Identification of Suitable Cobalt Application Method and Optimum Level for Enhancing of Chickpea Yield

In human beings at least 22 mineral elements are required through a balanced diet (Thacher et al., 2006). Currently, two billion people throughout the world are coming across micronutrient deficiency (Velu et al., 2014). Micronutrients are essential nutrients and to be supplied in very small quantity to crops. For the requirement of these micronutrients, plants depend on soil or micronutrients supplied to soil in the form of impurities through fertilizer and pesticides. Recently, micronutrient deficiencies are diagnosed more frequently. To achieve good crop yield, it is necessary to recognize and correct the deficiencies of micronutrients.
       
Cobalt is one of the beneficial element required in legume and non-legume plants for various physiological actions. Cobalt is an integral constituent of vitamin B₁₂ and essential in little amount for humans and other mammals (Mansour, 2016). Cobalt promotes cell elongation and auxin metabolism, activates the enzymes dehydrogenases and nitrate reductase, it increases chlorophyll content (Yagodin and Romanova, 1982). Cobalt deficiency occurs when dry hays cobalt content is <0.07 mg kg^-1 and animals suffer from cobalt deficiency (McDowell et al., 1983). Cobalt is reported as an essential elements for plants in lower concentration (Sinha et al., 2012). Low concentration of cobalt  is essential for nitrogen fixation in legume plants in the presence of root nodule bacterias (Witte et al., 2002) and it is also beneficial  for non-leguminous plants in trace amounts (Locke et al., 2000).
       
Anthropogenic activities and other sources have introduced heavy metals in biological system and causing health hazards. When cobalt is applied in higher doses to plants, it arrest plant growth and produce chlorosis in young plants (Adriano, 2001). Cobalt in high amount induces the formation of reactive oxygen species, when combined with H₂O₂ in cell free system, it also decreases the fidelity of DNA synthesis in some mammalian cells and prokaryotes, as cobalt is a heavy metal and responsible for the formation of harmful free radicals and thus buildup oxidative stress (Grant and Loake, 2000). Hence it can be said that cobalt is essential element for number of crops and animals in small quantity. Also it is found phytotoxic when higher dose is applied to plants. Hence in both cases i.e. deficient and excess, it is problematic. Identification of right method and quantity of cobalt application really turning out to be a challenge. If right method and quantity is identified, cobalt will no longer create the problem of shortage and excess. Agronomically cobalt can be applied through four methods namely seed priming, seed treatment, soil application and foliar application. In this experiment all four methods were employed with three levels of each to food legume chickpea to identify right method for and right dose of cobalt application.

The field experiment was conducted during rabi season of 2015-16 at Navsari Agricultural University, Navsari, Gujarat, India. The pre-planting composite soil samples were collected before commencement of experiment. The soil texture of the experimental site was clayey in nature with pH of 7.8 and electrical conductivity of 0.36 dS m^-1. The soil was low in elemental nitrogen (N) (188.16 kg ha^-1), medium in elemental phosphorus (P) (38.33 kg ha^-1), high in elemental potassium (K) (327.74 kg ha^-1) and elemental Co content was 0.32 mg kg^-1 (Jackson, 1973) i.e. cobalt content was found deficient in soil. The climate of the experimental site was typically tropical, characterized by humid, diurnal and warm monsoon with heavy rainfall, quite cold winter and fairly hot summer. The experiment was laid down in randomised block design with fourteen treatments with three replications. The experimental treatments onsisted of four methods of cobalt application with three levels of each i.e. seed priming of CoCl₂ at 0.5, 1 and 1.5 ppm (3.2 mg CoCl₂ dissolved in one liter of water), seed treatment at 1, 2 and 3 g kg^-1 seed, soil application at 50, 100 and 150 g ha^-1 and foliar application at 0.01, 0.025 and 0.05% twice at 30 DAS and just before flowering. Two control treatments, one absolute control and one was seed priming with water was also included in the experiment. Chickpea (GG-2) was seeded at 30 × 10 cm with RDF of 20:40:00 of N, P₂O₅ and K₂O kg ha^-1. On farm seed priming was done by dissolving CoCl₂ in water for two hours and air dried in shadow thereafter, seed treatmentwas given by mixing CoCl₂in viscous jaggery solution, soil application of CoCl₂ done through fertigation at 15 DAS at three leaf stage and foliar application at 30 DAS and just before flowering was done by dissolving CoCl₂ with water. Chlorophyll content was calculated by hand held SPAD meter, legheamoglobin content at flowering was determined by Appleby and Bergrsen, (1980) method. The experiment was conducted in randomized block design. The treatment effect was worked out on the basis of least significant difference (LSD) at 5% probability level (Gomez and Gomez, 1983).

Chlorophyll content
 
Perusal of chlorophyll content data (Table 1) at 30, 60 and 90 DAS showed that, soil application of cobalt at 50 g ha^-1 shown highest chlorophyll content 63.08, 85.90 and 46.63 during all three growth phases at 30, 60 and 90 DAS, respectively and it was followed by foliar application of Co  at 0.01%. Growing phase wise highest chlorophyll content recorded through hand held SPAD meter at 60 DAS, which is a peak flowering period in chickpea and it was decreased further at 90 DAS in all methods of cobalt application, which is a pod filling period and all photosynthates of legumes are generally diverted to pod filling, hence chlorophyll content might be decreased further after flowering up to crop maturity time. The percentage increase of chlorophyll content registered by soil application of Co at 50 g ha^-1 over absolute control is 57.75%. The most important compound for photosynthate production is chlorophyll (Mishra and Srivastava, 1983). Among all treatments, foliar application of cobalt at 0.05% has shown lowest chlorophyll content (40.19, 52.51 and 27.57), hence foliar application at 0.05% shown phytotoxicity to chickpea and the toxicity may have completely or partially stopped the process of nitrogen fixation. Hence through this experiment it can be concluded that seed priming of cobalt at at 0.5, 1 and 1.5 ppm were safe as it hasn’t shown any phytotoxicity symptoms and also 1 ppm concentration of cobalt has shown at par chlorophyll content with highest chlorophyll content treatment i.e. soil application of cobalt at 50 g ha^-1.
 

 
Nodule studies
 
Among each three level of all cobalt application methods, the least level of each method i.e. seed treatment  at 1 g kg^-1 seed (80.50, 2.95, 0.37 and 0.48), soil application at 50 g ha^-1(99.12, 3.38, 0.55 and 0.51) and foliar application at 0.01% (95.00, 3.02, 0.41 and 0.48), except seed priming at 1 ppm (78.50, 2.87, 0.29 and 0.42) of cobalt application resulted in a maximum elevation of all four parameters i.e. nodule count at flowering, nodule fresh and dry weight (g) and legheamoglobin content (mM), respectively (Table 2). Combination of CoCl₂ + beta amino butaric acid was treated at 3 mM to Brassica napus seeds and it is found that the combination of above treatment has increased root length over absolute control but did not shown its effect on shoot length (Rajaei and Mohamadi, 2013). Groundnut varieties M-522 and SG-84 were tested through foliar application with ethrel at 200 µg ml^-1 and CoCl₂ at 15 µg ml^-1 at 25 DAS i.e. vegetative stage and at 45 DAS. Cobalt may be regulating sugar metabolism as sugar works as an energy source during flowering development. Gynophores plant^-1 in M-522 increased through cobalt chloride till 65 DAS and reduced thereafter due to conversion it into pods.  (Kaur et al., 2011). Results of the present study indicates that nodule parameters and leghaemoglobin content increased significantly in all four cobalt application methods when little dose of cobalt was applied through seed priming (1 ppm), seed treatment (1 g kg^-1 seed), soil application (50 g ha^-1) and foliar application (0.01%). Cobalt allows cynocobalamine synthesis which is required for Lb synthesis. Hence nodule initiation/inhibition could be ascribed to deficiency of cobalt and recommended at little cobalt salts while fertilizing legume crops (Younis, 2011). Among all cobalt application methods foliar application of cobalt at 0.05% was found to be most toxic and it has severely affected the nodule parameters. Legheamoglobin synthesis is impaired directly under cobalt deficiency; it ultimately affects plants N fixing capacity. As nitrogenase enzyme gets protection of legheamoglobin from its exposure to oxygen supply (Hopkin, 1995). This experiment also confirms the same i.e. the least dose of cobalt through cobalt application methods namely priming, seed treatment, soil and foliar application shown conducive environment for nodulation and legheamoglobin synthesis and which finally increased chickpea seed yield.
 

 
Days to flowering and maturity
 
The exposure of chickpea plants to lowest concentration of cobalt by seed treatment (1 g kg^-1 seed), soil application (50 g ha^-1) and foliar application (0.01%) methods has took more days to flower and mature and  significantly delayed 50% flowering and maturity. As cobalt concentration increased the flowering and maturity period of the crop narrowed down (Table 3). Method wise order of most early flowering and maturity was observed in following levels, foliar application of cobalt at 0.05% (50.44 and 95.71), soil application at 150 g ha^-1 (53.22 and 99.08), seed treatment at 3 g kg^-1 seed (53.89 and 98.64) and seed priming at 0.5 ppm (55.67 and 98.28), it means these treatments attained early senescence and crop maturity. Ethylene synthesis is inhibited by cobalt chloride and this delays the chickpea flowers senescence and giving sufficient time for grain filling to chickpea at lower doses. Cobalt chloride expressed a dual role of increasing uptake of water and simultaneously increased vase life of flowers due to curtailing senescence of rose as a cut flower (Aslmoshtaghi et al., 2014). Rod et al., 2019 used soil application method for cobalt application through the sources CoSO₄ and CoCl₂ and they have noticed that soil application of CoCl₂ has increased days to 50% flowering and maturity by 8.33 and 17.22 days, respectively over CoSO₄.
 

 
Seed and stover yield
 
Application of cobalt through seed treatment (26.98 and 42.08 q ha^-1), soil application (27.80 and 42.59 q ha^-1) and foliar application (27.58 and 42.31 q ha^-1) methods has recorded highest seed and stover yield of chickpea at their first level itself, however it has shown a linearly decreasing trend from second to third level (Table 4). But seed priming of cobalt has shown an exceptional trend among all four cobalt application methods and gradually increased chickpea seed and stoveryield parameters from 0.5 (24.33 and 38.03 q ha^-1) to 1 ppm (26.67 and 41.23 q ha^-1), however lowest chickpea seed yield was exhibited by foliar application of cobalt at 0.05%. Faba bean was given foliar spray twice with  0.24 and 0.48 g L^-1 cobalt and seed priming once with 0.07 and 0.14 g kg^-1 seeds and the results showed that foliar spray of cobalt has significantly increased chickpea seed yield when cobalt spray level was increased from 0.24 and 0.48 g L^-1, however in case of seed priming chickpea seed yield was decreased when seed priming was doubled from 0.07 and 0.14 g kg^-1 seeds, hence foliar spray is found to be more efficient than seed priming (Attia et al., 2016).
 

       
Among all cobalt application methods studied through this experiment, application of cobalt through soil application at 50 g ha^-1 has exhibited significantly higher chickpea seed index (30.80), seed yield (27.80 q ha^-1), stover yield (42.59 q ha^-1) and harvest index (39.49) and which were found at par with all three levels of seed priming and first two levels of seed treatment, soil application and foliar spray of cobalt. Photosynthesis is susceptible to heavy metal toxicity at pigment synthesis and it alters chloroplast membrane and photosystems due to enzyme inhibition and degradation of stomatal functioning (Mysliva et al., 2004). The enzymes nitrate reductase and nitrite reductase may be inhibited due to oxidative stress of heavy metal like cobalt. Hence, photosynthesis is affected due higher dose of cobalt through seed treatment, soil application and foliar spray at second and third level and at third level of seed priming, leads to reduction of chickpea yield linearly with increase in cobalt dose.When required amount of cobalt is applied to leguminous crops they can synthesize sufficient vitamin B₁₂, that can ultimately synthesize adequate Lb. (Dilworth and Bisseling 1979). Looking to the heavy metal nature of cobalt, comparatively soil application of 50 g Co ha^-1 and seed priming of cobalt at 1 ppm has registered highest chickpea seed yield by spending least amount of cobalt chloride.
 
Corelation study of cobalt application methods
 
Perusal of the data for correlation studies (Table 5.1, 5.2, 5.3 and 5.4), it is revealed that among four methods of application of CoCl2, the two methods, namely soil application followed by seed treatment has registered significant and highly significant positive correlation with the highest number of parameters. Chlorophyll content in leaf showed its positive correlation with number of important chickpea parameters in all cobalt application method except seed yield of chickpea in seed priming. Hence cobalt helped to synthesize legheamoglobin, this further facilitated to fix nitrogen and nitrogen helped to synthesized surplus chlorophyll, this was favorable for growth, seed yield and protein yield by all four cobalt application methods. Also chlorophyll and cobalt were correlated with each other only with the method soil application of cobalt. It means nitrogen fixed by chickpea helped to synthesize good amount of chlorophyll, especially in soil application of cobalt. In all cobalt application methods, nodule count was correlated with legheamoglobin except in foliar application of cobalt, it means nodule formed in foliar application method have not effectively synthesized legheamoglobin and   hence this may be the reason that third level of foliar application method has registered lowest seed yield among all methods, even it was lower than control. Legheamoglobin has shown a strong and very strong correlation with important parameters in all methods except it was not correlated with seed yield in seed priming, but it was correlated with seed index, this may be the reason that second level of priming and first level of other three methods shown a at par seed yield of chickpea with each other. Seed yield was correlated with chlorophyll, nodule count and legheamoglin in all methods, except seed priming of cobalt, hence comparatively lesser amount of legheamoglobin content was recorded in the root nodules of seeds primed with cobalt and was luxuriant in first levels of all other remaining levels. Hence looking to the correlation studies, the preference of cobalt application method to chickpea can be in the following order soil application > seed treatment > seed priming > foliar application. 
 
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All methods of cobalt application and their different levels significantly influenced growth and yield of chickpea. Cobalt application through seed priming at 1.5 ppm, seed treatment at 2 and 3 g CoCl₂ kg^-1 seed, soil application of cobalt at 100 and 150 g CoCl₂ ha^-1 and foliar application of cobalt at 0.025 and 0.05% adversely affected chlorophyll content, nodule count, nodule fresh weight, nodule dry weight, legheamoglobin content in root nodules, days to 50% flowering and crops maturity, grain and stover yield and total uptake of chickpea. In general the third level of cobalt application through seed treatment, soil application and foliar application method was more damaging in comparison to second level. Method wise the least levels of cobalt i.e.  seed priming cobalt at 1 ppm, seed treatment at 1 g CoCl₂ kg^-1 seed, soil application of cobalt at 50 g CoCl₂ ha^-1 and foliar application of cobalt 0.01% shown significantly higher chickpea seed and stover yield and as these levels were increased chickpea seed yield linearly decreased thereafter. Among all cobalt application method and their levels soil application of cobalt at 50 g CoCl₂ ha^-1 has executed highest chickpea seed and stover yield and it was at par with all three levels of seed priming, first two levels of seed treatment and foliar application and  second level of soil application.