Effect of Rhizobium Inoculation and Tillage Practices on Fodder Cowpea (Vigna unguiculata) 

The agriculture and its allied sectors contributes to 16.1% of GDP and sustain livelihood security of 70 percent of rural households (Anonymous, 2019) and important role in achieving the goal of doubling farmers’ income. Livestock sector has been growing at an annual growth rate of 7.9 per cent during last five years and contribution of livestock is more than the crops sector. At present, India has largest animal population (536 million) in the world and hold first position in milk production (187.7 MT) but still productivity is very low. Due to the  increasing demands for food grains and cash crops, the area under fodder crops has been static for last 3-4 decades (8.4 m ha) resulted into a net deficit in dry and green fodder of about 10 and 35%, respectively making livestock rearing more challenging (Mallikarjun et al., 2018).
       
Cowpea (Vigna unguiculata) is one of the most important fodder crop in India due to its unique ability to drought tolerant, short duration, quick growing habits, higher protein content (16-18%) and good tonnage of fodder yield along with higher palatability particularly to ruminants (Mallikarjun et al., 2018). Its dual-purpose trait makes it a very attractive and being an indeterminate in growth habit also, it is highly shade tolerant and therefore, compatible as an inter-crop with maize, millets, sorghum and sugarcane. It has tap root system with a number of lateral roots occur in upper layer of soil coupled with these characters, its quick growth and rapid ground cover prevents soils erosion and root decay in-situ produces nitrogen rich residues and different nutrients that improve soil fertility and structure (Tamta et al., 2019).
       
Conventional agriculture has largely been characterized by intensive/conventional tillage which caused soil degradation through hasten mineralization of soil organic matter (SOM) and thus its loss over time besides negative impacts on soil physical and biological activity (Buchi et al., 2015; Ram et al., 2018). To mitigate these negative effects, resource conservation technologies (RCTs) likes zero tillage, bed planting and laser land leveling was tested and adopted to save substantial quantity of irrigation water, reducing the cost of cultivation in terms of land preparation, timely sowing, improved water and nutrient use efficiency, improves soil biological activity, increase porosity, produces stable soil aggregates, low organic matter oxidation and left indirect effect on mitigating the adverse effect of climate changes (Jat et al., 2014; Ram et al., 2018). Various researchers have revealed that negative, little or no difference in yields of different crops under ZT compared with CT (Krishna and Veettil 2014; Pittelkow et al., 2015).
       
Nitrogen (N) is an essential and vital plant nutrient and a major component of protein, enzymes, amino acids, nucleic acids, flavins, purines and pyramidine nucleotides, co-enzymes and alkaloids and plays a vital role in cell division and elongation by virtue of being an essential part of diverse type of metabolically active compound (Mallikarjun et al., 2019). Biological N₂ fixation (BNF) can make plants self-sustaining for N nutrition and avoiding the need for mineral N fertilization besides BNF is largest natural source them to sustain natural systems (Iannetta et al., 2016). However, BNF are energetically more costly to the host plant than N uptake from substrate (Ryle et al., 1984) and if sufficient quantity of fertilizer N is available; inhibit BNF as well as nodule establishment and development and nitrogenase activity (Eaglesham, 1989). Keeping these facts in view, the present study was undertaken to enhancing fodder availability by adoption of modern tillage practices and efficient N management.

The experiment was conducted during 2017-18 at Agronomy Research Farm, ICAR-National Dairy ResearchInstitute, Karnal, Haryana, located at 29°41' N latitudeand 76°58' E longitude with an altitude of 245 m above mean sea level in Trans Indo-Gangetic Plain of India. The experiment was  laid  out in the split  plot design having  tillage  practices such as zero tillage (ZT), conventional tillage (CT) and raised bed (RB) in main plots and six N management viz., N0, N75, N75+Rhizo, N100, N100+Rhizo and N125% in subplots. The soil of the experimental field was clay loam texture having pH 7.30, electrical conductivity 0.35 dS/m, medium in Walkley-Black organic carbon (0.63%), low in KMnO₄Oxidizable nitrogen (188.48 kg/ha), medium in 0.5 M NaHCO₃-extractable phosphorus (23.56 kg/ha) and 1N NH₄OAC-extractable potassium (271.12 kg/ha).
       
The recommended dose of fertilizer for fodder cowpea applied was 20:60:40 kg NPK/ha. Full dose of N, P and K was applied in the form of urea, SSP and MOP, respectively as basal application. As per treatments seeds of fodder cowpea were treated with rhizobium culture by manually using jaggary solution. A packet of 200 g biofertiliser used for treatment of 10 kg seeds in 5% solution of jaggary and a little quantity of water taken for the purpose of moistening the seed. The prepared slurry coated uniformly over the seeds and inoculated seeds are dried in shade and sown immediately.
       
The crop was harvested at 60 days after sowing and weighed for green fodder yield. The observations on plant height, number of branches/plant, leaf width and length and root length and nodulation count were recorded manually on fiverandomly selected representative plants from each plot as well as yield was recorded as per the standard method. Random chopped samples of green fodder were sun dried and placed in the oven at 65-70°C for 72 hours to estimate dry matter percentage. The plant samples were collected at harvest, oven dried (70°C), processed and analyzed for N content (Kjeldahl, method, described by Jackson, 1973). The N uptake was calculated by multiplying concentration with their respective yield. The nitrogen gain or losses was calculated by following formula:
   
Statistical analysis was done using analysis of variance in split plot design (Gomez and Gomez, 1984).

Effect of tillage and nitrogen management on root length and nodules per plant
 
Tillage practices had significantly influenced on root length of fodder cowpea (Table 1). Among tillage practices, significantly higher root length at 30 DAS was recorded in ZT over, RB and CT. While at harvesting, highest root length was recorded in ZT over, CT however, at harvesting ZT is statistically at par with RB. Higher root length under ZT may be due to better soil physical environment, aeration, better water regimes, lesser soil compaction, carbon sequestration, soil organic carbon and soil enzymatic activities were relatively more under ZT than CT. Similar results were also reported by Kayombo et al., (1991) and Dixit et al., (2019).
 

       
The nitrogen management options had significant effect on root length and nodules per plant through out the growth stages. It was observed that, at initial stage of plant growth (30 DAS) lowest root length was recorded in control. However, root length increased up to application of 75%N+rhizobium inoculation over rest of treatments but 75%N+ Rhizobium is statistically on par with 100%N and 100% N+rhizobium. At harvesting significantly higher root length was observed in 75%N+ rhizobium over 75%N and control. The root length was increased due to application of lower dose of chemical nitrogen (75%N)+rhizobium inoculation increased the biosynthesis of growth regulator such as auxin, cytokinin and GA which stimulate rhizosphere microbes, improved nutrient uptake and boost up biological nitrogen fixation resulted higher root length was recorded under lower level of N application these findings was supported by Jnawali et al., (2015) and Bullock et al., (1992).
       
Chemical N application had significant effect on root nodulation in cowpea. It was reported in present findings that negative effect on root nodules formation with successive increase the chemical N alone from zero to 125%. However, N application with inoculation of rhizobia has significantly increase number of root nodules up to 75% N+rhizobium application. The negative effect of chemical N application on root nodulation is due to inhibitory effect on symbiotic nitrogen fixation. Nodulation and nitrogen fixation by legumes is adversely affected by higher doses of fertilizer N. Fertilizer N especially nitrate, affects nodulation mainly by destroying indole acetic acid, reducing lectin production by the host, decreased root hair formation and root hair curling, limiting the attachment of rhizobia on root hair, decrease in lectin binding site; decrease in infection thread formation, inhibits initial cell division in the root cortex and integrity of bacteroids in the nodules due to the decreased supply of photosynthate to nodules, the loss of O₂ binding ability of leghemoglobin and there by inhibit the process of nitrogen fixation Dogra and Dudeja (1993) and Upadhyay and Singh (2016).
 
Effect of tillage and nitrogen management on growth attributes and fodder yield
 
The tillage practices had significant effect (ZT and RB) on higher plant height, number of leaves, number of primary branches and fodder yield (Table 2) over CT but ZT was statistically on par with RB. The increasing growth attributes resulted in increased green fodder yield in RB to the tune of 2.70 and 12.28% over ZT and CT, respectively. The higher growth and yield of cowpea due the compound effects of better nutrients, improved soil physical health, better water regimes, besides improved soil quality such as organic carbon, porosity, soil enzymatic activity, microbial biomass and structural stability under ZT and RB compared to CT (Singh et al., 2011; So et al., 2009; Ram et al., 2017) contradictory results also reported by Aikins and Afuakwa (2010). The nitrogen management options had significant effect on plant height, no of leaves, leaf width, leaf length, no of branches and yield (Table 2) of cowpea. The growth attributes and yield has increased with the increasing levels of nitrogen application up to 75% N with rhizobia inoculationover control and 75% N alone. However, successive increase N application had also increase no of leaves and leaf width up to 100% N with rhizobia application as a result increase the green fodder yield of cowpea up to 75% N with rhizobia application. The successive increase of nitrogen level and rhizobium inoculation increased the cell division, formation of co enzyme and enzymes resulted in cell elongation and chlorophyll content, which accelerated meristematic activity of plant cells that lead increase in internodes length and luxuriant vegetative growth besides better root development, high photosynthetic rate, better acceleration of carbohydrates and higher dry matter accumulation with amplify nitrogen levels. Similarly findings were also reported by Jnawali et al., (2015), Kumar et al., (2015) and Upadhyay and Singh (2016).
 

 
Effect of tillage practices and nitrogen management on soil health and available nutrients
 
Soil pH, EC and available nutrients (N, P and K) did not differ significantly between treatments (Table 3). A marginal decline in the pH was observed in all the treatments except RB comparison to initial pH (7.30). However, marginal increase in EC value as compared to initial EC (0.35) across the treatments. Similarly, marginal increase in SOC value in ZT condition. The initial available N, P and K status in soil was 188.45, 23.56 and 271.12 kg ha^-1. After 2 years, available N in soil was significantly increased in ZT and decline in CT and RB treatments in comparison to their initial value. However, the available P and K was almost similar value in all the treatments. The soil has buffering capacity to resist changes in physical properties like pH, EC and SOC. Similar findings also reported by Neugschwandtner et al., (2014). Higher dose of N application resulted in slightly decreased in pH was due to the soil acidification was greater with application of more than 120 kg N/ha then with no application (Lungu and Dynoodt 2008). However, reverse trend was observed in EC which increase slightly from 0.35 to 0.40 might be due to salt accumulation with application of chemical fertilizer. The slightly increase in SOC may be due to; the soil tilling breaking of soil aggregates and lower porosity in CT which resulted in higher losses of SOC and nutrients, due to increases organic matter decomposition and decreases carbon content by increasing organic matter oxidation, (Thomas et al., 2007) and Parihar et al., (2016). The biofertiliser application enriching the nutrient pools in addition it stimulates rhizosphere microbe’s activity, ultimately enhance available nutrients Bullock (1992) and Jnawali et al., (2015).
 

 
Effect of tillage practices and nitrogen management on nitrogen budgeting
 
The N budgeting was effected by tillage and nitrogen management practices (Table 4), higher actual N gain (2.49 kg/ha) over initial soil available nitrogen wasrecorded in ZT but actual loss was higher in CT (-15.61 kg/ha) and RB (-10.84 kg/ha). In CT have low SOC content due to higher soil aeration  under CT, destruction of soil macro aggregate to expose inaccessible organic matter, tillage increase soil CO₂ efflux due to aggregate distraction and biological mineralization of SOC and SOM (Kristensen et al., 2000; Aziz et al., 2015) besides increase the temperature of tilled soil due to organic material mixing resulted in to mineralization of crop biomass and rapid decomposition of buried  organic substances in tilled soils (Alvarez et al., 2001) resulted in to low available N under CT.
 

       
In ZT conditions more suitable environment due to better soil biological, chemical and physical conditions. ZT results  in the deposition of crop residues on upper soil surface may probably decompose at a slower rate, which reduce  moisture and energy exchange from soil to atmosphere resulted in to increase the moisture content and decreasesoil  temperature and aeration, thus allowing plant organic residues to stay intact for longer period of time and thereby facilitating more C accumulation and higher microbial  population with greater number of fungi and various microbes and earthworms under NT (Holland  and  Coleman, 1987; Dou and Hons, 2006; Aziz et al., 2015). Moreover, surface placement of crop residues may accelerates humification of organic matter due to greater atmospheric exposure and interactions, which facilitate greater microbial decomposition and thus favor C accumulation under NT over time (Aziz et al., 2015). Nitrogen application has also effect on nitrogen budgeting, higher actual gain was recorded in 100% N+rhizobia application (7.72 kg/ha) and 125%N (1.78 kg/ha) over initial N content (Table 4). The highest apparent gain was recorded in control might be due to the rhizosphere microbes utilized the maximum potential for regaining nutrient status (N) with supported from external and internal sources. Similar findings were also reported by Sharma and Banik (2012).

It can be concluded that zero tillage is better option in terms of crop productivity and soil health compared to conventional tillage besides play effective role in enhancing soil fertility and may decrease soil carbon losses. The application of rhizobia inoculation in fodder cowpea may substantial reduces the fertilizer nitrogen by 25% as compared to un-inoculates.