Agriculture, Ecosystems and Environment 80 (2000)

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1 Agriculture, Ecosystems and Environment 80 (2000) Integrated nutrient management and waste recycling for restoring soil fertility and productivity in Japanese mint and mustard sequence in Uttar Pradesh, India D.D. Patra, M. Anwar, Sukhmal Chand Central Institute of Medicinal and Aromatic Plants (CIMAP), P.O. CIMAP, Lucknow , India Received 4 November 1998; received in revised form 14 June 1999; accepted 14 March 2000 Abstract Supplementing the nutrient requirement of crops through organic manures plays a key role in sustaining soil fertility, and crop productivity and reducing use of fossil fuels. Field experiments were conducted for 2 years at two different locations (i.e. Lucknow and Pantnagar) in Uttar Pradesh, India. The objectives of the study were to assess the herb and essential oil yields of Japanese mint (Mentha arvensis cv. Hy 77), and its nutrient accumulation under single and combined applications of organic manures and inorganic fertilizers (NPK). Changes in physical and chemical characteristics of the soils (Fluvisols, Mollisols) were also determined. Eight treatments comprising different combinations of NPK through inorganic fertilizers and farm yard manure (FYM) were compared. The distilled waste of mint after extraction of essential oil was recycled to soils in the plots to supplement the nutritional requirement of the succeeding mustard crop (Brassica juncea cv. Pusa Bold). Herb and essential oil yield of mint were significantly higher with combined application of organic and inorganic sources of nutrients as compared to single applications. Accumulation of N and P was at par under full inorganic and combined supply whereas, K accumulation was higher with the former. Soil organic C and ph after harvest of mint did not significantly differ among the treatments, but the level of mineralizable N, Olsen-P and NH 4 OAc extractable K were higher in soil with integrated supply of nutrients. Significant increase in soil water stable aggregates, organic C, available NPK and microbial biomass, and decrease in soil bulk density were observed with waste recycling over fertilizer application. These benefits were reflected in the seed and stubble yield of mustard which succeeded mint. This study indicates that combined application of inorganic fertilizers with organics helps in increasing the availability of nutrients and crop yield and provides a significant effect to the succeeding crop. Similarly, recycling crop residues reduces the need for fossil fuel based fertilizer, and helps in sustaining and restoring soil fertility in terms of available nutrients and major physical and chemical characteristics of the soil Elsevier Science B.V. All rights reserved. Keywords: Essential oil; Japanese mint; Integrated nutrient management; Residue recycling 1. Introduction Corresponding author. Tel.: ; fax: address: cimap@flashmail.com (D.D. Patra) Improved cropping systems, involving major crops that rely on the use of high rates of inorganic fertilizers, continuously for several years often lead to unsustainability in production and also pose a threat to /00/$ see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S (00)

2 268 D.D. Patra et al. / Agriculture, Ecosystems and Environment 80 (2000) the environment. The major concerns are the development of multi-nutrient deficiency and fertilizer related environmental pollution (Prasad and Power, 1995). In response to these concerns, there is a concerted effort world wide to use green manuring, legumes and organic manures to produce the same amount of food with less fossil fuel based inorganic fertilizers. In general, there is a considerable move towards low input sustainable agriculture (Prasad, 1998; Ram and Kumar, 1996). In India, also, integrated supply of nutrients to plants through organic and inorganic sources is becoming an increasingly important aspect of environmentally sound sustainable agriculture (Meelu et al., 1995). For example, farm residues can be very efficiently recycled to supplement the nutrient requirement of crop(s), thereby sustaining productivity, reducing use of fossil fuel based fertilizers, and restoring soil fertility. Organic materials hold great promise due to their local availability, as a source of multiple nutrients and ability to improve soil characteristics. Mint (Mentha arvensis L. var. Piperascens) which is widely used in pharmaceuticals, food and cosmetic preparations, yields essential oil rich in menthol on hydro-distillation of fresh herb. The distillation waste which retains its nutritional values can be recycled to the soil as a potential organic manure for supplementing the nutritional requirement of the subsequent crop (Chattopadhyay et al., 1993). The present study was undertaken to evaluate the effect of combined applications of organic manure (FYM) and inorganic fertilizers (NPK) in different proportions on yield of mint as the test crop. In addition, the influence of incorporation of distillation waste of mint on the succeeding crop mustard (Brassica juncea L. Czern & Coss.) crop and influence of integrated supply of nutrients on soil properties were also determined. 2. Materials and methods 2.1. Experimental sites Field experiments were conducted for 2 years ( ) with Japanese mint at the CIMAP research farm at Lucknow, Uttar Pradesh, located at 26.5 N latitude, 80.5 E longitude and 120 m above m.s.l. Similar experiments were also conducted at Table 1 Initial properties of the soils at the experimental sites at Lucknow and Pantnagar, Uttar Pradesh Soil characteristics Lucknow Pantnagar Soil type Fluvisol Mollisol Texture Sandy loam Clayey loam ph (water) Organic C (g kg 1 ) Mineralizable N (KMnO extractable, mg kg 1 ) Phosphorus (0.5 M NaHCO extractable, mg kg 1 ) Potassium (N NH 4 OAc extractable, mg kg 1 ) Bulk density (Mg m 3 ) the CIMAP Field Station, Pantnagar, Uttar Pradesh, situated at 20 N latitude, 79.5 E longitude and 244 m above m.s.l. The details of the initial properties of the soils are presented in Table Treatments The trial was carried out in a randomized block design (6 m 8 m plot at Lucknow and 10 m 12 m at Pantnagar) with eight treatments having different combinations of fertilizers (NPK) and farm yard manure (FYM). Fertilizer NPK was applied through urea, single super phosphate and muriate of potash, respectively. Details of the elemental composition of FYM are presented in Table 2. The treatments comprising different combinations of organic fertilizer and NPK are presented in Table 3. All the treatments were replicated three times in a randomized block design. Suckers of Japanese mint were planted end to end in the last week of January in 5 cm deep furrows, 50 cm Table 2 Elemental composition (g kg 1 ) of farm yard manure (FYM) and mint distillation waste Nutrient element FYM Distillation waste Carbon Nitrogen Phosphorus Potassium Zinc Manganese Copper Sulphur

3 D.D. Patra et al. / Agriculture, Ecosystems and Environment 80 (2000) Table 3 Details of combinations of manure and fertilizers applied under different treatments Treatments a Fertilizer (kg ha 1 ) Manure (Mg ha 1 ) N P K a : Control; 2: fertilizer (full rate) and no FYM; 3: fertilizer (half rate) plus FYM at 5 Mg ha 1 ; 4: fertilizer (one-fourth rate) plus FYM at 10 Mg ha 1 ; 5: fertilizer (one-fourth rate) plus FYM at 15 Mg ha 1 ; 6: no fertilizer but FYM at 20 Mg ha 1 ; 7: no fertilizer but FYM at 15 Mg ha 1 ; 8: no fertilizer but FYM at 10 Mg ha 1. apart and covered with soil. Farm yard manure and inorganic fertilizers (full rate of P and K and one-third of N) were applied prior to planting. The remaining two-thirds of N was applied in two equal splits at 40-day intervals Harvesting of mint and sampling Above ground foliage of the crop was harvested during the last week of May. For plant sampling a 1m 1 m area in the middle of each plot was harvested 1 week before the final crop harvest. Extraction of essential oil was conducted on 200 g fresh samples following hydro-distillation for 3 h. For chemical analysis, a representative sample of 100 g was taken from the bulk sample collected for oil extraction. The plant samples were shade dried, oven dried, ground and chemically analysed for elemental composition following standard procedures Incorporation of distillation waste and growing of mustard The distillation waste (residues) of mint was recycled to half of the plots under each of the eight treatments. The elemental composition of the distillation waste and FYM is presented in Table 2. The materials after chopping were thoroughly mixed into the soil and followed by an irrigation (flooding). The waste materials were well decomposed, due to high temperature and adequate soil moisture, during August September. At the end of September plots were thoroughly prepared and mustard seed (cv. Pusa Bold) was sown to evaluate the residual effect of the treatments applied to mint and the recycled distillation waste. Nitrogen, P and K at minimum rates (25, 25 and 30 kg ha 1 N, P and K, respectively) were applied prior to sowing of mustard. The crop was grown to maturity and harvested at the end of February. The seed and total dry matter yields were recorded. A representative sampling for seed and stubbles was conducted and the samples analysed for elemental (NPK) composition Soil sampling and analysis Soil samples were taken after the harvest of mustard. Soils were sampled from three randomly selected sites (one in the middle and two from the opposite corners from each plot) to a depth of 15 cm with soil auger. The soils were mixed, homogenized, sieved (2 mm) and a composite sample of approximately 500 g was obtained. Soils were dried in shade and stored in plastic bags prior to analysis. For soil microbial biomass fresh samples after sieving were stored in bins at 5 C in the presence of soda lime prior to analysis. Microbial biomass C and N were estimated following the method of Jenkinson and Powlson (1976) and Vance et al. (1987), respectively. Shade dried soil samples were analysed for organic C (Walkley and Black, 1934), available N (Subbiah and Asija, 1956), 0.5 M NaHCO 3 extactable P, and 1 N NH 4 OAc extactable K (Jackson, 1973). Water stable aggregates were determined by the method of Kemper and Chepil (1965) Statistical analysis Data on all the observations were subjected to analysis of variance (ANOVA) and least significant differences (LSD) were calculated using T-method (Sokal and Rohlf, 1981). 3. Results 3.1. Herb and oil yields of mint At Lucknow, the herb yield of Japanese mint increased significantly with all treatment combinations,

4 270 D.D. Patra et al. / Agriculture, Ecosystems and Environment 80 (2000) Table 4 Influence of single and combined application of organic manures and inorganic fertilizers on herb and essential oil yield of mint at two locations in Uttar Pradesh Treatments a Lucknow Pantnagar Herb (Mg ha 1 ) Oil (l ha 1 ) Herb (Mg ha 1 ) Oil (l ha 1 ) LSD (P=0.05) FYM at 15 Mg ha 1 ; 8: no fertilizer but FYM at 10 Mg ha 1. over the control (Table 4). There was an increase of 36% in herb yield over control with the application of full rate of inorganic NPK. However, the extent of yield increase was 49, 22 and 42% over control with Treatments 3, 4 and 5, respectively. Herb yield increased by merely 8 and 3% over control with application of only FYM (without fertilizer) at 15 and 10 Mg ha 1, respectively. Herb yield with 20 Mg ha 1 application of FYM was at par with that of the combined applications of organic and inorganic fertilizers in different proportions. Oil yield followed a similar trend as the herb yield and it was highest with 15 Mg FYM applied with one-fourth NPK as inorganic fertilizer (Treatment 5). Oil yield with combined application of 5 Mg FYM and half rate of inorganic NPK was about 40% higher than that in the control. There was an increase of 38% in oil yield over control with supply of nutrients either through full rate of inorganic NPK (Treatment 2) or 20 Mg FYM (Treatment 6). For the Pantnagar site the response to fertilizer and organic matter application with respect to herb and essential oil yield was almost similar to that under Lucknow conditions (Table 4) The Pantnagar soil being more fertile than the Lucknow one, produced a higher yield of herb. The highest herb yield was recorded, for Treatment 5, which was about 67% higher than that with the control (Treatment 1). Herb yield with other treatment combinations were as follows: Treatment 6>Treatment 4>Treatment 3. The response to full application rate of inorganic NPK (Treatment 2) was a 35% increase in herb yield over the control. Application of FYM at 15 Mg ha 1 (Treatment 7) and 10 Mg ha 1 (Treatment 8) without any inorganic fertilizer resulted in an increase of 22 and 14% herb yield over control, respectively. The oil yield treatment response followed the identical trend as the herb yield. The oil yield with full rate of inorganic NPK (Treatment 2) was about 41% higher than that in control. The oil yield with a combined application of inorganic and organic nutrients (Treatments 3, 4 and 5), which was significantly higher than that of the control and Treatment 2 (full rate of NPK), were statistically similar Nutrient content and accumulation in mint Data in Table 5 indicates that the N content in mint at Lucknow site was significantly affected by the treatments. The highest N content in plants was found in Treatment 2, followed by the combined application of organic and inorganic nutrients (Treatments 3, 4 and 5). The N accumulation by the plants also followed similar trends. Accumulation of N with full inorganic NPK (Treatment 2) was about 35 and 53% higher than that in the control at Lucknow and Pantnagar, respectively, and it did not differ from that in Treatments 3, 4 and 5. Nitrogen accumulation with highest rate of manure (20 Mg FYM, Treatment 6) was greater than that with Treatment 7 (15 Mg FYM) and Treatment 8 (10 Mg FYM). Both P and K accumulation by

5 D.D. Patra et al. / Agriculture, Ecosystems and Environment 80 (2000) Table 5 Influence of single and combined application of organic manure and inorganic fertilizers on accumulation (kg ha 1 ) of major nutrients in mint at two locations in Uttar Pradesh Treatments a Lucknow Pantnagar Nitrogen Phosphorus Potassium Nitrogen Phosphorus Potassium (2.60) b 24.4 (0.52) 88.2 (1.88) (2.53) 24.5 (0.43) 85.6 (1.50) (2.93) 39.3 (0.70) (2.18) (2.80) 53.1 (0.68) (1.73) (2.75) 38.8 (0.68) (1.89) (2.69) 44.9 (0.54) (1.65) (2.85) 36.0 (0.64) (1.78) (2.54) 45.8 (0.54) (1.72) (2.83) 34.9 (0.60) (1.88) (2.48) 43.3 (0.52) (1.65) (2.63) 34.8 (0.67) 88.7 (1.71) (2.64) 45.3 (0.60) (1.55) (2.71) 35.0 (0.73) 84.0 (1.75) (2.58) 37.3 (0.58) 99.0 (1.54) (2.64) 33.8 (0.70) 84.0 (1.74) (2.60) 32.5 (0.54) 96.3 (1.60) LSD (P=0.05) 8.85 (0.12) 2.75 (NS) 4.92 (NS) (NS) 2.83 (NS) 5.86 (NS) FYM at 15 Mg ha 1 ; 8: no fertilizer but FYM at 10 Mg ha 1. b Figures in parenthesis represent nutrient content (g kg 1 ); NS: not significant. the plants were variable but no significant differences were found among treatments Status of N, P and K in soil after the harvest of mint Data presented in Table 6 indicates that the available N and P in soil after the harvest of mint were higher in all the treatments as compared to control (Treatment 1). The results also reveal that at both the locations, available N and P were higher under combined application of manure and fertilizers as well as under application of FYM alone as compared to that under full application of inorganic fertilizers (Treatment 2). No significant variation was observed with respect to available K under different treatment combinations. Table 6 Available N, P and K in soil after harvest of mint (kg ha 1 ) at two locations in Uttar Pradesh Treatments a Lucknow Pantnagar N P K N P K LSD (P=0.05) NS NS FYM at 15 Mg ha 1 ; 8: no fertilizer but FYM at 10 Mg ha 1 ; NS: not significant.

6 272 D.D. Patra et al. / Agriculture, Ecosystems and Environment 80 (2000) Table 7 Influence of integrated nutrient management on grain and stubble yield of mustard (as residual crop) at Lucknow Treatments a Seed yield (Mg ha 1 ) Stubble yield (Mg ha 1 ) Total dry matter (Mg ha 1 ) 1 MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS LSD (P=0.05) MS MS FYM at 15 Mg ha 1 ; 8: no fertilizer but FYM at 10 Mg ha 1 ; MS: without mint waste; +MS: with mint waste Seed and stubble yield of mustard grown after mint Data in Table 7 indicates that application of both organic matter in the form of manure and incorporation of distillation waste significantly increased the seed and total dry matter yield of mustard over the supply of inorganic nutrients alone. The influence was more pronounced in the treatments with either no fertilizers (Treatment 1), full rate of fertilizers (Treatment 2) and combined application of organic and inorganic sources of nutrients (Treatments 3, 4 and 5), and less in treatments which supplied organic manure only. Seed and total dry matter yields increased by 57 and 31%, respectively, due to waste incorporation under Treatment 1 (no fertilizer and manure during mint). The corresponding increase under Treatment 2 was 21 and 40%, respectively. The increase in total dry matter yield due to waste incorporation in the plots receiving the combined application of organic and inorganic sources of nutrients during mint growing period was in the order of 10 18% Changes in soil physico-chemical characteristics after the harvest of mustard Integrated supply of nutrients had significant influence on the physical and chemical characteristics of the soils (Table 8). Soil ph significantly decreased due to cropping and incorporation of waste. The highest reduction was with Treatment 5. Soil organic C increased in all the treatments. The extent of increase was, however, higher when manure was applied in combination with inorganic fertilizers. A similar trend was observed for soil bulk density, which decreased under integrated supply of inorganic nutrients and organic residues. Water stable aggregates (WSA), which are an important index of soil physical quality, significantly improved due to organic manuring and residue recycling. The extent of increase in WSA was 5% in plots receiving full supply of inorganic NPK (Treatment 2) and 19% when receiving 15 Mg FYM with one-fourth of the rate of inorganic fertilizer (Treatment 5). Data on available N (alkali KMnO 4 extractable), Olsen-P and 1 N NH 4 OAc extractable-k indicated

7 D.D. Patra et al. / Agriculture, Ecosystems and Environment 80 (2000) Table 8 Physical and chemical characteristics of the soil (15 cm depth) at Lucknow site, before and after harvest of mustard Treatments a ph (1:2.5 H 2 O) Organic C (g kg 1 ) Bulk density (Mg m 3 ) Water stable aggregates (%) Initial Harvest Initial Harvest Harvest Harvest LSD (P=0.05) NS FYM at 15 Mg ha 1 ; 8: no fertilizer but FYM at 10 Mg ha 1 ; NS: not significant. that integrated supply of nutrients significantly influenced the soil fertility with respect to their availability (Table 9). In general, available N and P were significantly higher in soils that received combined application of fertilizer and manure. Available N increased by 137% over the control and 44% over that receiv- ing full supply of inorganic NPK, with the combined application of 15 Mg manure with one-fourth rate of NPK (Treatment 5). The available N status was further enhanced with recycling of distillation waste. Almost an identical trend was observed with respect to Olsen-P after the harvest of mustard. Available K Table 9 Available N, P and K and microbial biomass (SMB) N and P in soil after the harvest of mustard, at Lucknow site Treatments a Nitrogen Phosphorus Potassium SMB-N SMB-P (kg ha 1 ) 1 MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS LSD (P=0.05) MS NS MS NS FYM at 15 Mg ha 1 ; 8: no fertilizer but FYM at 10 Mg ha 1 ; NS: not significant; MS: without mint waste and +MS: with mint waste.

8 274 D.D. Patra et al. / Agriculture, Ecosystems and Environment 80 (2000) was highest in soil receiving the full inorganic supply of NPK, but did not differ significantly among treatments. Estimates of soil microbial biomass after the harvest of mustard indicated a significant increase of biomass N and P due to addition of organic materials. Recycling of mint waste further enhanced the soil microbial biomass N and P as compared to that under non recycled conditions. 4. Discussion The results from the study indicate that combined applications of manures and fertilizers significantly influenced the herb and essential oil yield of mint. This could be attributed to a significant improvement in soil physical and chemical properties. Besides improvement in the available soil nutrients (Table 6), combined application of manure and fertilizers improved physical properties such as bulk density and WSA (Table 8). Similar observations on improvement of physical properties of soil like WSA, mean weight diameter, bulk density and infiltration rate were made by Meelu et al. (1995) under a rice (Oryza sativa L.)-wheat (Triticum spp.) cropping system through incorporation of crop residues and green manuring. Similarly, Prasad (1994) and Dudhat et al. (1997) in India observed a significant improvement in soil aggregation and hydraulic conductivity due to integrated supply of nutrients through inorganic NPK and blue green algae in rice-wheat system. Beneficial effects of combined application of organic and inorganic sources of nutrients on soil fertility and subsequently on productivity of agricultural crops like rice, wheat and maize (Zea mays L.) were also reported by Mandal et al. (1994). The recycling of distillation waste of mint has been observed to have significant influence in improving the soil fertility and productivity of succeeding crop mustard. The concept of recycling of distillation wastes of mints stems from the fact that herb after hydrodistillation retains its manurial value with respect to major as well as secondary and trace elements (Chattopadhyay et al., 1993). Distilled waste fortified with starter application of inorganic NPK has the potential to be a balanced source of available nutrients (Chattopadhyay, 1997). The residues on decomposition released nutrients specially N which are available for a longer growth period because of a slow but steady mineralization of different organic complexes as observed by McLaughlin and Champion (1987) and Chattopadhyay et al. (1993). Data on estimates of soil microbial biomass reveals that biomass N and P significantly improved due to combined application of manure and fertilizers and recycling of distillation waste. Soil microbial biomass is considered to be a significant repository of nutrients such as N, P and S in a form much more labile than the bulk organic matter and it plays a significant role in nutrient dynamics (Jenkinson and Ladd, 1981). Changes in soil microbial biomass can be used as an early indicator of changes in soil organic matter (Powlson et al., 1987; Jordan et al., 1995). Enhanced microbial biomass under the present study provides indication of sustainability of soil, with respect to organic matter and available nutrients like N and P. 5. Conclusions The findings from the present investigation reveal some distinct benefit of combined application of manure and fertilizers over a full supply of organic matter or inorganic fertilizers. Besides increasing the crop yield, such practices improve both soil physical and chemical properties. The possibility of sustaining the soil ecology and the environment cannot be ignored. These results indicate a significant increase in the soil microbial biomass. It is possible that by supplying nutrients to the plant in an integrated way, the use of fossil fuel based inorganic fertilizer can be reduced which in turn, can reduce the risk of fertilizer related environmental consequences (Anon., 1997). Acknowledgements The authors are thankful to the Director, CIMAP, Lucknow for providing necessary facilities and Scientist-in-Charge, CIMAP Field Station, Pantnagar for managing the field experiment at Pantnagar. References Anon., A Guide to Field Implementation of Integrated Plant Nutrition System. IFFCO, New Delhi, p. 106.

9 D.D. Patra et al. / Agriculture, Ecosystems and Environment 80 (2000) Chattopadhyay, A., Subrahmanyam, K., Singh, D.V., Recycling of nutrients in Japanese mint-assessment of soil fertility and crop yield. Fertil. Res. 35, Chattopadhyay, A., Nutrient enriched mint residue compost and its manurial value. J. Ind. Soc. Soil Sci. 45, Dudhat, M.S., Malavia, D.D., Muthukia, R.K., Khanpara, V.D., Effect of nutrient management through organic and inorganic sources on growth, yield, quality and nutrient uptake by wheat (Triticum aestivum). Ind. J. Agron. 42, Jackson, M.L., In: Soil Chemical Analysis. Prentice Hall, New Delhi, India. Jenkinson, D.S., Powlson, D.S., The effect of biocidal treatments on metabolism in soil. V. Method for measuring soil microbial biomass. Soil Biol. Biochem. 8, Jenkinson, D.S., Ladd, J.N., Microbial biomass in soil measurement and turnover. In: Paul, E.A., Ladd, J.N. (Eds.), Soil Biochemistry, Vol. 5. Marcel Dekker, New York, pp Jordan, D., Kremer, R.J., Bergfield, W.A., Kim, K.Y., Cacnio, V.N., Evaluation of microbial methods as potential indicators of soil quality in historical agricultural fields. Biol. Fertil. Soil 19, Kemper, W.D., Chepil, W.S., Size distribution of aggregates. In: Methods of Soil Analysis, Part I. Am. Soc. Agron., Madison, WI, USA, pp Mandal, S.S., Mandal, T.K., Dandapat, S., Sarkar, S., Effect of sulphur bearing fertilizers in conjunction with farm yard manure on growth, productivity and nutrient uptake of rice (Oryza sativa ). Ind. J. Agron. 39, McLaughlin, N.J., Champion, L., Sewage sludge as a phosphorus amendment for sesquioxide soils. Soil Sci. 143, Meelu, O.P., Singh, Y., Khera, T.S., Kumar, K., In: Proceedings of the 2nd Conference on Temperate Rice-Achievement and Potential. Yanco, Australia, pp Powlson, D.S., Brookes, P.C., Christensen, B.T., Measurement of soil microbial biomass provided an early indication of changes in total soil organic matter due to straw incorporation. Soil Biol. Biochem. 19, Prasad, B., Integrated nutrient management for sustainable agriculture. Fertil. News 39, Prasad, R., Fertilizer urea, food security, health and the environment. Curr. Sci. 75, Prasad, R., Power, J.F., Nitrification inhibitors for agriculture, health and the environment. Adv. Agron. 54, Ram, M., Kumar, S., Productivity and economic potential of cropping sequences with medicinal and aromatic crops in subtropical environment. J Herb Spice. Med. Pl. 4, Sokal, R.R., Rohlf, F.J., Biometry The Principle and Practices of Statistics in Biological Research, 2nd Edition. Freeman, New York. Subbiah, B.V., Asija, G.L., A rapid procedure for the estimation of available N in soil. Curr. Sci. 25, 259. Vance, E.D., Brookes, P.C., Jenkinson, D.S., An extraction method for measuring soil microbial biomass. Soil. Biol. Biochem. 19, Walkley, A., Black, I., An examination of the Degtejareff method for determining soil organic matter and a proposed modification of the chromic acid titration method. Soil Sci. 37,