh H and Joshi N 2020) whereas, on cucumber plant yield loss was approximately 26% (Ghongade 2020). Moreover, whitefly caused 54% yield loss in okra plant (Dhandapani et al. 2003). The development of the whitefly was seen higher on eggplant and cucumber, followed by tomato whereas slow on capsicum crop (Kamp and Lenteren 1981). Research indicated  that the whitefly prefers tobacco, cucumber, broad bean, and cowpea significantly more than paprika with more probing time on preferred hosts (Xu et al. 1994).

Aphids
The aphid species, M. persicae and A. gossypii are the most damaging insect pests of crops grown under protected conditions across the world because of their ability to transmit viruses to the plants. Their damage includes chlorosis, necrosis, fruit abortion, and stunted growth (Perdikis et al. 2008

Biological control by entomopathogenic fungi
Entomopathogenic fungi (EPF) are bioinsecticides with an ability to infect and kill arthropods. They are isolated from the naturally occurring soil and arthropod carcasses (Behie and Bidochka 2014;Litwin et al. 2020). These fungi belong to 6 fungal classes: Basidiomycota, Entomophtoromycota, Oomycetes, Chytridiomycota, Microsporidia, and Ascomycota. Until now, 238 species of Basidiomycota, 474 species of Entomophtoromycota, 12 species of Oomycetes, 65 species of Chytridiomycota, 339 species of Microsporidia, and 476 species Ascomycota were reported (Araujo and Hughes 2016). However, species of Ascomycota and Entomophtoromycota are more often in natural habitat. Metarhizium spp. (Metschnikoff) Sorokin are ubiquitous naturally occurring soil-inhabiting fungi (Meyling et al. 2011). In 1888, Elie Metchnikoff, a Russian microbiologist firstly discovered the EPF and named it as Metarhizium anisopliae. In 1965, Boverin, a Beauveria bassiana based mycoinsecticide was developed to control the Colorado potato beetle and codling moth (Litwin et al. 2020). Verticillium lecanii (Zimmerman) Viégas (reclassified as Lecanicillium lecanii (Petch) Zare and Gams as an insect pathogen that is used commercially to control greenhouse pests (Cuthberston and Walter 2005).

Virulence of entomopathogenic fungi against whitefly
All biopesticides containing insect pathogen B. bassiana, V. lecanii, and M. anisopliae were found effective in reducing the pest population. But the organic products and mineral oils were comparatively less effective. Therefore, a study Singh and Kaur Egyptian Journal of Biological Pest Control (2020)  was conducted to evaluate EPF against B. tabaci and T. vaporariorum, and obtained result showed that L. lecanii was more effective against B. tabaci and T. vaporariorum causing 89.3 to 96 and 79.3 to 95.6% mortality, respectively under greenhouse conditions. In addition to this, the infectious and epizootic potential of P. fumosoroseus on the susceptibility of B. argentifolii were not affected by the host vegetable species of tomato (Vidal et al. 1998). However, mortality was > 70%, the first week after treatment and increased further in the second week. Lecanicillium lecanii caused 86% mortality of B. tabaci on brinjal at 5 days after treatment (Phadke and Phadke 2000). Besides this, B. bassiana and P. fumosoroseus have strong potential for microbial control of nymphal whiteflies infesting cucurbit crops (Wraight et al. 2000). Thirty-five strains of L. lecanii originated from different hosts and geographical locations were tested as potential biocontrol agents against silver leaf whitefly, B. argentifolii (Gindin et al. 2000). Pathogenicity of B. bassiana (strain GHA, lot TGAI 97-10-1) and P. fumosoroseus (strain 612, lot 940917) with conidial concentration of 1.4 × 10 11 and 1.8 × 10 11 conidia/ml were tested against the 3rd-instar nymphs of T. vaporariorum reared on cucumber and tomato plants. Nymphs were highly susceptible to infection by both fungi after a one-time application of conidia onto cucumber plants. In contrast, insects reared on tomato plants were significantly less susceptible to infection (Poprawski et al. 2000). The whitefly mycoinsecticide products based on L. lecanii, P. fumosoroseus, and B. bassiana have the capacity to suppress and in some instances, provide good control of whiteflies in both greenhouse and field crops. Development of methods and strategies to enhance mycoinsecticide efficacy has been attempted and primarily focus on initiating treatments against the early stages of the pest to prevent population build up, targeting pest populations developing under moderate environmental conditions (e.g., during spring or fall growing seasons), selecting crops amenable to multiple, highly efficient spray applications and applying fungi asynchronously with incompatible fungicides (Faria and Wraight 2001). Pathogenicity of commercial EPF L. muscarium (Mycotal, Koppert Biological Systems Ltd, UK) was evaluated on sweet potato whitefly B. tabaci under laboratory and glasshouse cultivation and recorded that the application of L. muscarium against B. tabaci resulted in a significant increase in the mortality of B. tabaci under glasshouse cultivation and also no plant damage was recorded during the experiment (Cuthbertson and Walters 2005). Furthermore, the EPF L. lecanii caused 26.6 to 76.6% mortality against T. vaporariorum 7 days post treatment when applied with the concentration of 1 × 10 7 conidia/ml as reported by (Scorsetti et al. 2008). The efficacy of different bio-pesticide was reported against whitefly B. tabaci on tomato plant and result showed that biopesticides viz. Bio Magic (91.64%), Mealikil (93.55%), and Biopower (88.91) were highly effective in killing the nymphal whitefly population over control after 3rd spray (Budha et al. 2015). Some of the commercially available biopesticides are shown in Table 2. The most effective EPF that reduced the pest population were B. bassiana, V. lecanii, and M. anisopliae that was evaluated at three different concentrations (1 × 10 7 , 1 × 10 8 , and 1 × 10 9 spores/ml) against B. tabaci in laboratory and field cultivation, respectively (Abdel-Rahim and Ahmed 2017). They reported that the highest concentration (1 × 10 9 spores/ml) of the 3 EPF was highly toxic to adults of B. tabaci than the others. Under field cultivation also highest concentration (1 × 10 9 spores/ml) of V. lecanii was the best in managing the adult whitefly population. Virulence of entomopathogenic fungi against aphid Twelve different strains of EPF, viz., L. lecanii, P. farinosus, B. bassiana, M. anisopliae, Cordyceps scarabaeicola, and Nomuraea rileyi (Hypocreales: Clavicipitaceae) were evaluated against aphids on cabbage and cucumber crops grown under greenhouse conditions. Among these EPF's, L. lecanii 41185 strain was highly virulent than all other strains against M. persicae and A. gossypii because it germinates and grew well under a wide range of temperature and humidity as reported by Vu et al. (2007). Beauveria bassiana was used for the control of M. persicae on cabbage and it caused 76-83% mortality in 4 weeks after spray (Filho et al. 2011

Increased virulence by genetically engineered methods
The release of the cuticle enzyme was low during the initial penetration t and high during the degradation of protein (St. Leger et al. 1996). Beauveria bassiana and M. anisopliae caused infection against insect pests through their cuticle by release of cuticle degrading enzymes. By the use of phage display technology, hydrophobins were isolated from the culture of B. bassiana. Furthermore, cDNA library was built up by using RNA, in the presence of insect pests. Hydrophobins appears should be selectively enriched by melibiose agarose beads, which further yield two (hyd 1 and hyd 2) different hydrobhobin proteins. Results revealed that hyd 1 gene expressed well in aerial conidia, in vitro blastospores, submerged conidia, and cells sporulating on chitin and insect cuticle in contrast to hyd 2 gene (Cho et al. 2007). Additionally, modification of cuticle degrading and adhesion toxin intestinespecific virulence factor (Vip3Aa1) into B. bassiana isolate BbV28 in the cabbage transgenic plant against S. litura aids in digestion in the cytoplasm of insect after 18 to 36 h of conidial infection (Qin et al. 2010).
Systematically disruption of Cag8 gene is one of the alternative ways to increase the expression of gene Mrku70 (Xu et al. 2014) that helps to increase the pathogenicity of M. robertsii ARSEF2575 by 93% as compared to wild type (7%). Insect cuticle is mainly composed of chitin fibrils, to penetrate this barrier fungus produce chitin degrading enzyme such as proteases and chitinases (Zhao et al. 2016). Over expression of Pr1A enzyme in M. robertsii and CDEP1 Bbchit1 in B. bassiana can lead to increase the virulence of these fungi against major crop pests. Genes along with metabolic pathways to increase the virulence are described in Table 3.

Conclusion
Protected cultivation or greenhouse cultivations would be the modern approach to produce vegetable crops qualitatively and quantitatively in India. Due to the availability of favorable environmental conditions, various insect pests cause acute to severe crop damages under protected cultivations. Aphids and whiteflies are the major insect pests of crops grown under protected cultivations. The use of entomopathogenic fungi (EPF) is one of the alternatives to control various insect pests in India over a few decades. In the future, some novel techniques are required to increase the efficiency of various entomopathogens. Techniques like genetically engineering and cDNA probe will be one of the appropriate ones to decrease pest resistance. These techniques are well adopted by countries like the USA, China, and European countries but its use is limited in India and need to be improved in the near future.