3.1. Experimental Site and O3 measurement
The experiment was conducted in subtropical highland climatic region of Western Ghats at 11.4 °N, 76.7 °E at an altitude of 2520m mean sea level during September to December 2018 in the experimental Woodhouse farm of Horticultural Research Station, Tamil Nadu Agricultural University, Ooty, Nilgiris District.
The ambient ozone measurements were continuously monitored by using ozone analyzer (Thermo Fischer U.S.A Model 49i in the Fig.2) from February 2010 which is based on UV absorption. It measures the amount of ozone in the air from 0.05 ppb concentrations up to 200 ppm. The analyzer aspirates air from a height of about 5 m above the ground level through Teflon tube with a flow rate of about 1 L per minute. The minimum detection limit of the analyzer is about 1 ppb. Calibration is done at frequent intervals using zero air and known span values. The ambient ozone concentration in the experimental site of Ooty recorded from 2010 in which minimum of 10ppb and maximum of 120ppb were recorded. The highest values of ambient O3 are found in the year 2015 and 2016 in which it peaks from 100.446 to 123 ppb and 100.154 to 109.199 were recorded. The unforeseen increase in the O3 concentration may expected to escalate about 150ppb and 200ppb in the years ahead. Hence this intended the authors to study with elevated 150ppb and 200ppb of O3 levels.
3.2. Exposure design
The electric supply was provided to connect the corona discharge through ground cable for carrying out the experiment from the Ozone generator. For the uniform distribution of ozone, which is fumigated, the axial flow fans were fixed to the open top chamber. The Garlic had been grown in prepared pots and watered regularly. The plants were maintained uniformly up to the most critical stage of garlic outside the chamber. Only during the critical stages, the crops were exposed to 150 and 200 ppb (Elevated level of Ozone) of ozone and fumigated at the rate of 4 hours’ day-1 within the chamber.
Fig. 2. O3 Experimental design: 1) Potted garlic seedlings, 2) Garlic plants at critical stage i.e., bulb initiation stage, 3) Open top chamber- within which the potted plants were exposed to elevated levels of O3, 3a) Ozone Generator - fixed to the ground surface for stimulating elevated levels of ozone, 4) Ozone monitor- fixed inside the OTC for monitoring the O3 elevated levels (150ppb and 200ppb), 5) Measurement of photosynthetic rate, stomatal conductance using Portable photosynthetic system and chlorophyll content by using chlorophyll content meter, 6) Potted garlic plants after exposure to elevated levels of O3
3.3. Time of exposure
Growth of a garlic plant occurs in several stages viz., clove sprouting and establishment, bulb initiation, bulb development and bulb maturation. The bulb initiation stage of garlic is considered as the important critical stage since most of the significant physiological and growth effects occurred during the later stages of crop development. So, the bulb initiation stage of garlic has been taken for elevated levels of ozone (O3) exposures. Exposure of ozone between 40 - 100 ppb may cause different injury patterns in different crops according to its sensitivity. Based on this concept the following treatments were fixed.
3.4. Treatments details
The potted experiment of garlic treated with two different levels of ozone (150ppb and 200ppb) under open top chambers (OTCs) with twelve treatments arranged in factorial completely randomized block design (FCRD) with three replications are as follows
T1 - Ambient ozone level,
T2 - Elevated ozone exposure at 150 ppb,
T3 - Elevated ozone exposure at 200 ppb,
T4 - Ambient ozone level + foliar spray 3% Panchagavya,
T5 - Ambient ozone level + foliar spray 3% Neem oil,
T6 - Ambient ozone level + foliar spray 0.1% Ascorbic acid,
T7 - Elevated ozone exposure at 150 ppb + foliar spray 3% Panchagavya,
T8 - Elevated ozone exposure at 150 ppb + foliar spray 3% Neem oil,
T9 - Elevated ozone exposure at 150 ppb + foliar spray 0.1% Ascorbic acid,
T10 - Elevated ozone exposure at 200 ppb + foliar spray 3% Panchagavya,
T11 - Elevated ozone exposure at 200 ppb + foliar spray 3% Neem oil,
T12 - Elevated ozone exposure at 200 ppb + foliar spray 0.1% Ascorbic acid.
3.5. Chlorophyll content meter
The relative chlorophyll concentration is determined by using the instrument CCM-200 plus (chlorophyll content meter) designed by optic sciences (USA). The chlorophyll content meter readings were recorded regularly at the time of exposure of garlic to elevated levels of ozone.
3.6. Photosynthetic rate and Stomatal conductance
The fumigation study with elevated levels of ozone on plants in pots where the physiological responses of crop were considered as important criteria in crop growth and yield. Hence, LCpro-SD portable photosynthetic system (PPS), which designed by ADC BioScientific Ltd. was used to measure the physiological parameters i.e., photosynthetic rate and stomatal conductance. Daily readings were recorded every day at the time of exposure of crops under different concentration of ozone. The above characters were statistically analyzed by using SSPS version 16 in one-way analysis of variance (ANOVA) and the significant differences between the means were determined with Duncan’s multiple range test to assess the impact of tropospheric ozone on physiological response in garlic.
3.7. Pungency (pyruvate analysis)
To determine the content of naturally occurring pyruvate and carbonyl compounds, DNPH (2,4-dinitrophenylhydrazine) is used. The pyruvate content was determined by using a colorimetry spectrophotometer at 420 nm (Thermo Spectronic Genesys 20; ThermoFisher Inc., Waltham, USA) (Schwimmer and Guadagni, 1962).
3.8. Total Soluble Solids (0Brix)
Total soluble solids had been determined by using the hand-held refractometer after harvesting the bulbs. The refractometer was standardized by using distilled water. 1 to 2 drops of aliquot juice were dropped on to the prism and thus the reading were noted and washed with distilled water every time after each sample and dried with tissue paper