Comparative Effectiveness of Bio-inoculant and Enriched Compost on Lentil (Lens culnaris Medik.) Yield under Acid Soil

Lentil (Lens culnaris Medik.) yield and productivity are highly impacted (dipping by 71%) in acid soils by the reduced ability of symbiotic rhizobia (Rhizobium leguminosarum bv. viceae -pea cross inoculation group) to perform in low-pH conditions and its population (rhizobia) in the rhizosphere soil drop markedly when the soil pH goes below 6.0 (Jida and Assefa, 2014). The critical soil pH level for lentil growth is reported to be 5.7 and growth is severely reduced, stunted and necrotic leaves are formed at pH values below 3.7, 3.7 and 4.5, respectively; the highest numbers of nodules in lentil are formed above pH 5.5 and no nodulation occurs below pH values of 4 (Burns et al., 2017). So, despite being the nutritious food, the lentil crop is not popular among the growers in acidic soils and the reason is its difficulty in establishing under stress conditions and poor performance in terms of yield.
       
The cross inoculant group-specific Rhizobium biofertilizer is crucial for effective legume-Rhizobium interactions in acidic soils. This Rhizobium inoculant survivability in acidic soil conditions is the major drawback upon seed treatment alone, without providing a favourable environment for the growth and multiplication of Rhizobium in the rhizosphere region (Hartley et al., 2012). So, a strategy is framed to tackle these constraints of lentil production in the present research, which is by formulating the Rhizobium compatible enriched compost supplemented with cellulose degrading bacteria (CDB), phosphorus solubilizing bacteria (PSB) and fortified with rock phosphate (RP) to use along with the seed inoculation solely in the lentil crop and its cross inoculation group to increase the nodulation and nitrogen (N2) fixation and increase crop production as a whole keeping in mind the health of the soil too. The philosophy behind the addition of RP+PSB in the Rhizobium fortified compost is to support better phosphorus (P) nutrition in the crop rhizosphere for higher N2-fixation under phosphorus (P) deficient acid soil.

Enriched compost types
 
Five (5) types of enriched composts were prepared with the use of crop residues viz. rice straw, maize stalk and banana leaves mixed with the weed biomass viz. Eupatorium spp., Ambrosia spp. and broom grass following Berkley rapid composting method (Raabe, 2001) with slight modification according to the suitability for the region and availability of substrates. The efficient cellulose decomposer (CDB) was inoculated in all the compost pits. Phosphorus solubilizing bacteria (PSB) and R. leguminosarum (NR and ER) are inoculated in specific compost types. The compost types prepared were: (i) Enriched Compost 1 (EC1): Normal compost, (ii) Enriched Compost 2 (EC2): Rock phosphate (RP)+Phosphorus solubilizing bacteria (PSB) compost, (iii) Enriched Compost 3 (EC3): native Rhizobium (NR) compost, (iv) Enriched Compost 4 (EC4): RP+PSB+NR and (v) Enriched Compost 5 (EC5): RP+PSB+exotic Rhizobium (ER).
 
Rhizobium leguminosarum bv. viceae strains
 
Rhizobium leguminosarum bv. viceae CK1 strain, is the exotic strain (ER) used as reference was obtained from All India Network Project on Biofertilizers and Soil Biodiversity (AINP), Solan Centre, Dr. YSPUH and F, Solan, HP, India and it is suitable for acidic soil conditions. Rhizobium leguminosarum bv. viceae NR2 strain is the native strain isolated from acidic soils of North Eastern Hill region, India.
 
Preparation of liquid formulation for seed treatment
 
For preparation of liquid formulation for both native and exotic Rhizobium isolates (2 strains) YEMA (Yeast Extract Mannitol Agar) media (with composition of K₂HPO₄ 0.5 g, MgSO₄ 0.2 g, NaCl 0.1 g, Mannitol 10 g, Yeast extract 0.5 g and Agar agar 15 g and adjusted to pH 7.0, Vincent, 1970) was prepared where chemical amendments viz. polyninyl pyrollidone (PVP) as additives, carboxymethyl cellulose sodium salt as adjuvants, tween 20 as surfactants and ascorbic acid as an antioxidant were added. The liquid formulations were autoclaved and the Rhizobium strains were inoculated separately and incubated at 28±1^oC and from the 4^th day viable population count by serial dilution technique was carried out until the required population was achieved (108 cfu/ml). When the formulation was ready the required amount of seeds were washed with the tap water thoroughly and then 100 ml of liquid formulations were mixed with the 200 g of sterile compost and the seeds were mixed with the paste. Thereafter it was shade dried for 2 h and sowing was done.
 
Field experiment
 
The field experiment was conducted in acidic soils of ICAR-RC-NEHR farm, Umiam, Meghalaya located in 250 41' 18.36" latitude and 910 55'04.38" longitude and at the elevation of 1010m above mean sea level during rabi season of 2020-21. Nine (9) treatments in 3 blocks (replications) were laid out in RBD in the field experiment and lentil crop (variety-PL 8-140 days duration) was grown. Treatment combinations were: T1: 100% Recommended dose of fertilizer (RDF) @ 20:60:20 kg ha^-1 NPK, T2: 50% RDF @ 10:30:10 kg ha^-1 NPK, T3: Seed inoculation (SI) with native Rhizobium (NR2)+Enriched compost 1 (EC1)+50% RDF, T4: SI with exotic Rhizobium (ER)+ Enriched compost 1 (EC1)+50% RDF, T5: SI with NR2+Enriched compost 2 (EC2)+50% RDF, T6: SI with ER+Enriched compost 2 (EC2)+50% RDF, T7: SI with NR2+Enriched compost 4 (EC4)+50% RDF, T8: SI with ER+Enriched compost 5 (EC5)+50% RDF and T9: SI with NR2+Enriched compost 3 (EC3)+50% RDF. The layout of the field experiment is in RBD with 9 treatments and 3 replications. Enriched compost of 5 types was applied @ 2t ha^-1.
 
Plant growth and yield parameters
 
Plant growth parameters viz. plant height, leaf area, leaf area index, nodule number and weight were recorded following the standard procedure. The leaf area (LA) and leaf area index (LAI) in the lentil was determined by following the destructive gravimetric method (Gratani and Bombelli, 2000). Yield attributes viz. number of branches, number of pods, biomass yield and total yield were recorded.
 
Soil analysis
 
Soil samples were analyzed for pH (1:2.5 soil/water suspension) using a standard pH meter. Soil organic carbon (SOC) in air-dry soil sample was determined by following wet oxidation method described by Walkley and Black (1947). The alkaline permanganate oxidation method described by Subbiah and Asija (1956) was followed for the estimation of available nitrogen in soil (Avail. N). Available phosphorus (Avail. P) in soil was determined by method described by Bray and Kurtz, 1945. Available potassium (Avail. K) was determined in neutral 1N ammonium acetate by Hanway and Heidel (1952) method in flame photometer. DTPA extractable Zinc (Zn) was determined in an atomic absorption spectrophotometer (AAS) after shaking a soil in 0.005M DTPA (diethylene triamine penta acetic acid)+0.01M CaCl2+0.1M Triethanolamine (TEA) adjusted to pH 7.3 (Lindsay and Norvell, 1978).
       
Soil biochemical properties {dehydrogenase (DHA), acid-phosphomonoestrases (PHA) and urease activities} were determined at 60 days of plant growth. DHA {µg (TPF) g^-1 (dw) soil h^-1} in air dried soil samples was determined as per the method described by Casida et al., (1964). PHA {µg p-nitrophenol g^-1 (dw) soil h^-1} in fresh soil samples was determined as per the procedure described by Tabatabai and Bremner (1969). Urease activity {µg NH4 released g^-1soil h^-1} was determined by following the method described by Tabatabai and Bremner (1972).
 
Statistical analysis
 
All univariate analyses were performed using SPSS v21.0 (SPSS Inc., Chicago, IL, USA). Data generated from the field experiments were subjected to the statistical analyses of variance appropriate to the experimental design. Data were assessed by Duncan’s multiple range tests (Duncan, 1955) with a probability P≤0.05. Least significant difference (LSD) between means was calculated using the SPSS program.

Plant growth and yield parameters of lentils under field experiment
 
The addition of Rhizobium compatible enriched compost on lentil crops had a various positive effect in the present experiment under field conditions. Even though the experiment was conducted in the strong acidic soils with pH 4.11 at the initial crop growth the lentil plant performed well by attaining the medium height, good nodulations and yield as well as maintaining the soil fertility. The lentil crop attained a maximum height (Fig 1) of 9 cm (T6) tall in 30 days and a minimum of 4.47 cm height which was observed under the treatment T2 {50% RDF @ 10:30:10 kg ha^-1 NPK}.   


 
At 90 days the plant height of lentils was in the range of 20-27 cm and grows up to 30-33 cm height till 120 days, although a significant difference (P≤0.05) was not observed between the treatments in 90 and 120 days. Under field condition, the height of the lentil plants was taller compared to the pot experiment with seed inoculation alone (Sangma and Thakuria, 2020) but shorter than the average height attained in other parts of India (approximately 60 cm height, Kundu et al., 2017). A significantly higher number of branches (P£0.05) was observed under the treatment T6 (3.7 no.) followed by T8 treatment in 30 days (Table 1) of plant growth. At 90 and 120 days significantly higher branches were observed in the treatments T5, T6 and T7. The height of the lentil plant and the number of branches formed was reported to be affected by number of factors viz. the type of soil in the experiment, nutrient management system, Rhizobium inoculation and P nutrition to a greater extent as it is required for the cell division in the plants (Virk et al., 2024).


       
The effect of different treatments was visible on the number of nodules formed in the lentil crop (Table 2). The number of nodules form was found significantly higher in the treatment T5 {SI with NR2+Enriched compost 2 (EC2)+50% RDF} followed by the T7 {SI with NR2+Enriched compost 4 (EC4)+50% RDF} at 120 days of lentil crop growth. The fresh weight of the lentil was found maximum in the treatment T7 consecutively from 30 days to up to 120 days. The significantly higher nodulation in lentil, with the seed inoculation in combination with enriched compost and 50% of RDF, suggested that the adequate population of rhizobia was supplied to the crop through dual inoculation, which ultimately enhances the nodule formation. Similar results were obtained Singh et al., (2016). In the present experiment, the nodule formation was very low up to 60 days of crop growth and this delay in the nodule formation and the onset of N2 fixation even in the presence of adequate populations of rhizobia was many times due to the low temperature.


       
The leaf area (Table 3) of the lentil plant measured after every 30-day interval showed that after 30 days of plant growth, the significantly higher (P≤0.05) leaf area of 63.91 cm2 was observed in the T5 treatment followed by treatment T7. The leaf area in all the treatments was found to increase from 30 days to 90 days. The highest leaf area attained in the present experiment was 264.36 cm² in T7 treatment and a similar leaf area in lentil was observed by Veeresh (2003) with increased P nutrition in the soil. The leaf area index (LAI) also followed the same pattern as that of the leaf area. It was recorded highest in the treatment T5 in 30 days and in 90 days T7 showed the highest with 3.37. The leaf area (LA) and leaf area index (LAI) is affected by the nutrient management system and at optimum nutrient content, both LA and LAI attain the maximum value (Turuko and Mohammed, 2014). The inoculation of Rhizobium spp. specific to the crop was also reported to increase the photosynthetic rate of the plant by increasing the leaf area as well as the dry matter production in the crop (Thakur and Panwar, 1995).


       
The total dry biomass yield under the field experiment (Table 3) was found to range from 2.21 t ha^-1 to a maximum of 3.87 t ha^-1and found to be insignificant between the treatments (P≤0.05). The pod yield was found significantly higher in the treatments T5 (2.28 t ha^-1), T7 (2.26 t ha^-1) and T8 (2.23 t ha^-1) treatments. The maximum seed yield of 1.56 t ha^-1 was observed in the treatments T5 and T7 (1.56 t ha^-1) (Table 3). In the present experiment after observing the crop growth and yield parameters it can be concluded that the treatment T5 and T7 performed best in terms of nodulation and seed yield which explains that in acid soil the native isolates of Rhizobium play a very important role in the number of nodules formed and when it was combined with the enriched compost having native Rhizobium (NR2) in it nodulation is enhanced.
 
Soil nutrient availability under lentil crop in field experiment
 
Soil pH (Table 1) before crop and after the harvest of crop does not differ significantly under most of the treatments. In treatments T1, T3, T5, T8 and T2, soil pH was found to increase after the harvest of crop. SOC significantly increased (P≤0.05) under the treatments T7 and T3. Available nitrogen content significantly increased in all the treatments except T2 (174 kg ha^-1). Available zinc was also found to increase after the harvest of the crop and T8 (2.44 ppm) observed significantly higher content in Zn. Available potassium in the soil decreases in all the treatments after the harvest of the lentil crop. The increased in available N content after the harvest of the crop is due to the N2 fixation in field experiment under treatments with Rhizobium seed inoculation along with the Rhizobium compatible enriched compost application. The similar results were reported by Muhammad (2011). The lower available N in T2 treatment might have been caused by limited fertilizer application with no organic manures, uptake by plants, leaching and denitrification process. Significantly higher available phosphorus observed in T6 and T8. The observed high soil available phosphorus values in the present experiment after the harvest of the lentil crop explained that the phosphorus addition in the form of inorganic fertilizer and in enriched compost satisfied the phosphate demand by biological nitrogen fixation as well as for the yield of the lentil crop (Weria et al., 2013).
       
Soil enzymes play a vital role in nutrient mineralization and their activity is an excellent sensor in predicting the capacity of nutrient supply to plants (Akmal et al., 2012). Dehydrogenase (DHA) enzyme oxidizes soil organic matter by transferring protons and electrons from substrates to acceptors and is considered as an indicator of overall microbial activity because it occur intracellularly in all living microbial cells and is linked with microbial oxide-reduction processes (Stepniewska and Wolinska, 2005). The dehydrogenase activity (DHA) was observed (Fig 2) significantly higher (P≤0.05) in the treatments T7 {18.45 µg (TPF) g^-1 (dw) soil h^-1}. Enzyme phosphatases play an essential role in the cycling and availability of soil phosphorus in soil and they occur either extracellularly or within the living cell and their sources are the soil microbial community as well as plant roots and residues. The phosphatase activity (PHA) was observed significantly higher in the treatment T7 {79.94 µg p-nitrophenol g^-1 (dw) soil h^-1}. For all the enzyme activities the lower values were observed before the lentil crop was grown. Juma and Tabatabai (1978) reported that acid phosphatase (PHA) is predominant in acid soils and that alkaline phosphatase is predominant in alkaline soils. In present experiment also high amount of acid phosphatases were observed in almost all the treatments under study. This might as well be due to the application of higher doses of P fertilizers as well as the high content of P in the enriched compost. Enzyme urease is an extracellular enzyme involved in the hydrolysis of urea-type substrates and its activity is important in the transformation of urea fertilizer. Urease activity was recorded significantly higher in the treatments T3 (224 µg NH4 released g^-1 soil hr^-1) and T8 (213.5 µg NH4 released g^-1 soil hr^-1). Both DHA and urease enzymes were higher in treatments receiving seed inoculation, enriched compost and inorganic fertilizers treated plots and Akmal et al., (2012) also reported the similar results and stated that organic matter and N and P fertilizers often has the direct effect on the activity of various enzymes.

In acid soils the excess amount of H+, Al3+ and deficiency of P affects the crop by limiting the root growth and in turn nutrient absorption and ultimately, affecting severely the crop productions. So, resource manipulations and remedial measures play a very important role in these regions for effective crop productivity. The addition of Rhizobium compatible enriched compost on lentil crop along with the seed inoculation had a various positive effect in the present experiment under field conditions. Soil pH was found to increase in treatments viz. T1, T3, T5, T8 and T2. SOC significantly increased (P£0.05) under the treatments T7 and T3 after the harvest of the lentil crop. Available nitrogen and available phosphorus content significantly increased in almost all the treatments after the harvest of the crop. The experiment even though was conducted in the strongly acidic soils with pH 4.11, the initial crop growth of the lentil plant performed well by attaining a medium height, good nodulations and yield as well as maintaining the soil fertility.

The authors are thankful for the financial and the laboratory facilities provided by the College of Post Graduate Studies in Agricultural Sciences, CAU, Umiam and ICAR Research Complex for NEH Region, Umiam, Meghalaya, India.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.