Hybrid Pigeonpea (ICPH 2740) Nodular Activity and Biological Nitrogen Fixation as Influenced by Agronomic Practices

Pigeonpea (Cajanus cajan L.) is the second most important pulse crop after chickpea in India. Pigeonpea is grown in an area of about 4.5 m ha with production of 3.3 MT in the country. The national productivity of pigeonpea is only 729 kg ha^-1 give comparison to world productivity (Directorate of Economics and Statistics, GOI 2022). In Telangana, it is cultivated in an area of 2.96 lakh ha with production of 1.91 lakh tones and productivity of 647 kg ha^-1 (Directorate of Economics and Statistics, GOI 2022).
       
In India more than 90 per cent of pigeonpea area is under rainfed conditions (Tiwari and Namrata, 2020). Now a day’s due monsoon aberrations farmers unable to sow the pigeonpea in correct window as a result the length of vegetative period reduced (Susithra et al., 2019) so, to avoid this the concept of transplanting is came into picture. But with normal conventional method of crop geometry like 150 × 30 cm leads to severe interplant competition, improper expression and higher flower drops due to low ventilation (Pradeep et al., 2018). Transplanting hybrid pigeonpea under square system the distance between rows and plants is same which facilitates good branching, pod and seed set when compared to varieties. Due to higher branching ability of hybrid under transplanting conditions, more yields were obtained compared to normal dibbling (Ramanjaneyulu et al., 2017; Mallikarjun et al., 2015).
       
Biological nitrogen fixation is a process of great environmental and economic importance, performed by Rhizobium that will reduce atmospheric nitrogen (N₂) to ammonia (NH₃) and when in symbiotic association with plants, they provide NH₃, which is readily convertible to other forms, such as amides and ureides Rufini et al., (2016). Seed treatment with rhizobium before sowing in pigeonpea is suitable alternative for increasing yields. ICPH 2740 is the first commercial pigeonpea hybrid released in Telangana state as Mannemkonda kandi in 2015 and yield potential of this hybrid is ranges from 2407-3652 kg ha^-1 (Saxenaet_al2016). Among the different agronomic practices, suitable planting method, planting geometry and optimum nutrition is essential for enhancing productivity in hybrid pigeonpea.

Experimental site
 
A field study entitled with “Hybrid pigeonpea (ICPH 2740) nodular activity and biological nitrogen fixation as influenced by Agronomic practices” was conducted at BW5 block ICRISAT development center (IDC). International Crops Research Institute for Semi-Arid Tropics (ICRISAT), Patancheru, Hyderabad, during kharif, 2021 and 2022. The rainfall received during kharif, 2021 and 2022 experimental periods was 998.4 mm in 55 rainy days and 1000 mm in 53.1 rainy days respectively. Experimental soil is moderately alkaline in reaction with pH of 8.1 and EC of 1.94 dSm^-1. The N, P₂O₅ and K₂O and organic carbon content of soil was, low (231.1 kg ha^-1), medium (29.8 kg ha^-1) and high (350.5 kg ha^-1) and low (0.42%), respectively.
 
Treatment and experimental design
 
The experiment was laid out in split-split plot design with two planting methods in main plots i.e., M₁: Dibbling, M₂: Transplanting, three crop geometries in sub-plots i.e., S₁: 100 cm × 100 cm (10,000 plants ha^-1), S₂: 120 cm × 120 cm (6,944 plants ha^-1), S₃: 150 cm × 60 cm(11,111 plants ha^-1) (Normal transplanting) and five nutrient management practices in sub-sub plots i.e., N₁: Control, N₂:100% soil test-based (STB) NPK (25:37.5:8.5 kg ha^-1), N₃: 100% STB NPK + vermicompost + PSB + Seed treatment (ST) with Rhizobium, N₄: 150 % STB NPK , N₅: 150% STB NPK + vermicompost + PSB + ST with Rhizobium. The pigeonpea hybrid used for this investigation was ICPH 2740. The seedlings for transplanting were raised in protrays and sown on 28^th May in 2021 kharif and 30^th May in 2022 kharif, respectively. Transplanting of 24 days and 25 days old seedlings were done on 20^th June in 2021 kharif and 23^th June in 2022 kharif, respectively, on the same day of dibbling in the main field. Vermicompost 5 t ha^-1, PSB (Phosphorus solubilizing bacteria 5 kg ha^-1 added to vermicompost one week prior to application and seed treatment with rhizobium 500 g ha^-1 seed 2 hours before sowing. Based on soil nutrient statusthe recommendation was made such that, if nutrient status is low, 25% more dose on recommended dose, if it is high, 25% less than RDF and recommended dose is based on medium nutrient status.
 
Data collection and analysis methods
 
The plant were uprooted carefully by digging around the plant by crowbar and the plants were washed under running tap in sieve and the nodules were counted and expressed in (No. plant^-1) at active nodulation stages i.e. 30, 45 and 60 DAS/DAT.Nitrogenase reduces acetylene (C₂H₂) to ethylene (C₂H₄) and the ethylene formed can be very sensitively detected by gas chromatography. In this method detached root with nodules enclosed in air tight glass jar (700 ml) and 10% gas inside replaced by pure acetylene. After 60 minutes incubation at room temperature, 0.5 ml of gas inside is sampled using a hypodermic syringe and concentration of ethylene gas is analysed by gas chromatography in acetylene reduction assay (ARA). The amount of ethylene formed will proportional to nitrogenase activity. ARA is expressed µmol C₂H₄ plant^-1 hr^-1. By using this nitrogenase activity we can calculate biological nitrogen fixation (kg ha^-1) by area under curve method (Hardy et al., 1968). 3 is the theoretical conversion factor of 3 moles of acetylene reduced/mole of dinitrogen reduced (peoples et al., 2009).
 
Statistical analysis
 
Data on nodule number and nodular activity were subjected to analysis of variance procedures as outlined for spit-split plot design (Gomez and Gomez, 1984). Statistically significance was tested by F-value at p=0.05 (5%) level of probability and critical difference was worked out where ever the effect was significant. Treatment differences that were non-significant were denoted as NS.

Number of root nodules plant^-1
 
The effect of different agronomic practices on nodule number was studied during both years of experimentation (Table 1) and significant variation was found. Among the planting methods at 30 DAS/DAT significantly higher number of root nodule plant^-1 were recorded with transplanting (16.4) compared dibbling (12.4). Whereas, in planting geometry higher number of nodules plant-1 were recorded with 120 × 120 cm (6944 plants ha^-1), which was statistically on par with 100 × 100 cm (10,000 plants ha^-1) at all the stages of crop and with respect to the nutrient management options, significantly higher number of root nodules were recorded with control at 30, 45 and 60 DAS/DAT and which was on par with 100 % STB NPK + 5 t ha^-1 + vermicompost enriched with PSB + Rhizobium seed treatment. These treatments followed by application of 150% soil test based NPK 5 t ha^-1 + vermicompost enriched with PSB + Rhizobium seed treatment and on par with 100% soil test based NPK. Similar trend was observed during both the years of experimentation. Lower nodule number was observed with 150 % soil test based NPK during both the years at all the stages. As pigeonpea is a leguminous crop it able to fix the atmospheric nitrogen. Wherever, nutrient demand is more, the fixation ability increases to meet the required demand. This could be the reason for the higher number of root nodules formation in control plot and also influenced by integrated nutrient approaches (Mishra and Prasad, 2010). During the both years of experimentation the interaction effect of planting method, plant geometry and nutrient management practices were found to be non-significant with respect to the number of root nodules plant^-1 at 30, 45 and 60 DAS/DAT. As pigeonpea is deep rooted legume it has ability to draw the fixed phosphorus from deeper layers of soil by releasing certain root exudates and acids (Piscidic acid) but, the nitrogen requirement should be meet my biological nitrogen fixation (Singh et al., 2016). So, the recommended dose of nitrogen is very less, the required nitrogen demand of crop should be met by biological nitrogen fixation (Tigga and Singh, 2019), but this nodulation activity again depends upon the many external and management factors (Kumar and Dart, 1987). For successful nodulation certain agronomic practices at field level i.e. addition of rhizobium inoculum through seed treatment may help in the formation of effective nodule Patil and Padmani (2007); Mishra and Prasad (2010) and Rufini et al., (2016).
 

 
Nodular activity (Nitrogenase enzyme activity µmol C₂H₄ plant^-1 hr^-1)
 
As pigeonpea is leguminous crop nodule function and biological nitrogen fixation depends upon the nitrogenase enzyme activity. Nitrogenase activity was studied through acetylene reduction assay in kharif 2021 and 2022 (Table 2) and found that, significantly higher nitrogenase activity was recorded with transplanting at 30 DAT (36.8 µmol C₂H₄ plant^-1 hr^-1) compared dibbling (23.9 µmol C₂H₄ plant^-1 hr^-1). In plant geometry, higher nodule nitrogenase activity plant^-1 was recorded with 120 × 120 cm (6944 plants ha^-1), which was statistically on par with 100 × 100 cm (10,000 plants ha^-1). Among the nutrient management practices, significantly higher nitrogenase activity was found with control, which was on par with integrated nutrient management approach (i.e. 100 % soil test based NPK + vermicompost 5 t ha^-1 + vermicompost enriched with PSB + Rhizobium seed treatment). Lowest nitrogenase activity was found with 150% STB NPK during both the years experimentation. From 30 to 60 DAS/DAT there increase in nodular activity in all the treatments but, from 60 to 75 DAS nitrogenase activity was decreased in all the treatments. Interaction effect of planting methods, plant geometry and nutrient management practices was found to be non-significant. The nodule number is indirect indicators of nitrogenase activity, but the enzyme nitrogenase activity which is actually involved in the biological nitrogen fixation (Bidlack et al., 2001) which is forerunner for meeting nitrogen requirement of the plant (Peoples et al., 2009; Barber et al., 1976).
 

 
Biological nitrogen fixation (kg ha^-1)
 
In transplanting due to early establishment and vigorous growth of seedlings, at 30 DAT more nitrogen was fixed biologically compared with dibbling (Table 3). With respect to the plant geometry, 150 × 60 cm (11,111plants ha^-1) which was found on par with 100 × 100 cm (10, 000 plants ha^-1).  Significantly lower nitrogen was fixed with 120 × 120 cm (6944 plants ha^-1) on hectare basis. Among the nutrient management practices, 100 % soil test based NPK + vermicompost 5 t ha^-1 + vermicompost enriched with PSB + Rhizobium seed treatment was recorded higher nitrogen fixation and was found on par with control.  Lower nitrogen fixation was found with 150% soil test based NPK during kharif 2021, 2022. The average nitrogen was fixed about 6.12 kg ha^-1 in transplanting, 5.73 kg ha^-1 with 150 × 60 cm pant geometry (11,111 plants ha^-1) and 7.32 and 6.44 kg ha^-1 with control and 100% soil test based NPK + vermicompost 5 t ha^-1 + vermicompost enriched with PSB + Rhizobium seed treatment respectively. The higher root proliferation with transplanting, wider plant geometry leads to elevated root exudates secretions (Mark et al., 2009; Sekhon et al., 2018) might increase the microbial population in rhizosphere and proportionate biological nitrogen fixation (Tilak et al., 2006 and Dhaka et al., 2020).
 

Hybrid pigeonpea performed well under transplanting, 100 × 100 cm plant geometry and 100% soil test based NPK + vermicompost 5 t ha-1 + vermicompost enriched with PSB + Rhizobium seed treatment among nutrient management practices in terms of number of root nodules, nitrogen fixation which results required nutrient demand of the plant can be met for the growth and development of the crop.

Authors are very thankful to the technical and non-technical staff of International Crops Research Institute for Semi-arid Tropics, Patancheru, Hyderabad. And also special thanks to Indian Institute of Oil seeds Research for providing bus facility for smooth running of experiment.

This article publication is approved by all authors and tacitly or explicitly by the responsible authorities where the work was carried out and that, if accepted, it will not be published elsewhere in the same form, in English or in any other language.