Collection and laboratory culture of tea thrips
Adults and nymphs of thrips were collected from the tea fields of experimental plots of UPASI (United Planters’ Association of Southern India) (25.5° 55′ 0″ N, 87.5° 54′ 0″ E longitude) in India. Insects were released into a rearing chamber (a glass chimney of (15×16 cm2) provided with young tea shoots (each of 3 leaves and a bud) kept in small glass vials containing water. The culture of thrips was maintained in different wooden cages and glass chimneys in laboratory conditions (26 ± 3 °C; 80 ± 3% RH; 17L:7D photoperiod) on a susceptible tea clone, UPASI 17. Fresh tea leaves were provided on every alternate day as food. The susceptible cultures of insects maintained for more than 30 generations (from F1 to F30 without exposure to any pesticides) were utilized for different bio-efficacy evaluations.
Isolation and identification of entomopathogenic fungi
Extensive surveys were carried out in the tea gardens of the Anamallais (Tamil Nadu, Coimbatore District, South India) during 2015-2016. The field-collected cadavers of the insects/mites infected with fungi were used for isolation. For isolation of entomopathogens, PDA (potato dextrose agar) medium sterilized at 121 °C, 15 psi for 20 min was used. To isolate the fungi, the field-collected insects were sterilized by 5% NaClO and in 70% ethanol for 3 min and rinsed with sterile water for many times.
The diseased specimens were crushed in a Petri dish in a sterile condition, and a portion was transferred to a culture plate that contained the chosen medium and kept under constant observation for monitoring growth and development. The organisms were sub-cultured after 5 days for obtaining the pure culture. From the pure culture, slants of individual culture were prepared, and morphological characteristics of conidia and mycelium were observed under the microscope. Following Atlas of Entomopathogenic Fungi, the initial identification of fungi was made (Samson et al. 1988). Finally, the identified specimens were confirmed by molecular analysis carried out at Agharkar Research Institute, Pune, Maharashtra, India.
Biomass and conidial production
The biomass of the fungal isolate was determined using 100ml of sterile PDA amended with 1% yeast media. The liquid medium was inoculated using 1ml of fungal spore suspension (1×107 spores/ml) and incubated at 25+1°C, 75±2% RH and 14L: 8D photoperiod for 7 days in a growth chamber. To enumerate the biomass production, culture was agitated robustly and filtered, using Whatman filter paper after 7 days of growth. The same was dried at 40-45°C in an oven until it reached a constant weight. The biomass production observations were recorded in triplicate independent experiments.
The amount of conidia produced by the fungal isolate was evaluated on the 5th- and 8th-day-old cultures. A fungal culture of 1 cm diameter was suspended in sterile distilled water (5 ml) containing Tween-80(0.05% v/v). The conidia were harvested using a camel hairbrush and homogenized the suspension for 10 min on a magnetic shaker. Using the hemocytometer, the conidia were quantified, and the average number per milliliter was determined by following the formula of Lipa and Slizynski’s (1973):
$$ \mathrm{C}=\left(\mathrm{Cc}\right)\ \left(4\times 1060\mathrm{Df}\right)/80 $$
where C=number of conidia/ml, Cc=number of conidia counted, and Df =dilution factor.
Maintenance of fungal cultures and inoculum production
A loopful of inoculum from subcultured plates of L. lecanii was transferred to PDA and maintained as a pure culture. The fungus was cultured for 10 days at room temperature (26 ± 2°C) on sterilized PDA medium for laboratory studies. Using a small sterile metal spatula, conidia from the medium were harvested after complete sporulation and air-dried under laminar airflow, which was later stored in a small airtight screw cap vials (10 cm × 2.5 cm diameter) in the refrigerator at 4 °C for further studies. By plating technique, colony forming units (CFU) were estimated. After necessary serial dilutions, spore count was made under a phase-contrast microscope, using a double rolled Neubauer’s hemocytometer. For further studies, the required dilutions were made to obtain the required concentrations.
Laboratory bioassays of L. lecanii against 2nd instar nymphs of thrips, S. bispinosus
A total of 10 individuals of thrips (2nd instar nymphs) were released onto the fresh tea shoots, and the cut ends of the shoots were wrapped together by a piece of wet cotton. These shoots were kept in Petri dish (9 cm dia.) containing a round strips of tissue paper at the bottom. Treatment details were T1—L. lecanii (3.75 g/l), T2—L. lecanii + jaggery (for enhancement of the germination of L. lecanii) (3.75 g/l each), T3—Biocatch (commercial formulation of V. lecanii) (3.75 g/l), T4—Biocatch (commercial formulation of V. lecanii) + jaggery (3.75 g/l each), T5—NKAE @ 5%, and T6—Quinalphos 25 EC (Organo-Phosphatic insecticide) (2.5 ml/l). Neem Kernel Aqueous Extract (NKAE at 5%) was prepared, and spraying was done as described by Babu et al. (2008a). Using a glass atomizer, the spray fluids (T1 to T6) were sprayed on the shoots. Tea shoots with the 10 individuals of thrips were sprayed by distilled water and kept as control, and the bioassays were conducted using 5 replications. The treated tea shoots were kept in the round plastic containers sealed with transparent tape. The experiments were monitored at 24 h interval for 4 days for the mortality, and the percentage of bio-efficacy was calculated, and the corrected mortality was obtained using Abbot’s formula (Abbott 1925). Dead thrips were transferred to Petri dishes lined with moist filter paper for 5 days to observe mycosis for confirmation of their death by microscopic examination of mycelial growth on the surface of the thrips nymphs.
Assessment of the pathogenicity of L. lecanii conidial suspension
For assessment of the pathogenicity of L. lecanii strain against 2nd instar nymph of thrips, the prepared conidial suspensions of fungus were used by adopting the following different methodologies (direct spray, dipping, and leaf exposure methods).
Direct spray
A total of 10 individuals of 2nd instar nymphs of S. bispinosus were transferred on tea leaves kept in sterile Petri dish (9cm in diameter) and were sprayed by 3 ml conidial suspension of fungus (1×107 conidia ml−1) using a fine atomizer.
Leaf exposure
A piece of tea leaf (4 cm2) was placed in a Petri dish and was used for this experiment. Three milliliters of conidial suspension (1×107 conidia ml−1) was sprayed on each side of the leaf. The treated leaf materials were dried for 20-30 min on a clean laboratory bench. After that these leaves were provided to thrips for feeding for 24 h.
Dipping
A leaf containing a group of thrips nymphs (10 numbers) was dipped in a 3 ml conidial suspension (1×107 conidia ml−1) for 10 s and kept on a sterile Petri dish. After treatment, nymphs were maintained on a sterile Petri dish and were provided untreated leaves for feeding. Similarly, nymphs were provided by leaves treated with sterile distilled water containing a drop of Tween-80, served as control. The experiment was performed with 5 independent replications. The treatments were prearranged in factorial design consisting of 4 conidial concentrations and 3 exposure methods, and all the treatments were maintained at 25±1 °C. Mortality of thrips was observed at 24 h interval for 4 days using stereomicroscope (Olympus 1220; ×10). Dead insects were transferred on a moist Petri dish to observe the fungal growth.
Multiple-concentration bioassays of L. lecanii against thrips, S. bispinosus
A liquid suspension of the fungal strain L. lecanii was prepared for bioassays. The conidial suspension was mixed in 20 ml of liquid culture (2% sucrose and 5% peptone) containing 1 mg of the talc powder in 50 ml of water taken in conical flasks and incubated for 48 h in a rotary shaker (100 rpm). Before the application, the produced conidia from the formulations were examined for viability by spreading 0.01 ml of the culture onto PDYA medium. A stock of 1×108 conidia/ml was prepared, and 5 different concentrations (1×104, 1×105, 1×106, 1×107, and 1×108 conidia/ml) of conidial suspension were prepared in an aqueous solution containing 0.05% (v/v) of Tween 80 as the method of Butt and Goettel (2000). These suspensions were sprayed on tea leaves containing 10 thrips. The leaves sprayed with Tween-80 (0.05%) solution alone served as control. The thrips mortality rates were recorded, normalized with the natural mortality as observed in control (Abbott 1925).
Fungal development index (FDI)
The level of growth for L. lecanii on the nymphs and adult thrips were assessed as FDI. All assays were rated daily either until sporulation of fungus on the insect host or eclosion of the adult thrips. Control was used to assess the quantity of nymphs, and adult thrips emerged from these assays. Each nymph was assessed separately, under a compound light microscope, and the stage of fungal development on the nymphs and adult was recorded using the FDI.
Micro-plot field study
To evaluate the efficacy of L. lecanii against tea thrips, a micro-plot field trial was conducted in Paralai Tea Estate, Valparai (Coimbatore District, Tamil Nadu, India), The Anamallais. During the study period, the meteorological data were recorded. Treatments included were (1) L. lecanii W.P. at 1500 g/ha, (2) L. lecanii + Jagerry at both 1500 g/ha, (3) Biocatch at 1500 g/ha, (4) Biocatch at 3000 g/ha, (5) NKAE at 5.0%, (6) the recommended standard insecticide (Quinalphos 25 EC) at 1000 ml/ha, and (7) untreated control. Plots with ETL above 3% were selected and labeled based on treatments and replications. A hand-operated knapsack sprayer was used for spraying. To completely drench the bushes, special care was taken for better coverage and control. Thrips populations were assessed at weekly interval by collecting 10 shoots (3 leaves and a bud) at random from 10 bushes at each plot. Data were analyzed and the corrected percentage efficacy calculated according to Handerson and Tilton (1955) formula.
$$ \mathrm{Corrected}\ \mathrm{mortality}\%=1-\left[\left(\mathrm{Ta}/\mathrm{Cb}\right)\times \left(\mathrm{Tb}/\mathrm{Ca}\right)\right]\times 100 $$
where Tb and Cb represent the population density before treatment (pre-treatment) and Ta and Ca represent density after treatment in treated and control plots, respectively.
Large scale field study
Two large scale RBD field trials were carried out at estates in Paralai estate, Valparai (Coimbatore District, Tamil Nadu, India), The Anamallais (26° 54′ 0″ N, 88° 55′ 0″ E longitude), during two different seasons. The study was conducted in RBD (randomized block design) with 7 treatments, each replicated in 3 plots. Each plot contained 100 bushes. Spraying was carried out by maintaining a spray volume of 400 l/ha, as mentioned above in the micro-plot study. Both the pre- and post-treatment assessments were recorded as in a similar way described in the micro-plot study. Data were analyzed, and ANOVA was calculated.
Enhancement of virulence of L. lecanii with the addition of the additives
Seven commonly available vegetable oils were used to find out the impact on the condition and the growth of mycelia of L. lecanii. By following the poisoned food technique method, every 100 ml of sterilized PDA media was amended with 0.2% concentration (v/v) of selected vegetable oils, viz., groundnut oil (Arachis hypogaea L. Fabales: Fabaceae), sunflower (Helianthus annuus L. Asterales: Asteraceae) oil, coconut oil (Cocos nucifera L. Arecales: Arecaceae), paraffinic oil, neem oil (Azadirachta indica A. Juss. Sapindales: Meliaceae), and castor oil (Ricinus communis L. Malpighiales: Euphorbiaceae) under aseptic conditions. PDA medium without any oil was used as control. In a sterilized Petri dish, the media were poured for solidification. Using a cork borer, an isolate of L. lecanii grown on PDA for 2 weeks was cut into an 8-mm disk. The block was inverted and transferred onto the center of the vegetable oil + PDA-amended Petri dishes, placed gently on the surface of PDA, and then incubated at 25±1°C, 65 R.H. and a photocycle of 16 L: 8 D in a BOD incubator. Individual plant oil and the wetting agent were considered as a treatment, and each treatment was replicated 3 times. The effectiveness of L. lecanii was determined by measuring the radial growth of L. lecanii on the 5th, 10th, and 15th days after inoculation. Colony growth, spore germination, and conidial production were studied.
Effect of L. lecanii on natural enemies of thrips
Laboratory bioassays were conducted to study the infectivity of the fungal isolate, L. lecanii on the selected natural enemies present in the tea ecosystem, viz., Oligota pygmaea S. (Coleoptera: Staphylinidae), Stethorus gilvifrons M. (Coleoptera: Coccinellidae), Mallada boninensis O. (Neuroptera: Chrysopidae), and Neoseiulus longispinosus E. (Mesostigmata: Phytoseiidae). The predators were collected from the tea gardens at The Anamallais and were reared in the laboratory. Larvae of the respective predators were used for the study. Experiments were carried out by using a spore concentration of (1 × 107 spores/ml) (1×107 CFU/g in case of talc powder). All the experiments were replicated 3 times using 10 insects per treatment. Test insects sprayed with 0.05% Tween-80 solution served as control. Observation on mortality, pupation, adult emergence, and mycosis were recorded. Different methodologies were adopted in respect to each experiment, viz., (1) dipping of larvae in talc formulation (dip larva method), (2) dipping of larvae in spore suspension, (3) allowing of larvae to crawl on dried spore suspension (dry film method), (4) spraying L. lecanii talc powder on the larvae, and (5) dipping of larvae in only 0.05% Tween-80 solution as a control.
