Nutrient removal by rice–wheat cropping system as influenced by crop establishment techniques and fertilization options in conjunction with microbial inoculation

Nutrient uptake by the rice–wheat cropping system (RWCS) is an important indicator of soil fertility and plant nutrient status. The hypothesis of this investigation was that the rate and sources of nutrient application can differentially influence nutrient removal and soil nutrient status in different crop establishment techniques (CETs). Cropping system yield was on par in all the CETs evaluated, however, there were significant changes in soil nutrient availability and microbiological aspects. The system nitrogen (N), phosphorus (P), potassium (K) and zinc (Zn) uptake in aerobic rice system followed by zero tillage wheat (ARS-ZTW) was 15.7–17.6 kg ha−1, 0.7–0.9 kg ha−1, 7–9.8 kg ha−1 and 13.5–23.1 g ha−1 and higher than other CETs. The formulations of Anabaena sp. (CR1) + Providencia sp. (PR3) consortium (MC1) and Anabaena–Pseudomonas biofilm (MC2) recorded significantly higher values of soil chlorophyll and microbial biomass carbon and positively affected cropping system nutrient uptake and soil nutrient balance, illustrating the beneficial effect of microbial inoculation through increased supply of biologically fixed N and solubilised P. Zinc fertilization (5 kg Zn ha−1 through ZnSO4·7H2O as soil application) increased soil DTPA-extractable Zn by 4.025–4.836 g ha–1, with enhancement to the tune of 20–24% after two cropping cycles of RWCS. Our investigation recommends the need for change in the present CETs to ARS–ZTW, along with the use of microbial inoculation as a means of significantly enhancing cropping system nutrient uptake and soil nutrient status improvement.

In the present day scenario, changes in investigations on crop establishment techniques (CETs) and management practices in rice and wheat are getting more emphasis [1][2][3] . This is mainly because of variations in CETs with respect to their resource utilization 4 , energy requirements 5,6 , capacity to act as a mitigation strategy for climate change 7 can have far reaching implications in terms of yield and income to the farmers 8,9 , besides environmental health. Additionally, the adoption of new CETs and management practices are becoming increasingly significant to address the issues related to degradation of natural resources and increasing cost of chemical and agronomic interventions or resources.
Among the different CETs of rice, the system of rice intensification (SRI) is one which was invented in Madagaskar Island by the Father Hendra De Laolani in 1983 and promoted by researchers 10,11 over different part of the world. The SRI is getting momentum despite of different opinion about its superiority in term of yield 12,13 . In some places, only one or few components/recommendations of SRI are followed (modified SRI) which was found to be economically suitable 14 . Its adaptation is mostly promoted for its higher water productivity even though grain yield may remain same as that of conventional puddled transplanted rice 15 . The superiority of SRI in yield potential was not consistent over different part of the world and report of higher, lower as well as similar yield over conventional transplanted rice are accumulated and reviewed by several authors [16][17][18] . Another CET Scientific Reports | (2020) 10:21944 | https://doi.org/10.1038/s41598-020-78729-w www.nature.com/scientificreports/ uptake by 28.3 to 33.0 kg ha −1 . Zinc fertilization increased the cropping system N uptake by 34.5, 6.3, 33.9 and 36.0 kg ha −1 when applied along with RDN, 75% RDN, 75% RDN + MC1 + Zn and 75% RDN + MC2 + Zn, respectively in the first year and similar results were also recorded in the 2nd year. This showed that, application of Zn with 75% RDN + MC2 was better than the application with RDN. The increase in cropping system N uptake due to application of RDN was 38.3-39.7 kg ha −1 and 89.7-94.5 kg ha −1 over 75% RDN and control, respectively. In case of P, application of 75% RDN with MC1 or MC2 + Zn in ARS-ZTW had 5.4-6.2% and 6.5-6.9% higher P uptake than same treatment applied in PTR-CDW and SRI-SWI (Table 3). Similarly, for K, this increase was 21.9-26.5 and 25.4-29.1 kg ha −1 even though K application rate was remained same in all CETs and nutrient management treatments ( Table 4). The increase in P uptake due to application of RDN + Zn in ARS-ZTW was 0.8-1.0 and 0.7-1.0 kg ha −1 over the same treatment applied in PTR-CDW and SRI-SWI, respectively; while for K it was 6.8-7.8 and 9.4-12 kg ha −1 . The overall effect of this treatment superiority was reflected in significantly higher P and K uptake in ARS-ZTW.
Cropping system related Zn and Fe uptake. Among the treatment variables analysed, the highest enhancement in Zn uptake was recorded with rate of N and P application followed by microbial inoculation ( Table 5). The increase in Zn uptake in RWCS due to rate of N and P application, microbial inoculation, Zn fertilization and CETs were 101.4-282.7, 88.3-95.5, 76.8-79.3 and 18.3-23.1 g ha −1 , respectively. In case of Fe uptake, the rate of N and P application (457.8-1350.6 g ha −1 ) led to the highest contribution and CETs had the lowest contribution (42-47.5 g ha −1 ) for enhancing Fe uptake ( Table 6). The highest Fe uptake was found in PTR-CDW (5602.8 g ha −1 ) which was statistically at par SRI-SWI; while the values of uptake in ARS-ZTW (5559.8 g ha −1 ) remained inferior to other CETs.
Available soil status of NaHCO 3 -extractable P, 1 N ammonium acetate-extractable K and DTPA-extractable Zn after two cycles of RWCS. The soil P content (NaHCO 3 -extractable) at the end of two cropping cycles of RWCS was higher in RDN, 75% RDN + MC1 and 75% RDN + MC2 with and without Zn application and lower in the treatment 75% RDN with and without Zn and control (Table 7). An increase of 3-9 kg ha −1 was recorded, with the highest increase observed with application of 75% RDN + MC1 and the lowest with RDN + Zn. Application of RDN had 6.3-11.3 kg ha −1 higher available soil P compared to 75% RDN. Among CETs, both PTR-CDW and SRI-SWI had significantly higher available soil P after two cropping cycles, than ARS-ZTW and the increase in PTR-CDW and SRI-SWI was 1.84-3.12 and 1.75-2.64 kg ha −1 compared with ARS-ZTW.    (Table 8). The rates of N and P application had the highest effect on the soil available K content while Zn fertilization had the lowest effect; however the order of influence was of N and P application > microbial inoculation > CETs > Zn fertilization. The soil available K after first year cropping cycle decreased by 131-147 kg ha −1 ; while after second year, it decreased by 27.6-42.7 kg ha −1 over initial available soil K.
The soil Zn content (DTPA-extractable) was influenced by Zn fertilization, rate of N and P uptake, microbial inoculation and CETs with highest effect by Zn fertilization and the lowest with microbial inoculation ( Table 9). The increase in available soil Zn due to Zn fertilization ranged from 4284.2 to 5361.7 g ha −1 , with all the three CETs showing an increase in available soil Zn. The variation in Zn content among CETs was 191 and 649.9 g ha −1 in first and second year, respectively. The greatest increase in available soil Zn was found with application of 75% RDN + Zn (4950.2 g ha −1 ) applied in PTR-CDW and lowest increase in RDN + Zn (4024.4 g ha −1 ) applied in ARS-ZTW after two cycle of RWCS.

Soil chlorophyll and microbial biomass carbon (MBC).
All three CETs in rice differed significantly in soil chlorophyll and MBC with significantly higher values of both microbial parameters in SRI during the first year ( Fig. 1a,b). During the second year, SRI and PTR remained on par with each other and were significantly superior over ARS. Application of MC2 with 75% RDN led to significantly higher soil chlorophyll and MBC, as compared to the treatment 75% RDN + MC1 in first year and both microbial consortia remained on par in the second year. In wheat, ZTW was found significantly superior in first year and remained on par with other CETs in the second year. Application of 75% RDN + MC2 recorded the highest values of soil chlorophyll and MBC. All the four treatments receiving microbial inoculant had significantly higher soil chlorophyll and MBC than rest of the treatments in both rice and wheat. Both soil chlorophyll and MBC were positively correlated with biological yields (Figs. 2 and 3).
Partial factor productivity (PFP) and agronomic use efficiency (AUE). The cropping system PFP for N and P was found significantly higher in ARS-ZTW in both years of study over other CETs (Table 10). The increase in PFP of N in ARS-ZTW was 0.2-0.4 kg grain kg −1 N applied; while same for P was 0.8-2.1 kg grain kg −1 P applied over other CETs. The PTR-CDW had significantly higher AUE with increase of 1.53-1.55 and 7.1-7.2 kg grain increased kg −1 nutrient applied, respectively for N and P over other CETs. Among the nutrient management treatments, PFP was significantly higher in 75% RDN + MC (MC1 or MC2) + Zn than its counterpart without Zn application. All treatments with MC application had significantly higher PFP over RDN  Cropping system nutrient balance. Out of the total available P present in soil (soil initial available P + P applied through fertilizer) 36.9-40.8% was accounted by plant uptake and 7.6-8.7% contributed to NaHCO 3 -extractable soil P; while 50.4-55.3% was not extracted by NaHCO 3 (Table 11). This 50.4-55.3% P (not extracted by NaHCO 3 ) may be present in soil in fixed form or part of it might have been lost from the soil, due to leaching. The fixed P needs to be reutilized to make P fertilization in RWCS economical and application of microbial consortia is a suitable option. The difference between calculated and actual balance was 13.2-35.5 kg ha −1 and 24.3-32.0 kg ha −1 , respectively in the first and second year in treatments with application of RDN and higher values recorded in calculated balance in both years.
In the case of potassium, both calculated and actual balance was negative over initial soil available K in both years (Table 12). The total cropping system K uptake across different CETs in first year varied between 223 and 230.8 kg ha −1 , which was higher than K applied in RWCS with calculated and actual negative balance of 124-131 kg ha −1 and 131-148 kg ha −1 , over initial soil available K respectively. During the second year, total cropping system K uptake, calculated negative balance and actual negative balance were 216-225, 116.13 and 26-42 kg ha −1 , respectively. Out of the total available Zn present in soil (soil initial available Zn + Zn applied through fertilizer) across different CETs, only 11.6-18.5% were taken up by plants (Table 13); while 54.2-90.5% contributed to increase in soil DTPA-extractable Zn content of soil. The actual available Zn balance after completion of two cropping cycles of RWCS was lower by 1484-1927 g ha −1 than calculated balance.

Discussion
Biological yield of cropping system. The variation in biological yield at individual crop level was negligible which led to also the same at cropping system level. In case of rice, weed infestation problem and also the problem of seedling establishment due to high temperature and lower number of fertile tillers in ARS made it significantly inferior over PTR and SRI; while residual effect (especially of nutrients) of previous season ARS, better plant stand establishment and higher values of yield attributes made ZTW superior over other CETs of wheat. Among nutrient management options, the inherent soil nutrient status, nutrient application rate, yield enhancement due to microbial consortia and plant nutrient acquisition capacity influences the significance of applied treatments. This is clear from yield enhancement recorded in terms of cropping system in treatment with application of RDN by 1.43-1.24 Mg ha −1 and 3.35-3.62 Mg ha −1 over 75% RDN and control, respectively. The  www.nature.com/scientificreports/ positive effect of applying RDN and Zn fertilization on crop yield was also earlier reported by Shivay et al. 40 and Singh et al. 43 . The contributions of both types of microbial inoculation to cropping system yield was highest in ARS-ZTW (1.14 and 1.19 Mg ha −1 ) than the other CET systems in the first year; while during the second year, both microbial inoculants performed better in PTR-CDW with increase in cropping system yield by1.03 and 1.16 Mg ha −1 , respectively due to application of MC1 and MC2. One of the reasons for this difference was variation in rainfall and other weather parameters across years. The total rainfall received during rice growing season in first year was 1349 mm; while during the second year, it was 451.4 mm. This higher rainfall leads to higher water level in rice field, which may have favoured and positively influenced the survival and nutrient release/ acquisition capacity of the applied inoculants. This is validated from the higher values of microbial biomass carbon and soil chlorophyll during the first year ( Fig. 1a,b).
Cropping system N, P and K uptake. The cropping system N uptake in 75% RDN + MC1 + Zn and 75% RDN + MC2 + Zn was on par with application of 100% RDN, illustrating the significant role of microbial inoculation in nitrogen nutrition in RWCS. The superiority of RDN over 75% RDN signifies the role of optimal and balanced fertilization. The increased level of biological nitrogen fixation due to presence of optimum population of Anabaena sp., low available soil N and suboptimal N addition (75% of recommended) through fertilization are the important reasons for increasing N uptake in inoculated treatments. The nitrogen fixation in cyanobacteria (Anabaena sp.) takes place in specialized heterocyst cells. These cells create microanaerobic environment to form proper functioning of nitrogenase enzyme (enzyme involved in biological nitrogen fixation). The significance of microbial inoculation in increasing to nitrogen uptake in rice 44 and wheat 45 as well as contribution of microbial inoculation to growth and yielding ability of rice 46 and wheat 47 was reported. In present study, increase in N uptake in ARS-ZTW was same as that of PTR-CDW and SRI-SWI showing that, MC1 and MC2 also found better for application in ARS-ZTW (aerobic condition). The phosphorus is second most important nutrient after N; while need and significance of potassium nutrition in RWCS was also reported 48 . The conversion of applied water soluble P from fertilizer to the form which was not available to the plant and its fixation in insoluble form are the area where applied microbial inoculation work. The production of organic acids and lowering soil pH due to organic acid as the mechanisms by which soil fixed P was made available for plant growth by microbial cultures. The role of microbes in P solubilisation and mobilization was reported by Alori et al. 49 . This ultimately leads to improvement in crop growth, yielding ability and P uptake 50 as observed in our study. The P uptake was found higher in 75% RDN + MC1 or MC2 over 75% RDN even though rate of P application was remained same in these treatments. This was due to better nutrient acquisition capacity of well grown plant than nutrient stressed plant. In case of K, adoption of ZTW www.nature.com/scientificreports/ may increase the opportunity for incorporation and retention of straw 3 thereby reduces the problem of delay in sowing and burning of residue.
Cropping system Zn and Fe uptake. Both Zn and Fe uptake of rice and wheat are important considering their role in plant and human/animal health 51 . In present investigation, nutrient application rate of N and P had the highest contribution to increase in Zn uptake. This was due to their higher contribution to biological yield than rest factors. The soil Zn application rate was 5 kg ha −1 which is very high than cropping system Zn uptake; hence there will be sufficient Zn available for the plant uptake. In such conditions, the uptake capacity of plant (dry matter production) will decide the Zn uptake. The application of primary nutrient such as N and P has higher contribution to dry matter production; hence uptake of Zn in our study was mainly decided N and P application rate; even though concentration of Zn in rice and wheat in both years of study was found influenced mainly by Zn fertilization (data of concentration was not shown). This showed that dry matter production played major role in deciding Zn uptake than concentrations in present study. Another factors governing variation in Zn and Fe uptake is CETs. The variation in Zn uptake across studied CETs is due to change in hydrological regime 52,53 , variation in growth vigour and yield 15,27,54 , soil inherent nutrient availability and variation in conversion of applied Zn in different forms.

Soil available P (NaHCO 3 -extractable), K (NH 4 OAC-extractable) and
Zn (DTPA-extractable) status. The variation in soil NaHCO 3 -extractable P status in present study was contributed by higher uptake in ARS-ZTW, increase in soil available P (occluded P) under puddled condition in PTR and SRI, contribution of microbial consortia, rate of P application (100% and 75% RDN) and less vigorous growth of wheat in CDW and SWI leading lower P acquisition. The variation in available soil P status across CETs was also reported by Pradhan et al. 55 . The increase in soil available P with application of microbial inoculation 56 was significantly higher than RDN application even though uptake was remained on par. This showed that, microbial inoculations is sustainable strategy and have capacity to increase the contribution of fertilizer applied P and soil inherent but unavailable P to plant P uptake. At the same time, only 75% of RDN was applied with microbial inoculation and leads to higher nutrient use efficiency of P fertilizer 57 as yield level was same as that observed with RDN.
In rice and wheat 89-91% and 85-89% of K remained is straw; therefore, its recycling is possible either through residue retention or residue incorporation. The lower response of rice and wheat to K application 58 due to higher NH 4 OAC-extractable K content in trans and upper Indo-Gangetic plain 48 and higher subsidy on N and P than K are the possible reasons for lower K application in RWCS. This ultimately leads to imbalanced fertilization. This imbalance can be seen from nutrient application ratio (4.9:2.2:1; N:P 2 O 5 :K 2 O in rice and 11.7:4.9:1;  30 . In such condition, zero tillage wheat with residue incorporation and/or retention will be best option which helps in nutrient cycling and also ensuring timely sowing. The application of 10 kg Zn ha −1 through ZnSO 4 ·7H 2 O in single cropping cycle of RWCS increases the soil DTPA-extractable Zn along with increasing Zn uptake 59 . This increased DTPA-extractable Zn going to pay to next crops in succession. One of the possible reasons for increasing soil Zn content was the difference between Zn uptake in RWCS and amount applied. In present study, the uptake of Zn varied between 691 and 764 g ha −1 (6-9% of the total Zn applied in one cropping cycle of RWCS) in single cycle of RWCS. This showed that, 9200-9300 g Zn ha −1 remained unutilized; while increase in soil DTPA-extractable Zn content was 3100-3700 g ha −1 (42-54% of the total Zn applied in one cycle of RWCS). The remaining quantities either get fixed in the form which is not extracted by DTPA or form chemical compounds with other elements observed in soil 60 .
Soil microbial properties. The soil microbial properties respond quickly and significantly to change in tillage 61 , crop establishment techniques 29,62 , fertilization 63 and external inoculation of microbial culture 64 . In the present study, microbial consortia involving cyanobacteria for nitrogen fixation and bacteria for P solubilization were used. Their growth and activity in soil, illustrates their promise in competing with the inherent soil microbial population and responding to CETs and fertilization. The use of microbial consortia or microbial biofilms has immense significance as the synergy among the partners helps in efficient proliferation and functioning under different temperature-light regimes or environmental fluctuations, including flooding or dry conditions as they can grow attached to soil particles or plant roots or flood water [65][66][67] . Cyanobacteria are thoroughly investigated for their role as nitrogen-fixers, plant growth promoters and their benefits to neighbouring microflora and fauna, thereby, contributing to improved plant health and soil fertility. Bacteria such as Providencia, or Pseudomonas employ a variety of solubilization reactions, such as acidification, chelation, exchange reactions, and production of gluconic acid, to release soluble from insoluble P. Cyanobacterium-based combinations as consortia with Providencia, known for its nutrient-enriching potential in rice wheat cropping system 65,67,68 , and as biofilm with Pseudomonas sp. which has shown promise for its P mobilising and plant-promoting traits, is also well documented 66,67 . The superiority of SRI and PTR over ARS was might be due to better growth condition (puddled soil and continuous saturated condition of soil) for the applied microbial consortia than that of ARS; while in wheat, higher organic matter from stubbles of previous season rice crop and better soil physical conditions (no soil puddling in ARS) can be the important reasons for higher values of soil chlorophyll and MBC in ZTW. Significant improvement in the biological properties illustrates the potential of the applied microbes in mobilising nutrients and enhancing their uptake by plants. www.nature.com/scientificreports/ Partial factor productivity (PFP) and agronomic use efficiency (AUE) of N and P. The higher PFP in ARS-ZTW was due to higher cropping system yield with the same level of N and P applied. In case of AUE, the superiority of PTR-CDW can be due to lower yield in control plot, than in control plot of SRI-SWI and ARS-ZTW. This indicates higher availability of soil nutrients and overall contribution in SRI-SWI and ARS-ZTW systems. Among nutrient management options, AUE and PFP of N and P were significantly affected by the rate of nutrient application, microbial consortia and Zn fertilization. The significantly higher PFP and AUE with application of microbial consortia was due to lower rate of application over RDN, higher yield over 75% RDN and contribution of biological nitrogen fixation and P solubilisation to N and P uptake. The contribution of microbial consortia to increase in PFP of N and P was 2.1-3.0 and 9.8-13.9 kg grain kg −1 nutrient applied and same for AUE was 2.1-2.9 and 9.8-13.9 kg grain increased kg −1 nutrient applied. This improvement in PFP and AUE by microbial consortia without yield penalty is highly important as rice and wheat together consume 52.4% N and 48.4% of P out of total consumption in India 30 .

Nutrient balance.
Analyses of the balance of P, K and Zn showed that the application of recommended rate of P and Zn had a positive effect on their available soil status; while application of K at recommended rate is not sufficient for RWCS. The order of significance of applied treatments in increasing soil P after completion of two cropping cycle of RWCS over initial available soil P was-application of microbial consortia > rate of N and P application > CETs with respective contribution of 8.8-9.3, 5.7 and 0.2-3.3 kg ha −1 , respectively. This order of significance showed the important contributions of microbial consortia and their application towards efficient P nutrition of RWCS. The order of significance of applied treatments on K uptake and soil available K status was contradictory. The results showed that the variation in soil available K status was mainly governed by plant nutrient uptake. The application of Zn, with 75% RDN showed the highest increase in soil available Zn; while uptake was highest with RDN + Zn. The actual Zn balance after completion of two cropping cycles of RWCS was lower than calculated balance signifying the possible conversion of applied Zn into forms, not extracted by DTPA i.e. unavailable pool of Zn.
Our study showed that the uptake of nitrogen, phosphorus, potassium, zinc and iron in terms of cropping system was significantly influenced by CETs, microbial inoculation, zinc fertilization and rate of N and P fertilization in both the years of study. Among these factors, the rate of N and P application brought about the maximum effect, while effect of CETs was minimal for all nutrients. In case of nitrogen and potassium uptake, the order of significance was rate of N and P application > Zn fertilization > microbial inoculation > CETs; while for phosphorus and zinc uptake, rate of N and P application > microbial inoculation > Zn fertilization > CETs. The positive effect of Zn fertilization on soil DTPA-extractable Zn and nitrogen uptake along with increasing cropping Table 9. Influence of crop establishment techniques and nutrient management options on soil DTPAextractable Zn (g ha −1 ) after completion of first and second cycle of rice-wheat cropping system. PTR, puddled transplanted rice; SRI, system of rice intensification; ARS, aerobic rice system; CDW, conventional drill-sown wheat;   www.nature.com/scientificreports/ system yield and Zn uptake was also distinct in the present study. The superior performance of the microbial consortia used in the present study in terms of cropping system yield as well as for N and P uptake in ARS-ZTW, highlights their promise in actively participating and helping in nutrient acquisition under aerobic conditions. Experimental details. The rice variety 'Pusa Sugandh 5′ and wheat variety 'HD 2967' were planted in experiment which was conducted in split-plot design with treatment details as mentioned in Table 14 and all the treatments were replicated thrice.   of rice in main field for ARS and sowing of rice seeds in nursery for transplanting in both PTR and SRI were done on same date (16th and 17th June in first year and 19th and 20th June in second year). Similarly, sowing of wheat in all three CETs were also done on same date (15th and 16th November in first year and 17th and 18th November in second year). The details of crop management in different CETs are given in Table 15. The details of the preparation of microbial inoculants and their formulations are given by Prasanna et al. 65 , Nain et al. 66 and Prasanna et al. 67 . Both these formulations used in study (Table 14) were prepared by mixing with vermiculite (hydrous phyllosilicate mineral): compost (1:1) as the carrier. The paddy straw compost has C/N ratio of 16.22:1 and humus content of 13.8% (pH 7.34). The cyanobacterial and bacterial colony forming units in the formulations was 10 4 and 10 8 g −1 carrier, respectively, as optimized in earlier studies 68 .

Methods
Observations recorded. For measurement of above ground shoot dry matter, air dried plant samples were sun dried and further dried in a hot air oven at 60° ± 2 °C, till constant weight was obtained in both rice and wheat. The biological yield was calculated by weighing the harvest of net plots. For determination of concentration of nitrogen (Kjeldahl's apparatus), phosphorus (Vanado-molybdo-phosphoric acid yellow colour method, in nitric acid system), and potassium (flame photometer method) the procedure described by 69 were followed. The concentration of zinc and iron was determined by using Atomic Absorption Spectrophotometer (AAS) 69 . The soil NaHCO 3 -extractable P was determined by Olsen's method 70 ; while soil 1 N ammonium acetate-extractable K was determined by flame photometric method. The DTPA-extractable Zn and Fe content in soil was determined by Atomic Absorption Spectrophotometer (AAS) as described by 71 . The microbial biomass carbon (MBC) was determined by fumigation method 72 and soil chlorophyll was assayed using acetone: DMSO in ratio of 1:1 by using procedure given by 37 . www.nature.com/scientificreports/ The system partial factor productivity (PFP) and agronomic efficiency (AE) for nitrogen and phosphorus was computed using the following expressions: wherein, YN and Na refer to the cropping system grain yield (kg ha −1 ) and total nutrient (N or P) applied in cropping system (kg ha −1 ), Yt and Yac refer to cropping system grain yield (kg ha −1 ) in nutrient applied plots and in control plot (no nutrient), respectively. Data analysis. All the observations from the experiments were tabulated and observed to follow a normal distribution; hence the data was statistically analyzed using the F-test as per the procedure given by 73 . Least significant difference (LSD) values (p = 0.05) were used to determine the significance of difference between treatment means.     Table 14 Methods and timing of application: Incorporation of P, K and Zn just before transplanting and broadcasting of N in three equal splits at 5, 25 and 45 DAT Rate of application: as per the treatment details mentioned in Table 14 Methods and timing of application: Incorporation of P, K and Zn just before transplanting and broadcasting of N in three equal split at 5, 25 and 45 DAT Rate of application: as per the treatment details mentioned in Table 14 Methods and timing of application: drilling below the seed for 1/3 rd N and whole quantity of P, K and Zn at the time of sowing and broadcasting for top dressing of nitrogen1/3 rd N each at 30 and 60 DAS Rate of application: as per the treatment details mentioned in Table 14 Methods and timing of application: Drilling of 1/3rd N, complete dose of P, K and Zn below the seed at the time of sowing; top dressing of 1/3rd N each at 30 and 60 DAS in all CETs 6 Application of microbial inoculations The seeds were treated with slurry of respective microbial inoculant prepared using water and 1% CMC for 30 min and seeds were shade dried for 30 min