Isolation and identification of fungal species
The species of Trichoderma, Beauveria, and Metarhizium were isolated from soil through standard technique (Askew and Laing 1993; Ghanbary et al. 2009 and Qazzaz 2012) during 2014–2015. Purification of isolates was done through hyphal tip culture technique and stored in refrigerator. Then, pure cultures were grown on potato dextrose agar (PDA) plates, followed by incubation at 25 ± 2 °C for 48–96 h. The pure cultures were identified based on their colony characters like colony color, growth pattern and formation of conidial rings, and color of conidia. The shapes of conidiophores and phialides were observed using microscope. Later on, the identity of isolates was re-confirmed from Indian Type Culture Collection, Division of Mycology and Plant Pathology, Indian Agricultural Research Institute, New Delhi, India.
For isolation of pathogen, i.e., Fusarium solani, dieback diseased tender tea shoots were collected from tea bushes during 2014–15. The diseased shoots were cut in to small pieces, followed by surface sterilization with mercuric chloride (0.1%) and subsequent 2 washing with distilled water. Then, these pieces were inoculated in to PDA plates, and plates were incubated at 26 ± 2 °C for 1 week. Colonies developed in plates were purified, using PDA plate. After colony development, they were identified on the basis of morphological characteristics.
In vitro bioefficacy of fungi
Trichoderma spp.
Bioefficacy of Trichoderma spp. was assessed by dual culture technique (Stack et al. 1986). Five-millimeter discs of both fungi (Trichoderma spp. and F. solani) were transferred in to plates at equidistance. In control, only F. solani was inoculated for comparison. Each treatment was replicated 5 times. Plates were incubated at 25 ± 2 °C for 1 week. Pathogen’s mycelial growth was measured and its inhibition was worked out, using the following formula:
$$ \mathrm{Mycelial}\ \mathrm{growth}\ \mathrm{inhibition}\ \left(\%\right)=\frac{\mathrm{Colony}\ \mathrm{dia}.\mathrm{in}\ \mathrm{control}-\mathrm{Colony}\ \mathrm{dia}.\mathrm{in}\ \mathrm{treatment}}{\mathrm{Colony}\ \mathrm{dia}.\mathrm{in}\ \mathrm{control}}\times 100 $$
Beauveria bassiana
The in vitro bioassay of B. bassiana against H. theivora was carried out by employing the methodology of Amarsena et al. (2011) with slight modifications. To prepare spray suspension, 10 ml of distilled sterilized water was added in to 2 weeks old B. bassiana culture grown on potato dextrose agar (PDA) slant, and biomass was harvested, followed by filtration twice through muslin cloth. The conidial concentration (2 × 109) was determined, using hemocytometer and sprayed on the nymphs of H. theivora with the help of an atomizer. Each treatment was replicated 3 times. Insect mortality was recorded till 192 h, and percent mortality was corrected using Abbott’s formula (Abbott 1925).
$$ \mathrm{Corrected}\ \mathrm{mortality}\ \left(\%\right)=\frac{\mathrm{No}.\mathrm{of}\ \mathrm{live}\ \mathrm{in}\mathrm{sect}\ \mathrm{in}\ \mathrm{control}-\mathrm{No}.\mathrm{of}\ \mathrm{live}\ \mathrm{in}\mathrm{sect}\ \mathrm{in}\ \mathrm{treatment}}{\mathrm{No}.\mathrm{of}\ \mathrm{live}\ \mathrm{in}\mathrm{sect}\ \mathrm{in}\ \mathrm{control}}\times 100 $$
Different adjuvants, namely, Tween 20 (2 ml/l of water), glycerol (5 ml/l of water), and crude sugar/molasses (5 g/l of water), were in vitro studied to find out their role in enhancing the efficacy of B. bassiana against 2nd instar tea mosquito. B. bassiana conidial suspension in combination with these adjuvants separately was sprayed on tea shoots, and known numbers of nymphs were released on to these shoots. Each treatment was replicated thrice times, and observations on insect mortality were recorded and 24-h interval for a period of 1 week.
Metarhizium anisopliae
The pathogenicity of M. anisopliae was carried out against the red spider mite (O. coffeae) employing leaf disc technique (Plate 1). The fungal culture was mass multiplied, using potato dextrose broth (PDB). The conidial concentration (1 × 108 to 2 × 108) was determined, using hemocytometer and sprayed on the red spider mite with atomizer. Twenty insects per treatment were taken, and each treatment was replicated 4 times. The insect mortality was calculated by the following formula.
$$ \mathrm{Insect}\ \mathrm{mortality}\ \left(\%\right)=\frac{\mathrm{Number}\ \mathrm{of}\ \mathrm{dead}\ \mathrm{insect}}{\mathrm{Total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{insect}\ \mathrm{used}}\times 100 $$
The dead insects were collected and re-inoculated in the PDA (potato dextrose agar) plates followed by incubation at 26 ± 2 °C for 1 week to confirm insect mortality.
In vivo testing of fungi
The liquid fermentation technique was adopted with minor modifications for the mass production of Trichoderma atroviride, T. asperellum, T. harzianum, and B. bassiana using 25-l capacity fermenter (Bhat et al. 2009). The potato dextrose broth (Hi-media) was used as basal medium. The biomass was harvested after 11 days, and wettable powder formulations were prepared. M. anisopliae was mass multiplied on PDB medium. After 2 weeks, the medium and biomass was homogenized and used for field application. It was sprayed on tea stem and drenched uniformly in collar region during 2015–16.
Trichoderma spp.
Developed wettable powder (WP) formulation (2 × 108 cfu/g) of Trichoderma isolates and commercial formulation was tested against dieback disease in field during 2015–16. Two sprays at weekly interval were given immediately after plucking the shoots. Sixty bushes were taken per treatment, and each treatment was repeated four times. Pre- and post-spray observations on number of dieback shoots were recorded. One hundred shoots from plucking basket were taken, and infected shoots were counted. The disease reduction over control was calculated by the following formula.
$$ \mathrm{Disease}\ \mathrm{reduction}\ \left(\%\right)=\frac{\mathrm{No}.\mathrm{of}\ \mathrm{disease}\ \mathrm{shoots}\ \mathrm{in}\ \mathrm{control}-\mathrm{No}.\mathrm{of}\ \mathrm{disease}\ \mathrm{shoots}\ \mathrm{in}\ \mathrm{treatment}}{\mathrm{Number}\ \mathrm{of}\ \mathrm{disease}\ \mathrm{shoots}\ \mathrm{in}\ \mathrm{control}}\times 100 $$
The formulation was tested during cold weather in the first week of December on light pruned (LP) and deep skiffed (DS) teas at experimental plot of Tea Research Association North Bengal Regional Research and Development Centre. It was sprayed immediately after pruning operation. After 2 months, the number of bud break (five bushes) and shoot length (5 shoots of 5 bushes) was recorded from each treatment.
Beauveria bassiana
The B. bassiana formulation was evaluated during 2015–2016 at TRA NBRRDC plot in combination with different adjuvants, namely, Tween 20 (2 ml/l of water) and crude sugar (5 g/l of water). Randomized block design (RBD) with 3 replications was followed.
Metarhizium anisopliae
The in vivo bioefficacy of M. anisopliae was also assessed for the control of termite during 2015–2016. The broth culture at 5% v/w sprayed on the bush stem and drenched in the collar region properly two times at an interval of 3 months. Thiamethoxam was used as standard check for comparison. Pre- and post-treatment observations on number of bushes showed presence of earth runs, or live termite was calculated.
Statistical analysis of data
Statistical analysis of the data was carried out with the help of online statistical package OPSTAT of Chaudhary Charan Singh Haryana Agricultural University, Hisar, Haryana, India.