Lecanicillium spp. for the Management of Aphids, Whiteflies, Thrips, Scales and Mealy Bugs: Review

Lecanicillium spp. are potential microbial bio-control agent mainly used for the management of sucking insect pests such as aphids, whiteflies, scales, mealy bugs etc. and gaining much importance at present for management of pests. Due to indiscrimi-nate use of chemical pesticides which results in development of resistance, resurgence, outbreak of pests and residue problem, the farmers/growers are forced to use biopesticides for sustainable agriculture. Lecanicillium spp. is promising biocontrol agent against sucking insect pests and can be used as one of the components in integrated pest management (IPM). However, optimum temperature and relative humidity are the major environmental factors, for the performance of Lecanicillium spp. under protected/field conditions. The present review is mainly focused on nomenclature of Lecanicillium spp., mode of infection, natural occurrence, influence of temperature and humidity on the growth, factors influencing the efficacy, virulence/pathogenicity to target pests, substrates used for mass production, safety to non-target organisms, compatibility with agrochemicals and commercially available products. This review is mainly useful for the researchers/students to plan their future work on Lecanicillium spp.


Introduction
The increased use of conventional chemical pesticides over the years has not only contributed to an increase in food production, but also has resulted in adverse effects on the environment and non-target organisms. In view of these side effects, the necessity for sustainable crop production through ecofriendly pest management technique is being largely felt in the recent times. Few biopesticides are available in the market, among them Lecanicillium spp. based microbial bio-pesticide gaining much importance for sucking pests for organic and sustainable agriculture [1][2][3][4]. Myco pesticides are potential microbial alternative to chemical pesticides and offer a number of benefits such as facility of growth on a variety of substrates, high virulence, trans cuticular penetration, broad host range, less expensive, safe to humans, animals and the environment. Therefore, this review is prepared by compiling the research work done on Lecanicillium spp. by various research groups on various

Mode of infection
When L. lecanii conidia comes in contact with the host integument, it gets adhere to the epicuticle and germinate. Germinated conidia form germ tubes that penetrate cuticle directly or grow over the surface of the epicuticle. The germ tube penetrates by lysing both the epicuticle and the procuticle [8,9]. This is accomplished by the mechanical pressure exerted by appresorium (penetration peg) and secretion of enzymes viz., proteases, chitinases and esterase's which plays an important role during cuticle penetration of insect host and also serve as cuticle degrading enzymes. The fungus proliferates throughout the insect's body, draining the insect of nutrients, and eventually killing it in around 48-72 hours. The mycotoxins produced by L. lecanii are bassianolide [10,11], vertilecanin-A1, decenedioic acid and 10-hydroxy-8-decenoic acid) [12][13][14]. As the host nutrients are depleted, the blastopores' differentiate into elongated hyphae which extend outward from the body forming a mycelial mat of conidiophores over the surface of the integument resulting in mummification. Under favourable environmental condition, conidiophores mature giving rise to conidia which continues the disease cycle further.

R & D publications on different aspects of Lecanicillium spp.
The number of publications related to Lecanicillium spp. from 1971 to 2020 was presented in the Figures 1 and 2. The data clearly indicated that, during 1971-80 s  the publications were completely nil, but during 1981-91 s , the R&D work has been initiated in the entire world and the publications were increased gradually reaching 58% during 2001-2020 (Figure 2). While, considering the number publications on various aspects of Lecanicillium spp., more research work has been done on virulence and pathogenicity (Figure 3) followed by biotechnology and biochemistry as compared to morphology, diversity, ecology, mass production. The number of publications was meagre on effect of environmental factors (temperature and humidity), safety to natural enemies and compatibility with pesticides [15].

Natural occurrence of Lecanicillium spp.
Lecanicillium spp. is the most widely distributed and generally found on infected insects both in temperate and tropical areas throughout the world. There are number of reports on natural infection of Lecanicillium spp. on different insect pests but out of the reported insects and pests, maximum are sucking pests belonging to Hemiptera, Thysanoptera and Acarina which indicates its possible spectrum for use as a biocontrol agent for pest management. Reports of natural occurrence of Lecanicillium spp. on sucking insects presented in the Table 1.  L. lecanii (Is-6) Acrithosiphon pisum Israel [14] L. lecanii (R-1) T. vaporariorum Russia [14] Arthropods 4 6. Effect of temperature and humidity on the growth of Lecanicillium spp.
Temperature and humidity are the main factors influencing the growth of the fungus. Effect of different temperature on conidial germination, growth rate, colony size and mycelial growth was discussed and presented in Table 2

Temperature
Temperature affects the Lecanicillium spp. in different ways by influencing the germination, growth and viability of the fungus in the host insect and environment. High temperature inactivates the fungus before contact with the pest insect or may reduce or accelerate the growth within an insect depending on the temperature requirements of the fungus and the host insect. In contrast, low temperatures reduce or stop the germination and growth. Optimal germination and growth rates of Lecanicillium spp. range between 23°C and 28°C, growth rapidly slows >30°C and ceases at 34 to 37°C. Similarly, conidial germination is adversely affected by temperatures above 30°C. Temperature below 16°C increasingly slows germination and growth and thus affects efficacy in terms of a longer survival of the target population. Lecanicillium strains showed optimum growth at 25°C; the aerial conidia of Lecanicillium strains germinate in a broad temperature range (15-30°C) and L. lecanii 41,185 was the only strain with conidial germination at 35°C [16].
Effect of different temperature on conidial germination, growth rate, colony size and mycelial growth of L lecanii was discussed and presented in Table 2. At 25°C and 0.975 a w (water activity) conidial germination occurred in all the isolates ranging from 28.7 to 98% whereas isolate PFC 10 no conidial germination had. Per cent germination decreased from highest values at 25°C to the lowest trend at 10°C in Mycotol (20.6°C). Maximum germination of conidia was observed between 15 and 25°C [17]. Most of the isolates showed growth at 5 and 30°C and mean growth rate increased as temperature increased. Optimum growth rate occurred at 25°C (1.64 to 2.07 mm) for all isolates) [17]. Colony size of the fungus was influenced  23°C. Colony radial growth, conidial production and germination decreased with the reduction in water activity, while 32°C was extremely detrimental for all fungal isolates. However, some isolates were able to grow and produce conidia at low water activity and high temperature [20]. L. muscarium can multiplied in temperature range of 15-30°C but optimum temperature against M. persicae between 20 to 30°C [21].

Humidity
Humidity is another important environmental factor affecting the efficacy and survival of Lecanicillium. Spore germination on the insect cuticle and sporulation after outgrowth of the dead host insect require high moisture. Generally high humidity is required for germination of spores under in vitro, insects can become infected at much lower humidity. Under fluctuating humidity, daily saturated humidity requirement of at least 16 h for causing death in Trialeurodes vaporariorum (Westwood) infected with L. lecanii [22]. Several previous studies provided evidence that a threshold time period at high humidity was required for infection. Conidia of L. lecanii required at least 72 h at 100% RH and 20°C before removal to 70% RH to reach >90% infectivity of Myzus persicae (Sulzer) [23]. Similarly, at 25°C temperature and 75% relative humidity (RH), L. lecanii 41,185 showed highly virulent pathogenicity (100%) against M. persicae and Aphis gossypii Glover [16]. Application of L. longisporum against A. gossypii on cucumber in controlled environment (Temperature; 19-26°C and humidity; 80-98%) resulted in 100% mortality [23,24]. L. muscarium grow at optimum temperature but higher mortality observed against M. persicae between 55 and 90% humidity [21].

Factors influencing the efficacy of Lecanicillium spp. against sucking insect pests
The virulence and pathogenicity of Lecanicillium spp. vary with strain, stage of the insect and dose of the fungus.

Stage of the insect
Stage of host plays important role in the success of Lecanicillium spp. and not all stages of insect life cycle are equally susceptible to fungal infection. So, the fungal application can be successful against the particular pest when it can be done at the condition where the susceptible stage or weaker stages of the particular pest become dominant among population.

Dose/inoculums level
Fungal inoculum level is the important factor which affects the performance. It is general trend that the higher fungal inoculum level gives higher insect mortality. However, sufficient inoculum level should be worked out for the particular pest to prevent the over inoculum wastage and to achieve higher mortality. Higher dose of L. lecanii (1.2 x 10 9 conidia ha −1 ) was caused 92.30 and 80.93% mortality of Brevicoryne brassicae Linnaeus and Aleurodicus disperses (Russell) respectively at 10 days after treatment in the laboratory, whereas in field conditions L. lecanii (Vl3) at 2 x 10 12 conidia ha −1 ) causing 61.16% and 66.50% mortality of B. brassicae and A. craccivora respectively [2].

Efficacy of Lecanicillium spp. against sucking pests under laboratory/ greenhouse/field
Efficacy of Lecanicillium spp., against aphids, whiteflies, thrips, scales and mealy bugs in the laboratory/greenhouse/field conditions w.r.to its mortality, LC 50 and LT 50 values were presented in the

Safety of Lecanicillium spp. to parasitoids/predators/pollinators
The safety of any bio control agent to parasitoids/predators/pollinators is the important aspect which should be studied thoroughly before its commercialization to avoid the hazards and disturbance of ecological balance. Effect of L. lecanii on aphid parasitoid Aphidius colemani (Viereck) which showed the normal development (approximately 90% adult emergence) when its cotton aphid, A. gossypii host was treated with L. lecanii conidia 5 or 7 days after parasitization. Fungus exposure 1 day before or up to 3 days after parasitization, however, reduced Aphidius colemani (Viereck) emergence from 0 to 10%. They suggested that the parasitoid and fungus may be used together for aphid bio control [35]. L. lecanii showed pathogenicity against predatory mite, Phytoseiulus persimilis Athias-Henriot but its effect was lower than that of spider mite, Tetranychus urticae (Koch) [36]. L. lecanii is safer to predatory coccinellid, Coccinella septempunctata Linnaeus and predatory mites, Amblyseius ovalis (Evans) and Amblyseius longispinosus (Evans) under field conditions [37]. The fungus L. lecanii was not pathogenic to Chrysoperla carnea (Stephens), Coccinella septempunctata (Linnaeus), Episyrphus balteatus (De Geer) and Samia cynthia ricini (Boisduval), but was found to be pathogenic to Bombyx mori (Linnaeus). Parasitism, adult emergence and adult longevity of Trichogramma chilonis (Ishii) were affected by fungal treatments. Aphid mummification and Diaeretiella rapae adult emergence were affected by the fungus. Results suggest that L. lecanii is compatible with natural enemies of cabbage aphid, T. chilonis and is harmless to silk worm [38]. L. muscarium at 10 6 and 10 7 spores mL −1 was safer to predatory mite P. persimilis [39]. Number of parasitized larvae of Eretmocerus sp. nr. furuhashii survival decreased with increasing concentrations of L. muscarium and only 29% emergence of pupae was observed at a conidial concentration of 1 × 10 8 conidia mL −1 . Similarly, 67% emergence of adult E. sp. nr. Furuhashii was observed [40]. Parasitoid (Diaeretiella rapae) emergence was affected by application of L. longisporum before or after parasitism and longevity decreased in female F1 populations [41]. In the laboratory conditions, application of L. muscarium (1 x 10 8 conidia/ml) against A. colemani had not affected longevity and fertility of the female A. colemani. The combination of Aphidius colemani with L. muscarium reduced the aphid infestation in the semi field conditions as compared to A. colemani alone [21].
The Lecanicillium spp. is not harmful to humans during handling in the laboratory and field for the control of pests.

Compatibility of Lecanicillium spp. with agro chemicals
Chemical pesticides may have antagonistic or synergistic effect on the potentiality of Lecanicillium spp. and may disrupt natural epizootic. Under such epizootic condition, it is expected to enhance effectiveness through joint action of pathogen and compatible insecticides, which would reduce not only the cost of protection but also reduce the contamination of the environment. The literature on compatibility of Lecanicillium spp. with agrochemicals is lacking.

Commercial formulations
The commercial formulations based on Lecaniillium spp. are available in India and other countries are presented in Table 5. Number of manufacturers based on Lecanicillium spp. products is more in India however; the production is very low and not available to the farmers/stakeholders/growers on time as compared to synthetics due to dominant in pesticides market and lack of awareness to farmers/growers about biopesticides. In India, the efficacy of Lecanicillium spp., based products was less due to high temperature and low humidity as compared to temperate countries, even though in India, these products were used as one of the components in IPM and also used for the management of sucking pests of flowers and vegetables in greenhouse.

Country
Trade Name

Target pest Country Source
Lecanicillium spp.

Conclusions
Lecanicillium spp. is promising biocontrol agent and can be used as one of the components of integrated pest management under green house and field conditions against sucking insect pests. Lecanicillium is multiplying on commercially available media (potato dextrose agar and broth etc.) till date but it can be mass multiplied at cheaper rate on solid grain media of sorghum and rice; liquid media of sugar cane molasses. It can be used effectively in conjunction with other natural enemies and compatible pesticides.

Conflict of interest
Author declares that no conflict of interest is reported.  Table 5.