Isolation, identification, and selection of promising Trichoderma sp
Isolation of the antagonist was carried out following standard technique with slight modification [25]. Soil samples were collected from tea rhizosphere in sterilized polyethylene bags, brought to mycology laboratory, stored in the refrigerator at 4±10C. For isolation of antagonistic fungi, soil samples were homogenized processed following multiple serial dilution plate technique (MSDP). From 6th and 8th dilutions, 0.5 mL was drawn and uniformly distributed in solidified Trichoderma specific medium (HiMedia) plates in triplicates. Plates were then properly sealed with parafilm and incubated at 28±2 0C for 72-96 hours. Appeared fungal colonies were observed and antagonist’s colonies were identified based on its mycelia color and fast-growing character. Such colonies were transferred into another potato dextrose agar (PDA) plates for getting pure culture and re-incubated at 28±2 0C for 96 hours in the BOD incubator.
To screen the most potent Trichoderma isolate, seven isolates were studied through dual culture bioassay [26]. The mycelia bits (5 mm diameter) of F. solani and antagonists were inoculated in to same potato dextrose agar plate followed by proper sealing and incubation at 28±2 OC for one week. Three replications of each isolates were maintained. Colony diameter was observed and per cent growth inhibition was calculated using the formula (Per cent growth inhibition = colony diameter in control – colony diameter in treatment / colony diameter in control x 100).
For the re-confirmation and having accession number, the most promising isolate was sent to Indian Type Culture Collection, Division of Mycology and Plant Pathology, Indian Agricultural Research Institute, Pusa, New Delhi, and ICAR National Bureau of Agriculturally Important Microorganisms, Indian Council of Agricultural Research, Kushmaur, Mau, Uttar Pradesh, India.
Development of formulation: T. asperellum 2% Aqueous Suspension
The liquid formulation of T. asperellum was manufactured using liquid-state fermentation techniques by M/s Varsha Bioscience and Technology India Pvt Ltd, Hyderabad - 500059, Telangana, India using the slightly modified method [27]. The mother culture of the antagonist was sub-cultured on to plates containing Trichoderma Specific Media (TSM) followed by incubation at 28±2 °C for 5 days.
Then 10 liters of seed inocula was prepared by inoculating 120 hr old mother culture into 1000 ml conical flask filled with 250 ml autoclaved potato dextrose agar and incubated in an orbital shaker at 28±2°C and 180 rpm for seven days. From seed inocula, submerged large scale fermentation was done to scale up its quantity using another autoclaved medium containing 30g sugarcane molasses and 5g yeast extract per liter of water. One week old seed inocula (10%) was inoculated in the fermenter incubated for 7 days and the cultural biomass was separated by centrifuging the culture broth through the on-line centrifugation system and both the conidia, as well as mycelia were collected. Active ingredient (2x108 CFU/ ml) was determined by the MSDP technique in final formulation by adding required distilled sterilized water.
Multilocation field Bio-efficacy of T. asperellum 2% AS on dieback disease during the season I and II
The T. asperellum 2% AS formulation was tested under field conditions at Darjeeling, Dooars, and Assam zones against dieback disease caused by F. solani from 2016 to 2018. The plot size was kept 84 m2 for each treatment with 100 bushes. Experiments were laid out in randomized block design (RBD) with seven treatments in three replications. Common tea cultivars/clones of particular zone namely TRA-AV-2, TV-25, TV-26, and TV-1 of 28 to 54 years old were chosen for the study.
The plots having dieback disease incidence above 5% ETL were selected for field bio-efficacy study. The disease incidence was recorded by placing a 1 x 1-foot quadrate at 3 randomly selected spots per treatment then healthy and infected tea shoots were plucked. Then the first spray with hand operated knapsack sprayer fitted with NMD 450 nozzle was done immediately after the plucking (0-day). Observations on disease incidence were recorded on the 7th day of the first spray using the same quadrate. Subsequently, both healthy and infected shoots were plucked on the same day and weight of the fresh healthy shoots was recorded. The second spray was given on the 7th day after the 1st spray and disease incidence was recorded on the 7th, 14th, and 21st days of the second spray.
Effect of T. asperellum formulation on the yield of green leaves
Green leaf yield (kg/plot) was recorded from the first six rounds of plucking and at every plucking round it was converted into made tea per hectare using the formula [28].
Made tea Kg per hectare (KMTH) = Green leaf yield (Kg) x no. of bushes/ha x Conversion Factor (0.225)
Effect of the formulation on beneficial insects
The population of beneficial insect viz., Chrysoperla carnea, Oxyopes javanus, and Stethorus gilvifrons was recorded on 0 days (pre-spray), 7th day of 1st spray and 7th, 14th and 21st day of 2nd spray (post-spray). Visual observations were made from 30 randomly selected tea bushes per treatment to assess the population of C. carnea nymphs and O. javanus adults. Thirty leaves were collected at random per treatment and observed under a binocular microscope for assessing the population of S. gilvifrons.
Testing for phytotoxicity, tainting and organoleptic attributes
The developed formulation was sprayed at concentration of 4, 8, and 16ml per liter water to assess its phytotoxic effects on tea leaves. Three replications were maintained in 84 square meters area of the experimental plot. Observations were recorded on 0, 3, 7, and 14th day of spray for the appearance of leaf yellowing, stunting, necrosis, epinasty, and hyponasty type symptoms and the injury level (toxic level) was rated using the following phytotoxicity rating scale (PRS).
Crop response / Crop injury (%)
|
Rating
|
0.0
|
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
|
The percent phytotoxicity index (PPI) was computed using the following formula,
Sum of all numerical ratings
PPI = ------------------------------------------------------------------------------ x 100
Number of tea plant observed x Maximum phytotoxicity rating
Statistical analysis
The collected data were statistically analyzed to find out the critical difference among treatment at a 5% level of significance (p =0.05) through the online statistical package “OPSTAT” of Chaudhary Charan Singh Haryana Agricultural University, Hisar (www.hau.ac.in).