Pathogenicity, Ovicidal Action, and Median Lethal Concentrations (LC50) of Entomopathogenic Fungi against Exotic Spiralling Whitefly, Aleurodicus dispersus Russell

Biological control using entomopathogenic fungi could be a promising alternative to chemical control. Entomopathogenic fungi, Beauveria bassiana (Balsamo) Vuillemin, Metarhizium anisopliae (Metschnikoff) Sorokin, Lecanicillium lecanii (Zimmerm.) Zare and Gams, and Paecilomyces fumosoroseus (Wize) Brown and Smith, were tested for their pathogenicity, ovicidal effect, and median lethal concentrations (LC50) against exotic spiralling whitefly, Aleurodicus dispersus Russell. The applications were made at the rate of 2 × 109 conidia mL−1 for evaluating the pathogenicity and ovicidal effect of entomopathogenic fungi against A. dispersus. The results of pathogenicity test showed that P. fumosoroseus (P1 strain) was highly pathogenic to A. dispersus recording 100% mortality at 15 days after treatment (DAT). M. anisopliae (M2 strain) had more ovicidal effect causing 37.3% egg mortality at 8 DAT. However, L. lecanii (L1 strain) caused minimum egg hatchability (23.2%) at 10 DAT as compared to control (92.6%). The lowest LC50 produced by P. fumosoroseus (P1 strain) as 8.189 × 107 conidia mL−1 indicated higher virulence against A. dispersus. Hence, there is potential for use of entomopathogenic fungi in the field conditions as an alternate control method in combating the insect pests and other arthropod pests since they are considered natural mortality agents and are environmentally safe.


Introduction
The spiralling whitefly, Aleurodicus dispersus Russell (Homoptera, Aleyrodidae), the native of the Caribbean region of Central America [1], is a highly polyphagous pest, which has extensive host range covering 481 plants belonging to 295 genera from 90 families of vegetables, fruits, and ornamentals trees [2]. A loss of 80% in fruit yield recorded in guava infested by A. dispersus in Taiwan [3] and A. dispersus caused yield reduction up to 53% in cassava [4]. The nymphs are covered with heavy waxy flocculent materials and waxy threads offering a great defense against synthetic chemical insecticides and resulting in poor control of the pest [5].
One of the potential methods in A. dispersus management is the use of microbial biocontrol agents (MBCAs) as the natural enemies of the pest population devastate pests with no hazard effects on human health and environment. As the microbial biocontrol agents have complex mode of action, it is very difficult for a pest to develop resistance against MBCAs. The present MBCAs are viruses, bacteria, nematodes, and fungi and they are used throughout the world with great advantage and success. But fungal biocontrol 2 Journal of Pathogens agents are the most important among all the MBCAs due to easy delivery, improving formulation, vast number of pathogenic strains known, easy engineering techniques, and overexpression of endogenous proteins or exogenous toxins [6][7][8]. Similarly, the entomopathogenic fungi are important among all the biological control agents due to their broad host range, route of pathogenicity and their ability to control sap sucking pests such as mosquitoes and aphids [9][10][11] as well as pests with chewing mouthparts [12,13]. With this background, the research on entomopathogenic fungi was conducted to assess the pathogenicity, ovicidal effect, and median lethal concentrations (LC 50 ) of entomopathogenic fungi against A. dispersus under laboratory conditions.

Materials and Methods
Studies were conducted at Biocontrol Laboratory, Department of Agricultural Entomology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India. Bioassays were carried out to evaluate the pathogenicity, ovicidal effect, and LC 50 of entomopathogenic fungi. The insect used for the study was A. dispersus from the culture maintained in screen house.

Fungal Isolates and Culture
Maintenance. The entomopathogenic fungi, B. bassiana (B1, B2 strains), M. Anisopliae (M1, M2, M3 strains), L. lecanii (L1 strain), and P. fumosoroseus (P1 strain) were obtained from the National Bureau of Agriculturally Important Insects, Bangalore, Karnataka, India, the Department of Plant Pathology, Tamil Nadu Agricultural University (TNAU), Coimbatore, Tamil Nadu, India, and Sun Agro Biotech Research Centre, Porur, Chennai, Tamil Nadu, India, culture collections. Isolates were maintained in culture on potato dextrose agar (PDA) slants in universal bottles (30 mL) and stored at 4 ∘ C. Continuous cultures were maintained on slants with subcultures grown for 14 days at 25 ∘ C following which lids were tightly sealed and cultures stored at 4 ∘ C.

Pathogenicity of Entomopathogenic Fungi against A.
dispersus. Strains of different entomopathogenic fungi were assayed against A. dispersus nymphs by direct spray method in completely randomized design (CRD). Entomopathogenic fungi were sprayed with the help of automizer over the nymphs of A. dispersus with three replications. All the treated Petri dishes were maintained at 25 ± 1 ∘ C in an incubator. The nymphs were individually examined under a stereo zoom binocular microscope (Carl Zeiss Stemi 2000) at 40x magnification for verification of fungal infection. The mortality data were recorded by counting the dead cadavers and nymphs with fungal spores. Observations on the mortality of A. dispersus by entomopathogenic fungi were made at 3, 5, 7 10, 13, and 15 days after treatment (DAT). The mortality data were corrected using Abbott's formula [14]. The experiments were repeated for three times to confirm the pathogenicity of entomopathogenic fungi against A. dispersus.

Ovicidal Effect of Entomopathogenic Fungi against A. dispersus.
Entomopathogenic fungi were assayed using direct spray method to evaluate ovicidal effect on A. dispersus eggs. Uniform age of A. dispersus eggs were taken from eggplant (Solanum melongena L.) leaf placed on 1.5% agar in a Petri dish. Entomopathogenic fungi were sprayed with help of automizer over the eggs of A. dispersus with three replications in CRD. All the treated Petri dishes were maintained at 25 ± 1 ∘ C in an incubator and hatchability was recorded until no change for three consecutive days. Later, the eggs were individually examined under a stereo zoom binocular microscope (Carl Zeiss Stemi 2000) at 40x magnification for verification of fungal infection. Finally, all unhatched eggs were transferred to moist chambers for three days to observe fungal outgrowth if any, as an evidence of egg mortality due to fungal infection. Observations were made at 4, 6, 8, and 10 DAT. The experiments were repeated for three times to confirm the ovicidal action of entomopathogenic fungi against A. dispersus eggs.

Median Lethal Concentrations (LC 50 ) of Entomopathogenic Fungi against A. dispersus Nymphs.
Studies were conducted to find the median lethal concentrations (LC 50 ) of four entomopathogenic fungi, namely, M. anisopliae (M1 strain), B. bassiana (B1 strain), L. lecanii (L1 strain), and P. fumosoroseus (P1 strain), against A. dispersus nymphs. Five doses (from 2 × 10 5 to 2 × 10 9 conidia mL −1 ) were fixed for which dilutions were prepared with double distilled water. A. dispersus nymphs were treated starting from lower to higher concentrations, whenever different test doses the of same entomopathogenic fungi were used. Uniform age of A. dispersus nymphs was taken from eggplant leaf placed on 1.5% agar in a Petri dish. Five concentrations of each respective entomopathogenic fungi were sprayed with the help of automizer over the A. dispersus nymphs with three replications in CRD. All the treated Petri dishes were maintained at 25 ± 1 ∘ C in an incubator. The nymphs were individually examined under a stereo zoom binocular microscope (Carl Zeiss Stemi 2000) at 40x magnification for verification of fungal infection. The mortality data were recorded by counting the dead cadavers and nymphs with fungal spores. Observations were made periodically at 12 h interval up to 14 days and mortality data were recorded and corrected using Abbott's formula [14]. The median lethal concentrations (LC 50 ) and LC 95 values were estimated for A. dispersus [15].

Data Analysis.
Statistical analysis was done in completely randomized design. The percentage of mortality in both eggs and nymphs was collected and corrected with that in control by using Abbott's formula [14] as follows: where = estimated percentage of insects killed by fungus alone, = percentage of control insects living, and = percentage of treated insects that are living after the experimentation period.

Median Lethal Concentrations (LC
Since they are considered natural mortality agents and are environmentally safe, there is potential for the use of entomopathogenic fungi in the field conditions as an alternate control method in combating the insect pests and other arthropod pests. Additional testing of entomopathogenic fungi with other stages of A. dispersus and field evaluation of   entomopathogenic fungi must be conducted before ultimate conclusions are drawn.