In vitro virulence of three Lecanicillium lecanii strains against the whitefly, Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae)

Whitefly, Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae), is one of the most harmful pests in greenhouses and in open fields. The present study aimed to assess in vitro virulence of 3 entomopathogenic fungal strains (EPFs) of Lecanicillium lecanii 3 (V-3), 4 (V-4), and 5 (V-5) against the whitefly, Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae), by spray method. The 3 disparate bioassays were performed encompassed of conidial concentrations and fungal filtrate of the strains, V-3, V-4, and V-5, and also their binary combinations. In the filtrate bioassay, 2 ml of fungal filtrate of each strain was used. In the conidial bioassay, 3 disparate concentrations (1 × 105, 1 × 106, and 1 × 107 conidia ml−1) were used for each fungal strain, while in the binary combinations (1 ml filtrate + 1 ml conidia) of V-3 × V-3, V-4 × V-4, and V-5 × V-5 were used. Mortality rates against the whitefly were recorded on the 7th day. In the conidial bioassay, maximum mortality rates were found at V-3 strain (90.6%), V-4 strain (78.4%), and V-5 strain (83.6%) at the highest concentration (1 × 107 conidia ml−1) on the 7th day. In the filtrate bioassay, V-3 strain revealed a maximum mortality (93%), V-4 strain (85%), and then V-5 strain (87%) on the 7th day. Moreover, in the bioassay of binary combinations, the highest mortality rate of the whitefly was counted in V-3 × V-3 strain (84.6%), V-4 × V-4 strain (70.6%), and V-5 × V-5 strain (79.8%) on the 7th day. All treatments had the potential to control B. tabaci significantly. In all bioassays, the V-3 strain was the extreme virulent, and the filtrate application of V-3 strain was the utmost impressive against B. tabaci.


Background
Whitefly, Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae), is one of the most serious pests of crops that causes great losses (Oliveira et al. 2001). It causes direct destruction by sucking the plant sap, and also indirect destruction by encouraging the growth of black sooty mold on their honeydew secretions (Varma and Malathi 2003). It causes economic losses in many crops, including leafy vegetables, cotton, beans, and tomatoes (Fontes et al. 2012). The application of chemical pesticides leads to increase resistance and has a negative environmental impact that encourages the development of alternative pest management strategies, where microbial control plays a key role (Nazir et al. 2019b). Application of microbial control agents mainly entomopathogenic fungi isolates (EPFs) has been examined to control various orchard and field crop pests (Lacey and Shapiro-Ilan 2008). The use of EPFs in biological control is developing extremely due to food safety concerns, environmental awareness, and the failure of conventional chemicals (Shahid et al. 2012). EPF can play a significant role in biocontrol programs because of their pathogenic pathways, their wide range of hosts, and the control of economic insect pests such as the sucking insects (thrips, mites, aphids whiteflies, and mealybugs) (Khan et al. 2012). The use of biological control for pest control in greenhouses has been proven to be effective, and its application worldwide is growing steadily (Perdikis et al. 2008). The most promising EPF include Lecanicillium lecanii, Metarhizium anisopliae, and Beauveria bassiana (Saleh et al. 2016). L. lecanii is widely distributed and can cause a large-scale epizootic in tropical and subtropical regions as well as in humid and warm environments (del Prado et al. 2008). When the fungal conidia encounter the host, they attach to the epidermis through a hydrophobic mechanism and germinate under favorable conditions to form the germ tube (Inglis and Goettel 2001). During this process, the fungus produces several infection specific structures, including penetration pegs or appressoria, enabling the developing hyphae to enter the host integument (Ortiz-Urquiza and Keyhani 2013). L. lecanii has been documented to infect B. tabaci (Zhu and Kim 2011) causing a good mortality (Xie et al. 2019).
The present study was undertaken to evaluate the bioefficacy of L. lecanii strains against B. tabaci under laboratory conditions.

Insect rearing
Adults of the whitefly, B. tabaci were collected from Langfang station, the research area of Institute of Plant Protection (IPP), Chinese Academy of Agricultural Sciences (CAAS), Beijing, China. The pest was reared on cotton plants under a controlled greenhouse at 25 ± 2°C, 65% RH with a 14:10 (L:D) photoperiod.

Fungal culture and conidia suspension
The L. lecanii strains, V3, V4, and V5, were obtained from the key laboratory of Biopesticides Engineering, Department of Bio-Pesticides and Biocontrol, Institute of Plant Protection (IPP), Chinese Academy of Agricultural Sciences (CAAS). The strains were obtained from infected B. tabaci. The strains were grown on Potatoes Dextrose Agar in Petri dishes for 20 days at 25°C. Conidia were harvested after 21 days. The Petri dishes were flashed 20 ml with sterile water and filtered by using a sterile cheese cloth. Three different concentrations of conidia (1 × 10 5 , 1 × 10 6 , and 1 × 10 7 ) of spore suspension were determined using a hemocytometer. Earlier performing bioassays against B. tabaci, the viability test of conidia was accomplished, according to the procedures of (Hywel-Jones and Gillespie 1990).

Fungal filtrate
To get fungal filtrates, 4 ml of conidial suspension was prepared in 100 ml of Adamek liquid medium of all fungal strains. The culture was incubated 3 days in the dark for 150 rpm at 25°C. Secondary culture was prepared and added to 3 ml of primary culture in to Adamek liquid medium and incubated 7 days in the dark for 150 rpm at 25°C. The fungal filtrate was centrifuged 20 min, 11,000 rpm at 4°C. The supernatant was filtered 0.45 μm pore size filter.

Pathogenicity test
The effects of the fungal conidia, filtrate, and binary combinations ((filtrate + conidia) on the cotton whitefly were tested under laboratory conditions using a manual sprayer. Three different conidia concentrations (1 × 10 5 , 1 × 10 6 , and 1 × 10 7 ) were tested. Filtrate bioassays were evaluated by applying 7 days fungal filtrate. Binary combinations (1 ml conidia + 1 ml filtrate) were estimated, while sterilized water was used as a control. For this

Statistical analysis
Virulence of the 3 stains of L. lecanii as conidia, filtrate, and binary combination were analyzed, using factorial analysis of variance (ANOVA) Statistix software (Version 8.1) (Tallahassee, FL). Comparison of the treatment means was performed using Fischer's least significant difference (LSD) test at α = 0.05).

Filtrate bioassay
Outcomes of the filtrate bioassay showed that overall average mortality of the whitefly through the tested strains of fungus was more than the treatments of conidia. Filtrates of fungus, observation , and their interactions on B. tabaci mortality had a significant impact ( Table 2). The maximum mortality (93%) was noted on the 7th day of treatment of (V-3) and the minimum mortality (85%) was noted on the 7th day through the application of filtrate of V-4. Filtrate of fungal strain V-5 revealed an average whitefly mortality rate of 87% (Fig. 2).

Binary combination (conidia + filtrate) virulence
The pathogenicity bioassays executed by the binary combinations (conidia + filtrate) of three strains of fungus had a significant impact of all the treatments, observation time, and their interactions on whitefly mortality (Table 3). Significant and maximum whitefly mortality (84.6%) was witnessed at V-3 × V-3, while the minimum mortality rate of whitefly (70.6%) was noted at the combination of V-4 × V-4 on the 7th day of treatment. Binary combination of V-5 × V-5 affected (79.8%) whitefly mortality on the 7th day of treatment (Fig. 3). Generally, the study outcomes are in line with the earlier studies describing the efficiency of disparate isolates of L. lecanii and Beauveria bassiana against the sucking insect pests (Hesketh et al. 2008).
In all the bioassays, the mortality rate of B. tabaci was reliant on concentration and time, and it increased through increasing the conidia concentration and time interval after the application. The mortality of 90.6, 78.4, and 83.6% was reported after 7 days of conidial application of V-3, V-4, and V-5 at the highest concentration 1 × 10 7 conidia ml −1 whereas 93, 85, and 87% mortality rate was recorded after 7 days of filtrate application of V-3, V-4, and V-5 strains. One more experiment was executed and stated 84.6, 70.6, and 79.8% mortality of B. tabaci after 7 days by binary combination (conidia + filtrate) of V-3 × V-3, V-4 × V-4, and V-5 × V-5 fungal strains. Obtained results are in line with (Halimona and Jankevica 2011) who verified several concentrations of five EPFs against A. fabae and M. fuscoviride and found that the highest concentration 1 × 10 8 ml −1 of conidia exhibited the maximum mortality after 7 days. Also, the results are in accordance with (Hanan et al. 2020b) who demonstrated that in all the bioassays, L. lecanii V-3 strain was the utmost pernicious and application of its filtrate was found to be the utmost efficient against green peach aphid Myzus persicae (Sulz.) (Nazir et al. 2019a).  Mortality percentage of B. tabaci affected with the conidial concentration, exposure time and temperature (Keerio et al. 2020). Similarly, Wright et al. (2005) also confirmed that the aphids mortality increased through an increase of the concentration of conidia and time interval. The V. lecanii revealed maximum mortality in initial stages and minimum mortality in the older instars of B. tabaci (Zaki 1998). EPF demonstrated a worthy control of whitefly . The application of filtrate was more efficient than the application of conidia chiefly in the insects having a short life cycle to control the insect pests. There could be a possible reason for this that the conidia attachment to the cuticle of insects is less effective than the enormous penetration of the filtrates (Hanan et al. 2020a).
Regarding combined applications, acquired results were similar to Yun et al. (2017) who revealed the dual action of EPF, M. anisopliae, B. bassiana, and Botrytis cinerea against M. persicae, and observed that the practice of filtrate by blastospores presented the extreme mortality of M. persicae. The binary combination (conidia + filtrate) provided the lowest mortality rate as compared to the application of filtrate. Thus, it is clear that the application of fungal filtrate had the utmost virulence efficiency.

Conclusions
The in vitro study revealed the competence of 3 strains of L. lecanii against B. tabaci. V-4 exhibited the lowest mortality rate than the other both strains (V-3 and V-5), either alone or in the binary combinations (conidia + filtrate) of identical strain. The filtrate application was extremely suitable material to control the whitefly. Binary combination of conidia + filtrate had some of the invincible effect and cannot be used efficiently to control whitefly. However, the most characterization of L. lecanii strains (V-3, V-4 and V-5) and their prospective functional insecticidal elicitors is required to better clarify their approach of action.