ELICITATION OF PLANT DEFENSE AGAINST FUSARIUM OXYSPORUM F.SP. CICERIS IN CHICKPEA PLANT USING MARINE MICROCOCCUS SP

Chickpea is produced as well as consumed highest in India amongst the whole world but, production is seriously curtailed by Fusarium wilt, caused by Fusarium oxysporum f.sp. ciceris (FOC). In stressful conditions, the reactive oxygen species (ROS) production increases in plants which cause significant damage. To reduce this ROS, plants have inbuilt antioxidants that can detoxify ROS and protect cells of plants. The present study aimed to investigate the effect of Micrococcus luteus on the induction of defense enzymes in the presence and absence of Fusarium in chickpea. Talc-based bio-formulation of marine M. luteus was employed under pot trials to scrutinize mechanism of induced resistance against Fusarium by the improvement of defense enzymes like SOD, POX as well as accumulation of L-proline, total phenolic and pigments like chlorophyll and carotenoids. Pot trials were done using four different conditionsT1 (control), T2 (M. luteus treated), T3 (FOC treated), and T4 (M. luteus + FOC treated). Defense enzymes were studied and a maximum increase in M. luteus treated plants as compared to control and pathogen treated plants were noted. The chlorophyll and carotenoid content were improved in M. luteus treated plants to pathogen treated plants. This study indicated that M. luteus helps the plant to protect itself from free radicals as it showed significant induction of enhanced plant defense response against soil-borne pathogen to protect chickpea plant.


INTRODUCTION
Chickpea (Cicer arietinum L.) is one of the major legume crops widely cultivated for its edible seeds in the Indian sub-continent. Garbanzo beans are mainly used by humans and are an essential constituent of the Mediterranean diet. Seeds provide a decent and economical source of protein for essentially the vegetarian population, by choice or economic reasons, of developing countries especially in South Asia (Gaur et al., 2012). This plant has a significant role in agriculture but the overall crop productivity is sternly hampered by the incursion of soil-borne pathogens such as fungi, bacteria, and viruses. The first report of a soil-borne pathogenic fungus Fusarium oxysporum f.sp.ciceris (FOC) as a causative agent of Fusarium wilt in India and its correct etiology was determined by Butler in 1918and Padwik in 1940, respectively (Cunnington et al., 2007. Fusarium wilt considered a major exotic disease-causing wilting at any time from the seedling stage to podding. The fungus invades plant root through wounds, assault cortical region, and reach to the stele resulted in characteristic vascular wilt (Singha et al., 2011). Wilt in chickpea occurs in 32 countries across 6 continents (Singh et al., 2014). In response to the cross-talk between plant and pathogen, plants trigger a substantial array of defense mechanisms to ward off pathogens. A most decisive factor imparting successful warding of the pathogen is the swiftness of their defense response initiation which requires an apparent conception of the plant's ability to comprehend pathogen attack and control the expression of defense mechanisms. When plants are exposed to any biotic or abiotic stressful condition, the reactive oxygen species (ROS) production increases and it causes significant damage to the cellular components of plants. Plants have several inbuilt antioxidants that can detoxify ROS and inturn protect cellular components. Assorted defense-related genes chiefly encoding pathogenesisrelated (PR) proteins are present in plants, bestow resistance from pathogens attributed to their potential to ward off pathogens. Therefore, the resistance mechanism of the host can be scrutinized by measuring the alteration in defenserelated marker enzymes in response to external stimuli such as biotic and abiotic factors (Kavino et al., 2009).
The simulated response of plants is often complemented by the production of an array of host proteins with the ability to mount a defense against invader (Tahsili et al., 2014). Research on the elucidation of host defensive responses to pathogen invasion have determined catalase, superoxide dismutase (SOD), peroxidase (POX), polyphenol oxidase (PPO), phenylalanine ammonia-lyase (PAL) and "pathogenesis-related" (PR) proteins (Swarupa et al., 2014 andDehgahi et al., 2015). A multifaceted association of diverse signals regulates the plant's response for protection against pathogens (Datta and Lal, 2018). The number of chemical fungicides adopted to control fungal pathogens for a prolonged period, however, immoderate use of chemicals exert a detrimental effect on soil fertility and lead to a decrease in crop productivity. Moreover, the effectiveness of these fungicides remains for a stunted time during the croping season (Akram and Anjum, 2011). Plant growth-promoting bacteria were used to maintain the biogeochemical cycle of soil and helps to induce anti-oxidant property against reactive oxygen species (ROS) -used for detoxification against biotic and abiotic stress. The present investigation aimed to ascertain the effect of M. luteus on the induction of defense enzymes in the presence and the absence of Fusarium in chickpea plant. Yellow pigmented actinobacterial strain M. luteus was previously isolated from the marine environment and used as a talc-based bio-elicitor to induce plant defense under pot trials. Further, induced defense mechanisms were studied against Fusarium by enhancement of defense enzymes and markers like SOD, POX, L-proline, and total phenolic in chickpea. Chlorophyll and carotenoid content were also assessed in plants that were treated with M. luteus as compared to FOC treated plants.

Isolation of Marine bacteria
M. luteus strain (Accession No. JX679497) was isolated from the marine freshwater of Khambhat, Gujarat, India. M. luteus was inoculated on a nutrient agar plate and incubated for 37°C for 48 hours. The bacteria were analyzed by Chickpea is produced as well as consumed highest in India amongst the whole world but, production is seriously curtailed by Fusarium wilt, caused by Fusarium oxysporum f.sp. ciceris (FOC). In stressful conditions, the reactive oxygen species (ROS) production increases in plants which cause significant damage. To reduce this ROS, plants have inbuilt antioxidants that can detoxify ROS and protect cells of plants. The present study aimed to investigate the effect of Micrococcus luteus on the induction of defense enzymes in the presence and absence of Fusarium in chickpea. Talc-based bio-formulation of marine M. luteus was employed under pot trials to scrutinize mechanism of induced resistance against Fusarium by the improvement of defense enzymes like SOD, POX as well as accumulation of L-proline, total phenolic and pigments like chlorophyll and carotenoids. Pot trials were done using four different conditions-T1 (control), T2 (M. luteus treated), T3 (FOC treated), and T4 (M. luteus + FOC treated). Defense enzymes were studied and a maximum increase in M. luteus treated plants as compared to control and pathogen treated plants were noted. The chlorophyll and carotenoid content were improved in M. luteus treated plants to pathogen treated plants. This study indicated that M. luteus helps the plant to protect itself from free radicals as it showed significant induction of enhanced plant defense response against soil-borne pathogen to protect chickpea plant. morphology such as colony shape, size, opacity, elevation, and pigment production as portrayed in Bergy's manual of bacteriology Volume V (Goodfellow et al., 2012).

Preparation of talc-based bio-formulation
Adopting the method portrayed by Goswami et al., (2013), a talc-based bioformulation was prepared for M luteus strain. Briefly, 48 hrs grown culture was mixed with sterilized talc base. Talcum powder was taken and its pH was neutralized by 15 gm of CaCO3 per kg and 10 gm per kg of CMC was used as carbon source. The formulation was autoclaved followed by drying before further use. The chickpea seeds were surface coated with bioformulation by overnight soaking.

Treatment of Soil and Seed by talc-based formulation
Seeds were treated with 70% ethanol and HgCl2 for two and three minutes, respectively, under gentle shaking and finally washed with sterile distilled water to remove excess HgCl2 to achieve surface sterilization. The talc-based bioformulation was blended with sterile soil (0.5gm powder/kg soil), and seeds were surface coated with talc-based bio formulation (Table 1).

Seed application
Seeds coated with bio-formulation -1ml (10 6 cfu/ml)/ kg before sowing; the soil is treated with bioformulation during sowing.
Seeds coated with bio-formulation -1ml (10 6 cfu/ml)/ kg before sowing Watering As and when needed

Plant sample collection for biochemical analysis
Plants of four treatments and two applications were carefully uprooted. All the treated plants were pooled individually and rinsed under running tap water to strip off adhered soil and dust. Leaves and roots were homogenized in a respective buffer to perform biochemical analysis. Extracts were then subjected to centrifugation to get supernatant which was stored for further assay.

Estimation of Superoxide dismutase
Superoxide dismutase (SOD) catalyzes the dismutation of the oxygen-derived free radicals resulted from abiotic stress to plants. The activity of SOD was determined by following the method of Beauchamp and Fridovich (1971). The superoxide mediated reduction of nitro blue tetrazolium (NBT) was inhibited in the presence of SOD. The unit activity of enzyme represented by the amount of enzyme required to achieve 50% inhibition of NBT reduction measured at 560 nm. To derive specific activity (U.mg of protein -1 ), protein concentration present in each plant sample was determined using the Bradford assay procedure.

Estimation of Peroxidase
The assay was performed using pyrogallol as a substrate molecule. In the presence of peroxidase (POX) pyrogallol converts to purpurogallin, a brown compund. The color intensity was estimated at 420 nm, proportional to concentration of product formed, using a spectrophotometer (Shimadzu) (Kar and Mishra, 1976).

Estimation of Total phenolic compounds
Plant tissues were homogenized in methanol and the methanolic extract was separated and augmented with folin-ciocalteau reagent (1N) to form a blue color mixture. To determine the concentration, the color intensity was measured using a spectrophotometer at 725 nm against Gallic acid as a standard. The concentration of phenolic was expressed as µg catechol.gm -1 of fresh tissue (Zieslin and Ben-Zaken, 1993).

Estimation of L-Proline content
Adopting the method of Bates et al., (1973), L-proline content present in the leaves and roots was determined. The red-colored formazan formed upon mixing of L-proline and ninhydrin mixed under acid condition, solubilized in toluene, and read using a spectrophotometer at 520nm against toluene blank. To derive the concentration of L-proline, the standard curve of L-proline was prepared in the range of 5 to 100 μg.ml -1 concentrations.

Extraction of Chlorophyll and Carotenoids
Fresh plant leaves were homogenized in 80% acetone and centrifuged at 10000xg for 10 min. The supernatant was collected and analyzed for chlorophyll-a, chlorophyll-b, and carotenoids content using a spectrophotometer (Sumanta, 2014).

Statistical Analysis
To determine the effect of bioformulation treatment on the growth of plants in the presence and absence of a pathogen, the data collected after the germination of plantlets from all four treatments were analyzed statistically applying one way ANOVA.

Figure 1
The Structural morphology of M. luteus on a nutrient agar plate and gram reaction showed a coccoid structure.

Pot Study
M. luteus treatment showed a significant increase in growth more in seed+soil application than talc-based bioformulation applied directly to seeds. 19% growth observed in seed+Soil application and 3% growth in seed application while compared with pathogen treated and control plants after 20 days of sowing.

Estimation of SOD
The significantly induced activity of plant defense enzymes like SOD in the roots was observed in the plants which that were treated with M. luteus as compared to other treatments. SOD constitutes the first line of defense against ROS, it acts as antioxidant enzymes due to the dismutation of O2to H2O2. M. luteus treated plants showed maximum activity of SOD, 34.41% inhibition of mg protein -1 in leaves and 72.14% inhibition of mg protein -1 in roots in seed+ soil application (Figure 2A), while seed application showed 21.10% inhibition of mg of protein -1 in leaves and 67.57% inhibition of mg protein -1 in roots ( Figure 2B). Enhanced SOD activity in chickpea suggests that M. luteus helps the plant to suppress the stress as the function of SOD is to neutralize free radicals.  Figure 3A) whereas, 0.464 U.mg protein -1 of leaves and 0.666 U.mg protein -1 of roots in seed application ( Figure 3B). It suggests that M. luteus helps the plant to protect itself from the free radicals as well as cell wall strengthening.

Estimation of L-proline content
Accumulation of L-proline was observed in plants that were treated with M. luteus as compared to other treatments. M. luteus treated plants showed maximum accumulation, 0.962 µmoles.gm -1 of fresh tissue of leaves, and 0.23 µmoles.gm -1 of fresh tissue of roots in seed + soil application ( Figure 4A). While seed application showed 0.24 µmoles.gm -1 of fresh tissue in leaves and 0.42 µmoles.gm -1 of fresh tissue in roots ( Figure 4B).

Figure 4
Estimation of L-proline from chickpea plant after applying four treatments in (A) seed + soil application and (B) seed application.

Estimation of total phenolic compounds
There was a significant increase in total phenolic content in the plant roots which were treated with M. luteus as compared to pathogen treated plant in seed+soil application. The maximum total phenolic content in seed+ soil application was 0.058 mg catechol.gm -1 of fresh tissue in leaves and 0.08 mg catechol.gm -1 of fresh tissue in roots ( Figure 5A). While seed application showed 0.081 mg catechol.gm -1 of fresh tissue in leaves and 0.091 mg catechol.gm -1 of fresh tissue in roots ( Figure 5B). Increased phenolic activity by M. luteus signifies that it protect cellular components of plants in stress condition.
Table2 Extraction of chlorophyll and carotenoids from plants after seed + soil application and seed application.  VanLoon et al., 1998). Plants can accumulate different types of small compounds, often called secondary metabolites. It has been reported that plants harbor an inherent capability of co-ordinated defense network of biochemical reactions, which are generally inducible required appropriate stimuli (Jones and Dangl, 2006). The present investigation assessed the elicitation capability of a marine M. luteus as talc-based bioformulation to stimulate systemic resistance in chickpea plant by provoking defense enzymes like superoxide dismutase, peroxidase, induction of total phenolic, accumulation of L-proline and extraction of pigments like carotenoids and chlorophyll against Fusarium. The rationale for preparing talc-based bioformulation was the ability of CMC present in the formulation act as a binding agent and allowed bacterial cells to adhere to the surface of the seed coat. Besides, talc-based bioformulation enhances the viability of bacterial cells by providing the environment and in turn, improves the shelf life of the biofertilizer. SOD, a metalloenzyme, constitute the first line of defense against ROS. The superoxide radicals are dismutated into hydrogen peroxide (H2O2) and molecular oxygen (O2) by SOD and consequently bestow an important defense mechanism against the toxicity of superoxide radicals. In the present study, we have found enhanced SOD activity in plants treated with M. luteus, in turn, helps plants to fight in stress condition because SOD is an illustrious antioxidant enzyme accumulates during any type of stress in plants to counter oxidative stress. Besides upon challenge by pathogens, two enzymes involved in phenylpropanoid biosynthetic pathway, peroxidase, and polyphenol oxidase, enhances the lignification of the plant cell wall and triggered hypersensitive response. According to Ali et al. (2011), POX is the enzyme that neutralizes the free radicals generated due to stress in the plant. In this study, we found an increase in POX activity in M. luteus treated plants. Phenolic compounds are secondary metabolites that possess the antioxidant property and they are adequately present in plant tissues (Bartwal et al., 2013) Proline is generally accumulated under salt stress or drought stress and it may also get accumulated in some type of biotic stress too. Proline maintains structures of cells and buffering cell redox potential under stress conditions. There is a strong relationship between the accumulation of proline and stress experienced by the plants. More the stress experienced by plants greater the amount of proline accumulate. Hence, proline could be used as a stress marker. Proline exhibit three main functions, as a metal chelator, an antioxidative defense, and a signaling molecule. While experiencing the stress conditions by plant proportionally proline concentration increase. These over produced proline, in turn, imparts stress tolerance through maintaining turgor pressure by balancing osmotic pressure, stabilizing membrane resulted in prevention of electrolyte leakage, and help to reduce ROS concentration to normal range, which ultimately prevents oxidative burst in the plant (Hayat et al., 2012). In this study, we found a maximum accumulation

CONCLUSION
The present investigation reveals the role of a marine Micrococcus luteus as an elicitor which induces defense in chickpea plants which can aid in plant protection against a fungal pathogen Fusarium oxysporum f.sp. ciceris.