In vitro pathogenicity of the fungi Beauveria bassiana and Lecanicillium lecanii at different temperatures against the whitefly, Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae)

The whitefly, Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae), is one of the most damaging pests in field crops as well as in greenhouses. The present study aimed to evaluate the in vitro pathogenicity of 2 fungus strains of Beauveria bassiana (BB-72 and BB-252) and one strain of Lecanicillium lecanii (V-2) against the whitefly (B. tabaci) at 4 different temperature degrees 20, 24, 28, and 32 °C, using a spray method. Three different bioassays were carried out comprised of conidial concentrations and filtrate of fungal strains, BB-72, BB-252 and V-2, and their binary combinations. The 1.5 ml of fungal filtrate was used in filtrate bioassay for each strain of fungus. Three different concentrations (1 × 106, 1 × 107, and 1 × 108 conidia ml−1) were used in conidial bioassay for each strain of fungus, whereas in bioassay for binary combination (1 ml conidia + 1 ml filtrate) of BB-72 × BB-72, BB-252 × BB-252, and V2 × V2 were used for these strains of fungus. According to the outcomes, the maximum mortality against whiteflies was observed on 12th day post-treatment. In conidial bioassay, the maximum mortality of B. tabaci was observed in BB-72 isolate (84%), BB-252 isolate (77%), and V-2 isolate (67%) at the highest concentration (1 × 108 conidia ml−1) at 24 °C, and minimum mortality was recorded in BB-72 isolate (33%), BB-252 isolate (29%), and V-2 isolate (19%) at the lowest concentration 1 × 106 conidia ml−1 at 32 °C on 12th day post-treatment. Infiltrate bioassay, BB-72 isolate exhibited maximum mortality (92%) at 24 °C, and V-2 isolate showed minimum mortality (34%) at 32 °C on 12th day post-treatment. Furthermore, in binary combination bioassay, the highest whitefly mortality was recorded in BB-72 × BB-72 isolate (87%), BB-252 × BB-252 isolate (73%), and V2 × V2 isolate (65%) at 24 °C and the lowest mortality in BB-72 × BB-72 isolate (57%), BB-252 × BB-252 isolate (50%), and V2 × V2 isolate (39%) at 32 °C on 12th day post-treatment. In all bioassays, the BB-72 isolate was the utmost virulent, and application of its filtrate was found to be the most impressive against B. tabaci.


Background
The whitefly, Bemisia tabaci (Genn.) (Hemiptera: Aleyrodidae), is one of the most destructive pest in the field crops as well as in greenhouses. It directly damages the crops causing heavy losses of the crops (Oliveira et al. 2001). Entomopathogenic fungi (EPF) have the ability to individually affect their host directly through the inward penetration in an insect (Lacey et al. 1996). Beauveria bassiana, V. lecanii, and Metarhizium anisopliae proved to be the most promising fungi in this respect (Wraight et al. 2007). For the control of whitefly, the entomopathogenic fungi have been used almost all around the world (Jackson et al. 2010). The potential strains best for utilization as pest management tools can be categorized into two groups: one group includes Hyphomycetes in Deuteromycotina and the other belongs to Entomophthorales (EPF) in Zygomycota (Feng 1998).
Very few of these EPF, viz., Nomuraea rileyi, Beauveria bassiana, Paecilomyces fumosoroseus, Metarhizium anisopliae, B. brongniartii, and Verticillium lecanii have been registered as products to control insect pests (Shah and Goettel 1999). B. tabaci showed resistance against the chemical insecticides such as nitenpyram, imidacloprid, acetamiprid, and pyrethroid (Horowitz et al. 2008). In china, many recent studies have revealed that most of the commonly available pesticides have lost their effectiveness in the control of whitefly (Wang et al. 2009). Therefore, the development of alternative sources of control is urgently needed. Biological control has now become key area of research in the management of B. tabaci over the past 2 decades (Cuthbertson et al. 2010). A number of microbial bio-pesticides based on entomopathogenic organisms like bacteria, nematodes, viruses, and fungi have been playing key roles in plant protection and were used against a broad range of insect pests (Majeed MZ et al. 2017). Entomopathogenic fungi like M. anisopliae, Isaria fumosorosea, B. bassiana, and L. lecanii proved to be impressive, target-specific, and environment-friendly biocontrol agents against several species of sucking insect pests (Cabanillas and Jones 2009). After germination of spores connected to the target insect's cuticle, fungal hyphae enter the body of the insect and cause death of host within a day (Mora et al. 2017). In addition, EPF have less or no residual activity or mammalian toxicity and are specific to the target pest, as well as there is less risk for developing resistance (Zimmermann 2007).
B. bassiana and L. Lecanii are the most studied EPF and known as a virulent biocontrol agent of a wide range of forest, field crops, and green house pests. These entomopathogens were equally effective in desert, agricultural, and forest habitats (Annamalai et al. 2016 andDogan et al. 2017). Lately, (Nazir et al. 2019) demonstrated the toxicity of filtrates and conidia derived from different isolates of L. lecanii and B. bassiana against the aphid species, Myzus persicae (Sulz.).
The strains of both the entomopathogens can be easily isolated from the infected soil dwelling pests or from vegetation pests (Freed et al. 2011). As with other organisms, the growth rate of EPF varies by temperature. They are usually cultured nearby 25°C, even depending on the strain in a single species (Sato 1993). The aim of the present study was to assess the pathogenicity and virulence of EPF, i.e., L. lecanii and B. bassiana against B. tabaci under controlled environmental conditions.

Materials and methods
Whitefly stock culture Bemisia tabaci individuals were collected from the seedlings of tomato (Solanum lycopersicum) grown in the greenhouse at Chinese Academy of Agricultural Sciences (CAAS) Beijing, China. The population of whitefly was reared on the seedlings of same tomato grown in pots kept in cages at 25 ± 2°C and 50 60% RH with photoperiod of 16:8 h light:dark (L:D). In the entire study period, the plants were replaced weekly with new ones.

Effect of temperature on fungus virulence against Bemisia tabaci
Evaluation of the role of temperature, the pathogenicity bioassays of B. bassiana and L. lecanii against the nymphs of whitely, was carried out at 20, 24, 28, and 32°C.

Fungal filtrate
Adamek's liquid medium (40 g yeast extract (Difco, Detroit, MI, USA), 40 g dextrose and 30 g corn steep liquor (Sigma-Aldrich, Saint Louis, MO, USA), was used for primary culture of fungal filtrate by adding 4 ml of conidial suspension into 100 ml of medium for all the strains of fungi. Cultures were incubated for 72 h at 150 rpm, and then for secondary culture, 5 ml from primary culture was added into 500 ml of Adamek's liquid medium and incubated at 26°C and 150 rpm for 6 days for each strain. The samples were then centrifuged at 12000 rpm and 4°C for 30 min. After that, the supernatant was filtered through 0.45 μm pore size filter (Millipore Corp., Billerica, MA, USA) to get filtrate, and pH was sustained at 6.

Pathogenicity bioassays
To evaluate the efficacy of filtrate, conidia, and binary combinations, different bioassays were carried out. For binary combination bioassays (conidia + filtrate), 1 ml of the highest concentration of conidia 1 × 10 8 conidia ml −1 of each fungal isolate was combined with 1 ml filtrate of the same fungal isolate. The combinations were BB-72 × BB-72, BB-252 × BB-252, and V2 × V2. For all treatments ,the 90mm Petri dishes were used with a skinny layer of 1.5% agar, and 50 mm diameter of leaf discs of tomato was placed in Petri dishes. In the control treatment, the leaf discs were sprayed only by 0.01% Tween 80®. Ten apterous adult of B. tabaci were released on the leaf discs at each Petri dish, and 1.5 ml sample of each of the treatments was sprayed on B. tabaci with the help of small sprayer and then these Petri dishes were incubated at 20, 24, 28, and 32°C and 50-60% RH with photoperiod of 16:8 h light:dark (L:D) for 12 days. Ten replications of each treatment were used. B. tabaci mortality was recorded after 3, 6, 9, and 12 days posttreatment.

Data analysis
Virulence of the two strains of B. bassiana and one strain of L. lecanii as filtrate, conidia, and binary combination (conidia + filtrate) were analyzed, using factorial analysis of the variance (Statistix software (version 8.1) (Tallahassee, FL)). Comparisons of the treatment means were performed using Fischer's least significant difference (LSD) test at a = 0.05.  Result and discussion
Obtained results are in accordance with the previous studies describing the effectiveness of different isolates of L. lecanii and B. bassiana against sucking insect pests (Hesketh et al. 2008). In all bioassays, the  73, and 65% mortality of whiteflies after 12 days through binary combination of conidia + filtrate of BB-72 × BB-72, BB-252 × BB-252, and V2 × V2 isolates. These results are in accordance with (Wraight et al. 2000) who reported that B. bassiana caused up to 97% mortality, and V. lecanii showed 100% mortality of Chilo partellus. Obtained results are in accordance with (Halimona and Jankevica 2011), who tested various concentrations of B. bassiana against A. fabae and Metopeurum fuscoviride and found that the highest concentration of conidia 1 × 108 ml −1 showed maximum mortality after 7th day of application. The percentage of mortality was affected by temperature, conidial concentration, and exposure time (Ansari et al. 2004). B. bassiana showed a great effect on the B. tabaci and Aphis craccivora infesting cucumber (Maniania 1991). The V. lecanii showed high mortality in early stages of B. tabaci and less mortality in old instars (Zaki 1998). EPF showed a good control of B. tabaci (Abdel-Raheem et al. 2016). The filtrate application was more effective to control the insect pests than the conidial application mainly in the case of short life cycled insects. There may be a possible cause that the less attachment of conidia to the insect cuticle as compared to huge infiltration of filtrates (Hanan et al. 2020a b).
Concerning the combined application obtained results is identical to (Yun et al. 2017) who studied the double behavior of EPF B. bassiana and M. anisopliae against M. persicae and B. cinerea and witnessed that the usage of filtrate with blastospores provided the maximum mortality of M. persicae. Furthermore, the mortality percentage was near to the combination of filtrates with blastospores; however, the combination of conidia and filtrate gave the minimum mortality as compared to filtrate application. So, it is clear that the fungal filtrate application had extreme virulence efficiency. Moreover, EPF are considered safe and environmentally friendly than the chemical pesticides (Goettel and Jaronski 1997); so, they are suggested as a control agent against destructive insect pests, like whitefly B. tabaci.

Conclusion
The in vitro study exhibited the efficiency of 2 strains of B. bassiana and one strain of L. lecanii against B. tabaci. L. lecanii showed lower mortality than both strains of B. bassiana either singly or in binary combinations (conidia + filtrate) of same strain at optimum temperature. The application of filtrate was utmost appropriate material to control B. tabaci. The binary combination of filtrate + its conidia had some irreconcilable effect and cannot effectively be used to control B. tabaci. Further studies are still needed to assess the role of EPF against B. tabaci, especially under field conditions.