Physiological Response of Garlic (Allium Sativum) to Elevated Tropospheric Ozone in High Altitude Region of Western Ghats, Tamil Nadu, India


 A pot culture study was conducted at Horticultural Research Station, Ooty to assess the effect of ground level ozone on physiology in garlic plant and to find out the suitable remedial measures against ground level ozone. The potted soil was found to be acidic in nature with very low salt concentration, very high in organic carbon, medium in nitrogen, phosphorus and high in potassium. Elevated ozone levels (150ppb and 200ppb) had significantly reduced the garlic plant chlorophyll content, stomatal conductance, photosynthetic rate, pungency and total soluble solids. The highest chlorophyll content (33.97µmolm-2) was observed under ambient ozone level (T1) and the lowest chlorophyll content (12.68µmolm-2) was observed in elevated ozone exposure at 200 ppb (T3), the highest stomatal conductance (0.45mmol m-2s-1) was recorded in Ambient Ozone level + foliar spray 3% Panchagavya (T4), and the lowest stomatal conductance (0.11mmol m-2s-1) was observed in elevated ozone exposure at 200 ppb (T3). Since, the elevated ozone had significant reduction in photosynthetic rate in garlic, the lowest was observed (0.82µmol CO2 s-1m-2) in T3- Elevated ozone exposure at 200 ppb and the highest photosynthetic rate (3.02µmol CO2 s-1m-2) was observed in treatment T4- Ambient Ozone level + foliar spray 3% Panchagavya after a week. When coming to quality of garlic bulbs, the highest pungency content was found in Ambient Ozone level + foliar spray 0.1% Ascorbic acid and the lowest was observed in Elevated ozone exposure at 200 ppb (T3) furthermore, in observing the garlic quality where total soluble solids (TSS) showed that the treatment Ambient Ozone level + foliar spray 3% Panchagavya (40.00°Brix) as highest and the treatment Elevated ozone exposure at 200 ppb (T3) recorded the lowest. Thus the tropospheric ozone has detrimental impact on physiological responses, which will reduce crop growth and yield. The ozone protectants helped in scavenging the O3 from apoplast of the crops and among the ozone protectants, neem oil acted as a good ozone scavenger followed by ascorbic acid and panchagavya to improve the physiological response of garlic plant under elevated tropospheric ozone levels.


Introduction
Air pollution, the major threat to humans, crops, animals whereby the emitter source is largely by human beings. The primary air pollutants are the precursors for the formation of secondary air pollutants such as surface or tropospheric ozone (O 3 ). The primary air pollutants are sulfur dioxide(SO 2 ), carbon monoxide(CO), methane(CH 4 ), non-methane volatile organic compounds(NMVOC) and nitrogen oxides(NO X ) which formed tropospheric ozone as a secondary air pollutant by the oxidation of CO, CH 4 or NMVOCs in the presence of NO X (nitrogen oxides) (Unger et al., 2006) and the concentration of ozone increased due to industrialization (Grulke and Heath, 2020). Ozone is a short lived pollutant, which signi cantly reduced the growth, yield and productivity of crops globally (Ainsworth, 2017), when its concentration increased in the troposphere of atmosphere. The ozone enters into the plants through stomata and forming reactive oxygen species (ROS) directly by reacting with organic molecules present in the apoplast, which will cause cell damage and cell death (Ainsworth, 2017;Li et al., 2017). There will be signi cant decrease in soybean yield and quality, when the crop is being exposed to long term higher concentration of ozone (Morgan et al., 2006). According to the effect of ozone few crops has been classi ed so far as sensitive (Wheat, water melon, pulses, cotton, turnip, tomato, onion, soybean and lettuce), moderately sensitive (Potato, sugar beet, potato, oilseed rape, tobacco, rice, grape, broccoli and maize) and tolerant (Barley, plum and strawberry) (Emberson et al., 2009). The crops that are being exposed to higher concentration of ozone shows some common symptoms on foliar i.e., ozone injury. Acute injuries are ecking and stippling, whereas the chronic injuries are bronzing, chlorosis and premature senescence. The estimated wheat loss due to ozone is 39.7%, 31.8% and 26.8% in Developed Countries, Upper Middle Income Countries and Territories and Lower Middle Income Countries and Territories respectively (Mills et al., 2018) In increasing radiative forcing, O 3 as a greenhouse gas modi es water and carbon exchange between ora and atmosphere in regionally as well as globally by affecting transpiration and photosynthesis of plants (Lombardozzi et al., 2012). The potential to destruct several ways of photosynthesis happens when O 3 enters the leaf via stomata, once the O 3 enters the leaves, it oxidizes the cellular membrane by altering mesophyll cells thus lowering Rubisco activity and carbon xation. This results in reducing chlorophyll content (Kulshrestha and Saxena, 2016). The regulation of photosynthesis and transpiration is modi ed by stomatal conductance. The direct way of affecting the plants is by changing guard cell turgor pressure and signaling pathways and indirectly by stomatal closure in case of rise in intercellular CO 2 concentration (Lombardozzi et al., 2013). In few studies with respect to chronic O 3 exposure there was the increase in stomatal conductance (Lombardozzi et al., 2013). Photosynthesis is directly proportional to chlorophyll content and net assimilation rate where these are the photosynthetic related parameters (Kobayakawa and Imai 2017). The decline of photosynthetic rates resulted in lowering of biomass in crops (Chen et al., 2018).
The increase in the concentration of ozone causes leaf injury, senescence, abscission, stomatal closure leading to reduction of photosynthesis, reduction of root growth, reduction of leaf growth, reduction of plant biomass and reduction of phloem translocation e ciency, which resulted in signi cant decreased in crop yield (Wilkinson et al., 2012). The ozone sensitivity varies across species due to species-speci c biochemical, physiological and morphological traits (Li et al., 2016), stomatal conductance and to the leaf antioxidant capacity (Brosche et al., 2010; Fares et al., 2013). Increase in the concentration of ozone suppressed the photosynthetic activity and stomatal conductance, which resulted in premature leaf fall, reduction in biomass content that showed changes in physiology and growth of crops (Tetteh et al., 2015). The net photosynthesis reduction in wood plants also occurred due to elevated levels of ozone concentration (Li et al., 2016). The negative effects of elevated tropospheric O 3 concentration include a decreased plant growth and alter the plant metabolism that reduced the crop yield signi cantly (Emberson et al., 2003). Reductions in stomatal conductance(gs), net photosynthetic CO 2 assimilation and carboxylation e ciency had all been associated with O 3 exposure (Morgan et al., 2003). A decline in the photosynthetic rate in O 3 exposed plants was associated with damage to the photosynthetic machinery that leads to reduced xation, and increased CO 2 concentration (Ci), resulting in reduced stomatal conductance (gs) (McKee et al., 2000).
The ozone protecting studies are also being carried out globally to reduce the ozone impact on agricultural crops and to improve crop yield. Ascorbic acid as a source of vitamin c plays an important role to reduce the ozone impact in plants such as radish (Madddison et al, 2002), Soyabean (Jiang et al., 2018), Snap bean (Burkey and Eason, 2002) and tobacco (Sanmartin et al., 2003). Application of ascorbic acid, neem oil and panchagavya were done for assessing their capability of reducing the ozone impact on garlic crop in this study. Thus the current study intended to study on physiological responses and quality of garlic to elevated different levels of ozone.

Status Of Trace Gas Level At Ooty
The study area, Ooty located in the District of Nilgiris, a fertile mountain range in Indian state of Tamil Nadu., represents a fresh, clean and pleasant environment with highly subdued human activity. Dense forests, lofty mountains, extensive tea and coffee plantation and sprawling grasslands characterize the location. The Nilgiris Hills form a part of a larger chain of mountains known as the Western Ghats along the western side of India, which is one among the eight hottest hotspots of biological diversity in the world. Due to anthropogenic activities the climate is changing drastically at Nilgiris District. Seasonal variation has been observed based on the meteorological data recorded. The maximum temperature in the summer has increased to 26 0 C (Normal 24 0 C) and winter temperature 4 0 C (Normal 2 0 C). Black

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 ow fans were xed 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.

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.

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 BioScienti c 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 signi cant differences between the means were determined with Duncan's multiple range test to assess the impact of tropospheric ozone on physiological response in garlic.

Pungency (pyruvate analysis)
To determine the content of naturally occurring pyruvate and carbonyl compounds, DNPH (2,4dinitrophenylhydrazine) 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 The chlorophyll content of garlic was not signi cantly differed initially up to four days after exposure and from fth day onwards the signi cant difference was observed in chlorophyll content of garlic to elevated ozone exposure (Fig. 3).

Photosynthetic rate:
The signi cant reduction was observed in garlic photosynthetic rate due to elevated ozone level from the rst day after exposure (Table 2.). The highest photosynthetic rate was observed in ambient Ozone level + foliar spray 3% Panchagavya (T 4 ) (4.68 µmol CO 2 s -1 m -2 ) followed by T 6  There was a signi cant difference among the treatments with respect to pungency content in garlic cloves due to elevated tropospheric ozone. Under the ambient condition the highest value was recorded in Ambient ozone level + 0.1% Ascorbic acid (74.29µmol g -1 ) followed by Ambient ozone level + 3% Neem oil (73.21µmol g -1 ) treatment, Ambient ozone level + 3% panchagavya (72.45µmol g -1 ) treatment and Ambient ozone level (59.73µmol g -1 ). As the elevated levels of tropospheric ozone causes ill effects to the crops, in garlic the treatment with Elevated ozone exposure at 200ppb (38.93µmol g -1 ) recorded the lowest value followed by the treatment Elevated ozone exposure at 150ppb (39.87µmol g -1 ).
The ozone protectants help in nullifying the effects of ozone on crop. In the garlic, the pungency in garlic bulbs after exposure with elevated levels of ozone with ozone protectants where the highest value recorded in the Elevated ozone exposure at 150ppb + 0.1% Ascorbic acid (59.19µmol g -1 ) followed by Elevated ozone exposure at 150ppb + 3% panchagavya (58.97µmol g -1 ), Elevated ozone exposure at 150ppb + 3% Neem oil (54.71µmol g -1 ). The lowest values are recorded in the treatment Elevated ozone exposure at 200ppb + 3% Neem oil (44.65µmol g -1 ) followed by the treatment Elevated ozone exposure at 200ppb + 3% Panchagavya (47.85µmol g -1 ) and Elevated ozone exposure at 200ppb + 0.1% Ascorbic acid (50.57µmol g -1 ).

Total Soluble Sugars
The total soluble sugars in the cloves of garlic showed signi cant difference among the treatments in view of elevated tropospheric ozone in different concentrations were shown in the Fig. 4. Among the treatments under normal conditions, the highest total soluble sugar content was recorded in Ambient Ozone level + foliar spray 3% Panchagavya (40.00°Brix) treatment followed by Ambient Ozone level + foliar spray 3% Neem oil (37.33°Brix) and Ambient Ozone level + foliar spray 0.1% Ascorbic acid (37.33°Brix) treatment and the least value been recorded in Ambient ozone level (35°Brix) treatment.
Since the elevated tropospheric level had negative effect on the quality of garlic cloves the lowest value was observed in Elevated ozone exposure at 200 ppb (28.00°Brix) treatment followed by Elevated ozone exposure at 150 ppb (29.67°Brix) treatment.

Discussion
The present study revealed that the physiology and quality of garlic was greatly affected by increased tropospheric ozone level. The chlorophyll content of garlic plant was signi cantly reduced by elevated ground level O 3 of 150 and 200 ppb (69.67% and 77.29%) compared to the present ozone level. The signi cant chlorophyll reduction will certainly affect the plant photosynthesis and reduce the garlic growth and yield (Gayathri et al., 2019). The chlorophyll reduction might be due to production of reactive oxidant species (ROS) in apoplast by encountering apoplastic antioxidant and killing the cell. Our results on line with Kumari et al., 2013 reported that the signi cant reduction was observed in total chlorophyll content by 55.9% and 50% reduction in total chlorophyll of soybean (Caregnato et al., 2013) under elevated ozone. Formation of superoxide radicles and hydrogen peroxide in leaf margin due to ozone exposure leads to trigger the injury signal by causing necrotic lesions and thus causes irreversible damage to the photosynthetic system that will lead to reduction in photosynthetic rate. In the same way, potato crop exposed under the elevated ozone resulted in chlorophyll content decline, more rapidly than the control, which triggered leaf senescence during crop growth (Donnelly et al., 2001). Similar studies on cauli ower showed that higher levels of ozone concentrations decreased chlorophyll content, which directly reduced the crop growth and yield (Sethupathi et al., 2018). The ozone will produce ROS within the cells and will evoke hypersensitive response (HR) phenomena and cause necrosis and death of cell in plants (Iriti and Faoro, 2008). The ozone induced ROS formation in cantaloupe plant reduced by the production of super oxide dismutase will scavenge the superoxide (Zhang et al., 2020). The enzymatic and non-enzymatic leaf antioxidants can't be able to maintain the intercellular redox homeostasis due to higher ROS production in the cell, which leads to oxidative damage of membrane lipids ( The experimental results showed that the increasing concentration of ozone reduced the stomatal conductance of leaf (79.07% in 150 ppb and 86.05% in 200 ppb) from the rst day after exposure. Though it is the immediate plant adaptive mechanism to escape from the ozone injury, but the reduced stomatal conductance has direct effect on carbon dioxide exchange and transpiration rate, thereby reducing the growth and yield of garlic plant. Our results in line with ndings of Noormets et al., 2010, reported that when the plants exposed to elevated ozone decreased the stomatal conductance, which reduced photosynthesis and internal CO 2 concentration. Under elevated ozone concentration, reduction in the stomatal conductance and leaf area index observed in soybean (Bernacchi et al., 2006). The longterm exposure of ozone leads to loss of stomatal control, thus decreases the stomatal conductance and carbon dioxide xation and increases the photorespiration and phosphoglycolate production (Skarby et al., 1987).
Since, the photosynthetic rate is directly in uenced by elevated ozone level, signi cant reduction of photosynthetic rate was observed in our experiment nearly 75.06% and 81.24% in 150 and 200 ppb, which is in line with results of Vandermeiren et al., 2005 in which they observed the decreased net photosynthesis in potato crop. In palak (Lettuce) crop the reduction in the capacity of photosynthetic rate due to the leaf area reduction was observed under elevated ozone level compared to ambient ozone level (Kumari et al., 2013). The reduction in photosynthetic rate and stomatal conductance were observed along with extensive visible leaf damage under ozone exposure (Sanmartin et al., 2003). Similarly, the greater accumulation of ROS in leaf is the result of reaction between ozone with symplastic and apoplastic cell components (Cho et al., 2011), caused an oxidative damage to photosynthetic membrane and death of photosynthetic mesophyll cells that takes place due to ozone effects which works through excess ROS production, so reduction in rate of photosynthesis and chlorophyll loss both occurs in sequence (Chen et al., 2005). Membrane integrity of the chloroplasts affected by ozone, initially but afterwards it affects mesophyll cells which affects photosynthesis in this whole process, so effect on mesophyll cells bring both reductions in chlorophyll content which consequently leads to reduction in photosynthesis rate (Tiwari et al., 2018). Excess ozone will affect the gas exchange (Black et al., 2007) which bring both effects of reduced photosynthesis and chlorophyll content. When the C 3 plants exposed to 100 ppb of ozone, decreased rate of photosynthesis and stomatal conductance and increased rate of respiration were observed Feng et al., 2008).
The quality parameter such as pungency ( were observed in soybean by Broberg et al., 2020 stating that the 200 kg/ha of seed protein was found to be decreased due to current ozone levels. The quality reduction is mainly due to more allocation of carbohydrates for maintenance and repair process by producing more secondary metabolites (Biswas et al., 2008), which reduced the availability of photosynthate for economic parts and suppressed the quality improvement in crops.
The production of ascorbic acid by plants is a defense mechanism to alleviate ROS and allied stress. Exogenous application of ascorbic acid stimulates growth and photosynthesis of wheat (Malik and Asraf, 2012 (Maddison et al., 2002). Similarly, Venkatesh and Park, 2014 stated that ascorbate plays a major role in cellular ROS scavenging activity and also in uence many stress responsive enzyme activities through synergetic action with the other antioxidant such as glutathione and α-tocopherol to protect cell from oxidative damage.

Conclusion
Hence the present study revealed that the elevated tropospheric ozone showed deleterious effects on the physiology and quality of garlic which would regulate the growth and yield. With the above observations we conclude that the elevated tropospheric ozone with increased concentration showed detrimental effects on physiological responses (chlorophyll content, photosynthetic rate and stomatal conductance) and quality parameters such as pungency and total soluble sugars on garlic crop, nevertheless ozone protectants such as panchagavya, neem oil and ascorbic acid stimulated the plant defense mechanism, which generated the counteracting mechanism in protecting the plant tissues against O 3 toxicity. Among the ozone protectants, neem oil performed well followed by ascorbic acid and panchagavya. Though the tropospheric ozone is short lived air pollutant, which have been identi ed as one of the potent greenhouse gas recently by Intergovernmental Panel on Climate Change (IPCC), the future research may be focused to study the tropospheric ozone impact studies on different food crops in detail and identify the better ozone protectants and its mode of action for nullifying the negative impact of tropospheric ozone to sustain agriculture production and food security for ever increasing population with less land and limited water resources under changing climate. Tables Table 1. In uence of tropospheric ozone on stomatal conductance of garlic In uence of tropospheric ozone on chlorophyll content of garlic Figure 4