Larval rearing
To investigate the sensitivity of H. talaca, the second instar larvae of this looper caterpillar were originally collected from Dooars region, West Bengal, India. The collection site was free of pesticides since 6 months and the larvae were carried with leaves to reduce their transportation disturbances. They were reared in the laboratory of the Plant Protection Department, North Bengal Regional Research and Development Centre, Tea Research Association (TRA), for three successive generations at 28 ± 2 °C, 72 ± 3% relative humidity and a 13L:11D photoperiod. Fresh tea leaves were provided as food after sterilizing with 10% formalin for 5 min and rinsed with sterile double-distilled water. These larvae were used for viral propagation and bioassays. The larvae were also examined daily to eliminate the secondary infection.
Viruses
The HytaNPV samples, used in the PCR study, were collected during 2012–2013 from geographically distinct localities covering eastern Dooars, western Dooars, central Dooars and Terai region of West Bengal, India. The average rainfall of Terai and Dooars are approximately 3500 and 3160 mm, respectively, situated between 26° 16′ and 27° 12′ north latitudes and 87° 59′ and 89° 53′ east longitudes. The virus samples were collected from ten tea estates located across these four distinct geographic regions (Table 1). From each collection site, 50–60 virus-infected larvae were sampled. The virus-infected larvae were collected as samples considering the primary signs of typical NPV infection in larval stage like flaccidity, rupturing of the cuticle and hanging upside down by their abdominal legs. Later, the presence of HytaNPV was ascertained by microscopic view and PCR study. The individual larva was collected in 1.5 ml centrifuge tube and the collected samples were kept at −20 °C for future use.
Table 1 Details of the survey site location
Purification of HytaNPV
For molecular analysis, each of aqueous homogenate of viral samples containing polyhedral occlusion bodies (POBs) was purified by two rounds of centrifugation. The putrefied samples were homogenized with pestle and mortar, and the concentrates were diluted with sterile double-distilled water. The crude filtrate was initially centrifuged for 5 min at 600 rpm to remove larger contaminants. The supernatant was collected in a new centrifuge tube for further centrifugation at 8000 rpm for 20 min to pellet the virus and then washed with sterile double-distilled water for three times. The pellets were finally dissolved in 1 ml sterile double-distilled water and conserved at −20 °C. The quantification of POBs was carried out by using Neubauer hemocytometer (Marienfeld, Germany) under phase-contrast microscope (Olympus BX 51). For bioassays, the viral samples were purified by following the same method.
Virus propagation
Four HytaNPV isolates (SMB, RJB, WSB and TLP), belonging to four different geographic regions, were taken for bioassay tests and multiplied in second instar H. talaca larvae for mass propagation. In case of each isolate, the multiplication of HytaNPV was conducted by isolation of POBs from infected cadavers. For multiplication of each isolate, 150 larvae were fed on tea leaves dipped with virus suspension in a concentration of 107 POBs/ml. After 2 h of starvation, they were exposed to virus-contaminated leaves for 24 h. Virus-induced larvae were kept as 30 larvae per group and reared in the laboratory at 28 ± 2 °C, 72 ± 3% relative humidity and a 13L:11D photoperiod. Microscopic view confirmed the presence of POBs in the cadavers of virus-induced larvae.
Viral DNA extraction and PCR amplification
After following the purification method, viral DNA was extracted from POBs of each of ten field collected HytaNPV samples and purified with QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s protocol. The final DNA was obtained after cleaning several times with ethanol and diluted wash solution. Later, DNA was eluted and re-suspended in 20 µl molecular-grade water (Himedia). The isolation of DNA was confirmed by electrophoresis in 1% agarose gel and quantified with BioPhotometer (Eppendorf).
A highly conserved region of polyhedrin gene from HytaNPV isolates was amplified. The PCR was performed using the degenerate primer (F: 5′-GGACCSGGYAARAAYCAA AAA-3′ and R: 5′-GCRTCWGGYGCAAAYTCYTT-3′) designed according to Antony et al. (2011). The PCR reaction was carried out taking 50–100 ng of viral DNA in a 25 µl reaction solution containing 1X PCR buffer (Invitrogen, USA), 1.5 mM MgCl2 (Invitrogen, USA), 0.5 mM dNTPs (Bangalore Genei, India), 1 U Platinum Taq DNA polymerase (Invitrogen, USA) and 0.5 µM of each primer. PCR amplification was performed in a DNA thermal cycler (Veriti Thermal Cycler, Applied Biosystems, CA, USA). The conditions used were initially denaturation for one cycle at 94 °C for 5 min, followed by 35 repeated cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 30 s, and the final extension cycle at 72 °C for 7 min. The amplified products were resolved in 1.5% agarose gel stained with ethidium bromide.
Cloning and sequencing of the polyhedrin gene
The positive PCR product of the SMB isolate was eluted from the gel using HiPura Agarose Gel DNA Purification Spin Kit (Himedia, India) following the manufacturer’s protocol. The eluent was ligated into the pGEM-T vector (Promega, UK) in a 3:1 (insert: vector) molar ratio with T4 DNA ligase as described by the manufacturer. The ligated products were transformed into Escherichia coli DH10β competent cells (Invitrogen, USA). Following amplification of recombinant clone with M13 universal forward and reverse primers, the sequencing was performed using the BigDye Terminator v1.0 cycle sequencing kit (Applied Biosystems) in ABI 3500 Genetic Analyzer (Applied Biosystems).
Sequence comparison and phylogenetic analysis
The sequence data were assembled into contig by DNA Dragon Version 1.5.6 (SequentiX, Germany) followed by the multiple-sequence alignments construction with the highly similar DNA and protein sequences using the Clustal W program (Thompson et al. 1994). The obtained nucleotide sequence was blasted to the GenBank database (blastx) to retrieve similar nucleotide and amino acid sequences, which were used for phylogenetic tree construction. The phylogenetic tree of aligned amino acid sequences was generated by the neighbor-joining (NJ) algorithm (Saitou and Nei 1987) using Molecular Evolutionary Genetics Analysis (MEGA) v6.06 (Tamura et al. 2013). To estimate the confidence limits of nodes, 1000 bootstrap samples were generated. To reduce the impact of partial sequences of polyhedrin available in the GenBank database and to maximize the use of the available information, the pairwise comparison of amino acid sequences was performed by ClustalW with the default parameters.
Bioassays
The biological activity of four HytaNPV isolates, viz., SMB, RJB, WSB and TLP (belonging to four different geographic regions), was tested against early second instar H. talaca larvae. Bioassay tests were conducted using purified viral suspension by leaf-dip feeding technique. The virus concentrations were quantified with a phase-contrast microscope and a Neubauer hemocytometer. The concentrations of each tested isolates were prepared from the following stock concentrations in POBs/ml: 3.6 × 109, 5.7 × 109, 2.9 × 109 and 8.1 × 109 for SMB, RJB, WSB and TLP isolates, respectively. The experiments were performed using six concentrations of each virus isolate containing 1 × 103, 1 × 104, 1 × 105, 1 × 106 1 × 107 and 1 × 108 POBs/ml. Larvae were taken from the virus-free rearing culture and starved for 2 h before feeding viruses. Leaves were dipped in viral suspension, air-dried and fed to larvae for 24 h prior to feeding on fresh foliages until death or pupation. In the control treatment, the virus suspension was replaced by double-distilled water. For each isolate, three replicates each of 30 larvae were used for each virus concentration and three replicates (30 larvae per replication) of double-distilled water-treated leaves served as control. Larvae were incubated at 27 ± 1 °C, 72 ± 3% RH and a 13L:11D photoperiod. Virus-induced cumulative mortality was recorded daily till death and the mortality response data were analyzed on the basis of mortality on day 7 post-inoculation. The diseased cadavers were collected and kept in −20 °C for further analysis. Later virus infections were confirmed by the presence of POBs in the cadavers, when viewed under phase-contrast microscopy.
Statistical analysis
The mortality of larvae was tabulated daily for all NPV isolates vs. dose combinations (4 × 6). Probit analysis was performed using IBM SPSS release 23.0.0.0 on the basis of mortality data obtained after 7 days of post-inoculation. The median lethal concentrations (LC50) for second instar were obtained from the SPSS probit model. Median lethal time (LT50) was also determined for each concentration using the equation (Biever and Hostetter 1971):
$${\text{LT}}_{50} = a + e(c - b)/D$$
where ‘a’ is the number of hours from the initiation of the test until the reading made immediately before the 50% mortality was recorded; ‘b’ is the total number of larvae dead at the reading immediately before the 50% mortality was recorded; ‘c’ is the 50% of the total number tested (in our case, it is 45); ‘D’ is the number of larvae dying in 24 h during which the 50% mortality was reached; and ‘e’ is the number of hours between mortality counts (24 h in this case).