Collection and laboratory culture of the TMB
The adults of TMB of mixed populations were collected from the experimental plots of North Bengal Regional R&D Centre (NBRRDC), Nagrakata (26° 54′ 0″ N, 88° 55′ 0″ E longitude), West Bengal, India. The culture of TMB was reared on a susceptible tea clone, TV1 in wooden cages containing glass chimneys under the laboratory conditions (25 ± 2 °C; 75 ± 5% RH; 16L: 8D photoperiod). The fresh tea leaves were provided to the adults every alternate day as food supplements. The cultures of the insects were used for the efficacy assays maintained for more than 10 generations (from F1 to F10 without exposure to any pesticides).
Isolation and identification of the fungus
Soil samples were collected with the help of auger from the rhizospheres of the tea plantations of Dooars and Darjeeling regions in pre-sterilized zip bags, separately in 2018 and were kept in a container with an ice bag (Ogunmwonyi et al. 2008). The collected samples were brought to the laboratory, and the fungus, B. bassiana was isolated through the serial dilution method (Morris and Rideout 2005). One ml aliquot of the soil sample from the 10–4 dilution of the soil in the sterilized distilled water was plated on the Potato Dextrose Agar (PDA) plates in 3 replicates and these plates were incubated in a BOD (Biological Oxygen Demand) incubator at 28 °C for the isolation of target entomopathogen. After 6 days of incubation, the colony of B. bassiana was isolated from the plates and purified by single spore isolation method. The culture of the EPF was maintained on the agar slants at 4 °C for further use.
The fungal isolates BKN20 and BKN1/14 (collected from Dooars and Darjeeling regions respectively) were identified as B. bassiana by studying cultural and morphological characteristics, as well as by using a fungal key. The cultures of both isolates were sent to the Indian Type Culture Centre (ITCC), Indian Agriculture Research Institute New Delhi, India for long term preservation. The isolates BKN20 and BKN1/14 were also identified by sequencing the ITS regions (ITS1, 5.8S and ITS2) of the nuclear rDNA. The gDNA (genomic DNA) from each isolate was extracted, following the CTAB method of Moller et al. (1992) and quantified with the help of NanoDrop1000 spectrophotometer (Thermo Scientific). The rDNA gene cluster was amplified by PCR, using universal primer pairs ITS1/ITS4 (White et al. 1990). The amplified PCR products of each isolate were separated by electrophoresis on a 2% agarose gel, and the obtained bands were excised and purified (UniPro Gel extraction kit) for sequencing (Macrogen, Inc., Korea.). BLASTn was used to match B. bassiana sequencing results of each isolate with known sequences of B. bassiana strains accessible at the public database GenBank.
Preparation of the fungal inoculum
For the preparation of the fungal suspensions of both isolates of B. bassiana (BKN20 and BKN1/14), each isolate was grown separately on the Potato Dextrose Broth (PDB) in conical flasks (500 ml) for 15 days at 28 °C in a BOD incubator. After the stipulated incubation, the fungal mycelial mat (conidia plus mycelia) was harvested from each flask and ground in a grinder with 50 ml of double-distilled water under aseptic conditions to prepare the spore suspension. The homogenized spore suspension was filtered through a muslin cloth to make the stock suspension, and spore density of stock suspension was adjusted to 2 × 107 conidia/ml using a hemocytometer (Singleton and Sainsbury 1981). Then, from the stock spore suspension of each isolate (BKN20 and BKN1/14), different concentrations such as 2.5, 5.0, and 10 ml (each concentration containing 2 × 107 conidia/ml) were mixed in one liter of sterile water in the pre-sterilized glass containers to make the liquid formulations separately. Similarly for field trials, a stock solution of effective entomopathogen’s spore suspension, i.e., isolate BKN20, was prepared and conidia were adjusted to 2 × 107 conidia/ml. From this stock solution, different amounts such as 600 ml, 800 ml, 1000 ml, and 1200 ml (each concentration containing 2 × 107 conidia/ml) were taken and mixed in the requisite amount of water for field applications to assess their bio-efficacy, separately.
In vitro bio-efficacy of the spore suspensions of B. bassiana against TMB
The bio-efficacy test was carried out against the second instar nymphs of TMB, collected from the laboratory reared culture, which were released on the tea leaves (TV 1 clone) kept inside a glass chimney (Roy et al. 2011). The spore suspensions of both isolates of BKN20 and BKN1/14 of different concentrations were sprayed on the tea leaves through a glass atomizer, separately. In control sets, tea leaves were sprayed with distilled water. A group of 20 TMBs of mixed populations were used for each treatment, and the experiments were carried out in 5 replicates in a complete randomized design (CRD). The mortality of TMB was recorded for 5 days at 24 h intervals. The number of dead insects was counted, and the percent mortality was calculated. During the laboratory experiment, the BKN20 isolate of B. bassiana was more pathogenic on TMB than the BKN1/14 isolate; hence it was selected for detailed field study.
Micro-plot field study
For the field experiment, a liquid formulation of BKN20 isolate containing 5% aqueous spore suspension was prepared, following the methodology of Godonou et al. (2000). A micro-plot field trial was conducted to evaluate the efficacy of BKN20 5%AS (aqueous suspension) against TMB. The trial was conducted in 3 replicates in a randomized complete block design (RCBD) in the experimental field of NBRRDC in 2018. Each plot (37.5 m2) was consisting of 50 tea bushes. The treatments included BKN20 5%AS @ 1000 ml/ha (T1, containing 2 × 107 conidia/ml), the recommended standard insecticide (Thiamethoxam 25%WG) @ 120 g/ha (T2), and untreated control (T3). The plots with ETL above the 5% were selected and labelled for each treatment. Spraying was done, using a hand-operated knapsack sprayer (ASPEE Agro Equipment Pvt. Ltd.) using a spray volume of 1.5 l/plot. Care was taken to drench the bushes for better coverage and control. Plucking of the shoots was carried out before initiation of the treatment spray, and the percentage damage caused by TMB was computed from the randomly selected 100 shoots per replicate in each treatment. The first treatment spray (01.04.2018) was applied immediately after the harvesting. The second treatment spray (08.04.2018) was applied after plucking the leaves on the 7th day after the first spray. The percentage damage caused by TMB on 7th day after the 1st treatment spray, followed by the 7th day, 14th day and 21st day after the 2nd treatment spray (post-spray) was recorded from the randomly selected 100 shoots per replication. The percent shoot damage due to TMB was calculated, using the formula: C − T/C × 100, where, C = Pre spray assessment and T = Post spray assessment (Sarmah and Bhola 2015).
Large-scale field study
The large-scale field trials were also conducted to evaluate the efficacy of the BKN20 5%AS in the 2 consecutive years of 2018 and 2019 in the tea gardens of 2 locations namely, Sungma Tea Garden, Darjeeling (26° 94′ 5″ N, 88° 17′ 43″ E longitude), and the experimental plot at the NBRRDC, (26° 54′ 0″ N, 88° 55′ 0″ E longitude). Each trial was conducted in a randomized complete block design (RCBD) in three replications. There were total 7 treatments [BKN 20 5%AS @ 600 (T1), 800 (T2), 1000 (T3), 1200 (T4) ml/ha (each treatment containing 2 × 107 conidia/ml), B. bassiana 1.15%WP @ 2500 gm/ha (T5- a market product), recommended standard insecticide (Thiamethoxam 25%WG) @ 120 gm/ha (T6), and untreated control (T7)] per replication, and each plot (75 m2) contained 100 tea bushes. The market product of B. bassiana (1.15% WP) was purchased from the local vendor. The pre-and post-treatment assessments were carried out as described earlier in the micro-plot study.
Yield estimation of harvestable shoots from the large- scale field trials
The yield estimation was also carried out during the experiments. By maintaining a standard plucking round (7 days interval), yield (green tea leaf) was recorded for the first 6 rounds of plucking. Average yield was expressed in kg/plot. The green leaf yield recorded at every plucking was converted into processed tea for one hectare as described by Ponmurugan and Baby (2007), using the formula: Green leaf yield (kg) × no. of bushes/ha × conversion factor (0.225).
Effect of BKN20 5%AS on the non-target organisms
Laboratory bioassays were conducted to study the infectivity of the BKN20 5%AS the most common insect predator in the tea ecosystem. Larvae and adults of the predators were collected from the tea gardens at the NBRRDC and reared in the laboratory. Experiments were carried out by spraying the different concentrations of BKN20 5%AS @ 600 (T1), 800 (T2), 1000 (T3), 1200 (T4) ml/ha (each treatment containing 2 × 107 conidia/ml), and untreated control (T5) per replication. All the experiments were replicated 3 times, using 30 insects per treatment. Observations on larval mortality, larval period, pupation period and adult emergence were recorded by following the methodology of Leatemia and Isman (2004).
Phytotoxic effect and organoleptic evaluation
To evaluate the phytotoxicity effect (yellowing, stunting, necrosis, epinasty, hyponasty, etc.) of the BKN20 5% AS (at ‘X’ ‘2X’ and ‘4X’ concentration) on tea leaves, field experiments were carried out in three replicates at 84 square meters per replication in a RCBD design at Nagrakata, West Bengal. There were in total 3 treatments; 2.5 ml/l (T1), 5 ml/l (T2), 10 ml/l of water (T3), and an untreated control. The conidial density of each treatment solution, i.e., T1, T2 and T3 was 2 × 107 conidia/ml. The spraying was applied by a hand-operated knapsack sprayer, using a spray volume of 400 l/ha. Observations were recorded on 0 day (pre-treatment) and day 3, 7 and 14 (post-treatment) on yellowing, stunting, necrosis, epinasty, hyponasty, etc., and the injury levels were graded, using the Phytotoxicity Rating Scale (PRS) as follows: no crop response/crop injury = 0, 1–10% = 1, 11–20% = 2, 21–30% = 3, 31–40% = 4, 41–50% = 5, 51–60% = 6, 61–70% = 7, 71–80% = 8, 81–90% = 9, 91–100% = 10 (Babu et al. 2008).
Data obtained through laboratory and field experiments were analyzed through ANOVA (analysis of variance) using SPSS17. All the data were angularly transformed prior to the statistical analysis and the differences among the treatments in pre-spray percent incidence and percent reduction of shoot damage were assessed through Tukey’s post-hoc Honestly Significant Difference (HSD) test to separate the means at 95% confidence interval. For the green leaf yield, the treatments were compared with untreated control, using Tukey’s post-hoc Honestly Significant Difference (HSD) test to separate the means at the 95% confidence interval. DMRT F-Test was carried out to compare the effect of different concentrations of BKN20 5%AS on non-target beneficial organism.