72 J. Hortl. Sci. Vol. 15(1) : 72-80, 2020 Original Research Paper Soil and Plant Analysis - A Strategic Tool to Diagnose Micronutrient Imbalance in Lime and Sapota Orchard in Tablelands of Chambal Ravine Region of India Rashmi I.1*, Meena H.R.1, Somasundaram J.2 and Radha T.K.3 1ICAR-Indian Institute of Soil Water Conservation, RC, Kota, 2ICAR-Indina Institute of Soil Science, Bhopal; 3ICAR-Indian Institute of Horticulture Research, Bengaluru - 560 089, India Email : rashmimenon109@gmail.com ABSTRACT Micronutrient imbalance in lime and sapota fruit crops result in unstable fruit yield, fruit shedding and degrade quality of the produce. A study was therefore conducted to evaluate micronutrient statusoflime and sapota orchard by analysing soil and plant samples. Soil samples were collected from surface (0-15cm) and sub-surface (15-30cm)depth representing whole orchard. At the same time, plant samples including 35-40 each for leaves and petiole samples each from lime and sapota field was also collected.Available micronutrients from soil samples were extracted using diethylenetriaminepenta acetic acid (DTPA) and it was in the order of manganese (Mn)> iron (Fe)> zinc (Zn)> copper (Cu) in both lime and sapota plantations. DTPA- extractable Zn and Cu showed low status, marginal status of Fe and sufficient level of Mn in soils of sapota plantations. In plant analysis, high concentration of Cu (869 mg kg-1) and Zn (411mg kg-1) was observed in lime leaves; however, in sapota crop Cu and Zn content was 8.25mg kg-1 and 16.7mg kg- 1 respectively. Similarly, Fe and Mn content of lime leaves was 197 and 43 mg kg-1 which was slightly higher than sapota leaves that recorded 128 and 49mg kg-1 of Fe and Zn respectively. In sapota plants, higher Mn and Cu concentration in leaf resulted in Zn deficiency symptoms such as shortened internodes or rosette disorders of sapota plants. Thus, correcting micronutrient deficiency is pre-requisite for qualitative and quantitative fruit production in tablelands of India. Keywords: Copper, Iron, Leaf analysis, Manganese, Micronutrient deficiency, Sapota, Zinc INTRODUCTION Ravines are typical examples of land degradation covering approximately 2.06 Mha and gully formation occurs in 8.31 Mhaarea in India (ICAR-NAAS, 2010). Generally, these ravine lands also known as badlands are situated near rivers and typically know for deep ravines cutting with extension overnearby arable lands (Pani and Carling 2013). Cultivation of crops is practised on the top slope called tablelands and adjacent undulating topography of gully eroded areas of Chambal ravines. Fruit crops like lime is of great significance in semi-arid regions of Rajasthan due to its hardy nature and low water requirement. However, sapota crop is recently introduced in the r egion a nd ther efor e infor ma tion on a rea a nd production of sapota in Rajasthan is not readily available. Lime per unit production in Rajasthan is 4.0 t ha0-1 which is low against India’s national average of 8.33 t ha”1 (Srivastava and Shyam, 2008). The area under sapota in India is estimated to be 1.77 lakh hectares, with an annual production of 1.74 million metric tonnes and productivity of 9.91 Mg ha -1 (Sharma, 2015). Major sapota growing state includes Andhra Pradesh, Gujarat, Karnataka, Maharashtra, Tamil Nadu, Kerala, Punjab, West Bengal, and Haryana. In citrus crops necrosis, die back, chlorosis symptoms are commonly observed in the region due to nutrient deficiency r esulting in decline of lime yield (Somasundaram et al., 2011). Nutrient imbalance in sapota crop is visualised by poorfruit setting,quality, shedding of fruitsand low productivity. Guvvali (2016) This is an open access article d istributed under the terms of Creative Commons Attribution-NonCommer cial-ShareAl ike 4.0 International License, which permits unrestricted non-commercial use, d istribution, and reproduction in any med ium, provide d the original author and source are credited. 73 J. Hortl. Sci. Vol. 15(1) : 72-80, 2020 Micronutrient imbalance in lime and sapota reported thatonly 10-12% of the total fruits set, and retains until maturity in sapota crop. Lower fruit production in north western India is mainly due to nutr ient imba la nce or disor der s which ca use considerable yield reduction with huge economic loss (Somasundaram et al., 2011; Guvvali and Shirol, 2017). Subsequently, orchards provide sub optimal fruit yield with increasing gap between the amount of nutrient added and demand of crop (Srivastava and Singh, 2006). In Rajasthan, about 57, 34, 28 and 9% soils are deficient in zinc (Zn), iron (Fe), manganese (Mn) and copper (Cu) respectively (Shukla, 2018). Micronutrients are required by plants to perform specific biochemical reactions, metabolism required for its growth and productivity. Thus in order to avoid yield and quality loss, nutrient requirements of lime and sapota crop need to be carefully monitored through soil a nd pla nt a na lysis for evolving nutr ient management strategies. Besides soil analysis, leaf sample analysis is considered a more direct method of plant nutritional status evaluation, especially, for fruit crops as these differ from seasonal crops in nutrient requirement due to their size, population density, rate of growth and rooting pattern (Motsara and Roy, 2008). In ravine landforms, very scanty information is available on micronutrientdeficiency in fruit crops (Somasundaram et al., 2011; Meena et al. , 2019). T her efor e, the pr esent study wa s conducted with the hypothesis that diagnosing micronutrient disorders of sapota crop is vital to achieve optimum fruit yield so as to improve orchard efficiency with advancing age of crop. The objective of this study was to analyse micronutrient deficiency or sufficiency level through soil and plant analysis in lime and sa pota cr op a nd its ma na gement for sustainable productivity in semi-arid regions of ravine ecosystem. MATERIAL AND METHODS Brief description of experimental site The study area comprises two distinct landscapes, the agricultural tablelands and the ra venous lands a djoining Chamba l r iver. T he physiogr aphy is constituted of gently sloping (<2% slope), moderately well-drained tablelands in the immediate vicinity of ravines. The experimental orchard area is a tableland located at Research Farm, ICAR- Indian Institute of Soil and Water Conservation, Research Centre, Kota, situated at 25º 11' N latitude and 75º 51' E longitudes at an elevation of 256.9 meters above mean sea levelwith fairly levelled topography. According to Koppen’s climate classification subtype, the climate of Kota is semi-arid type (Mid latitude steppe). More than 90 per cent of rainfallis received during mid-June to September with scanty showers during winter months (Nov-Dec). This region is characterized by mild and dry winters and hot summers with average rainfall of 740mm (mean of last 5 years) of which most of the r a infa ll is r eceived dur ing July month (300 mm). Lime (Kagzi lime variety) crop planted at 4.5 x 4.5 m (row x plant) during 2001; sapota cv. ‘Kalipatti’ trees planted at 8 x 8 m spacing (row x plant) during 2008. The study was carried out during 2018-2019at the research farm of Indian Institute of Soil and Wa ter Conser va tion, Resea r ch Centr e, Kota , Rajasthan. The soils are brown to dark grey brown in colour, generally non calcareous occurring on flat gently sloping land with less than 2% slope. The soils of the region are moderately well drained fine textured soils classified as Typic Chromoustert belonging to Kota soil series. The region comprises of two diverse geology namely sandstone quartzite, silicaceous limestone and dolomite where a vast area is formed from the alluvium brought down by Chambal and its tributaries passing through the residual hillocks and gently sloping rocky plateau (Shyampura and Sehgal, 1995). The physico-chemical properties of the orchard soil are given in Table 1. The irrigation water used in sapota orchard contained bicarbonate, calcium a nd ma gnesium of 600, 66. 8 a nd 33.5 mg l -1 respectively, with pH of 7.6 and electrical conductivity (EC) of 2.76 dSm-1. Orchard Management The experimental trees were managed with uniform cultural practices as per the standard recommendations with respect to manures and fertilizers, irrigation and pla nt pr otection mea sur es, etc. Nutrients wer e regularly supplied to lime crop duringcritical period of crop growth for better production and explained in Ta ble 2. Recommended dose of nitr ogen (N), phosphorus (P2O5), and potash (K2O) was applied beyond a 30-cm radius from the tree trunk of lime. After ten years, fertilizer were mixed @ 750g N, 450g P and 750g K was applied to each lime plant every year. Fertilizers were applied to each tree in two or 74 Rashmi et al. three split doses when soil is moist. In sapota orchard, recommended doses of fertilizer were mixed @ 1000 g N, 500 g P and 500 g K per plant for ten-year-old sapota plants. For the application of full recommended dose of NPK,2174 g urea (1000 g N), 3125 g single super phosphate (500 g P) and 833 g murate of potash (500 g K) per plant were applied from 6 th year onwards. Full amount of phosphorus, potash and half dose of nitrogen in various treatments were applied as basal dose before vegetative sprouting in the month of June. Remaining half dose of nitrogen was applied after fruit set in the month of December. Collection of plant and soil samples A systematic survey of lime and sapota orchard was conducted to assess the micronutrient status in 15 and 10 year old plantation covering an area of 2 and 0.4 ha respectively. For leaf sample collection, uniform area was selected in the orchard and 30 trees were selected as shown in the Fig 1. In both lime and sapota orchard, recently matured leaf were collected from north, south, east, and west quarters of thetrees (Reuter et al. , 1997) dur ing September a nd October.About 35-40 fully developed leaf samples were collected, from which petioles samples were separated.The sampling pattern is shown in figure 1 omitting the border plants. From the 35-40 leaf samples collected, petiole samples (40) separated, J. Hortl. Sci. Vol. 15(1) : 72-80, 2020 Soil parameters Lime Sapota Depth (cm) 0-15 15-30 0-15 15-30 pH(1:2.5) 7.74 7.51 7.81 7.62 EC (dS m-1) 0.57 0.62 0.55 0.59 OC (g kg-1) 4.7 3.6 4.5 3.4 Available nutrients (kg ha-1) Nitrogen (N) 365.7 304.4 342.3 288.7 Phosphorus (P) 17.4 13.9 15.41 11.4 Potassium (K) 436.5 412.4 386 344.5 Exchangeable cations (cmol p+ kg-1) Na 4.9 5.7 3.2 3.5 Ca 18.2 17.9 17.7 17.8 Mg 7.6 6.1 7.5 6.4 Cation Exchange Capacity (CEC) 27.6 22.5 33.4 32.8 (cmol p+ kg-1) Soil texture (%) Sand 27.8 29.3 27.2 29.7 Silt 42.2 41.5 44.3 42.3 Clay 30 29.2 28.5 28 Table 1. Soil properties under lime and sapota orchard 75 shade dried and grounded to fine powder for nutrient analysis. For nutrient analysis, 1g of sample was digested with tri acid mixtures (nitric, sulphuric and per chloric acid a t 9:2:1). Micr onutrients wer e estimated by directly feeding the filtered tri acid extract of the plant sample to a calibrated atomic absorption spectrophotometer using respective hollow cathode lamps for each element (Fe, Mn, Zn and Cu). Micronutrient concentration was expressed in mg kg-1 on dry weight basis. Soil samples were collected from four quadrants at two different depths (0-15cm and 15-30cm) of the lime and sapota orchard (15 composite samples from each depth). Soil samples were air dried, grounded and passed through 2mm sieve and subjected to analysis of available micronutrients, namely Fe, Mn, Zn and Cu. For the soil analysis 20g soil samples was shaken with 40ml 0.005M DTPA extractant for 2 hours (Linday and Norvell, 1978). The filtered extract was directly read on AAS (model Thermo M6 series; Thermo Scientific, Waltham, Mass.) for micronutrient analysis of iron (Fe), manganese (Mn), copper (Cu) and zinc (Zn). RESULTS AND DISCUSSION Micronutrient concentration in soil samples Micronutrient concentration of soil samples under sapota plantations are shown in Table 3. Among Micronutrient imbalance in lime and sapota J. Hortl. Sci. Vol. 15(1) : 72-80, 2020 Table 2. Fertilizer management in lime and sapotaorchard Age (years) Nitrogen (g/plant) P2O5 (g/plant) K2O (g/plant) Lime 1 75 40 75 2 150 80 150 3 225 120 225 4 300 160 300 5 375 200 375 6 450 240 450 7 525 280 525 8 600 320 600 9 675 360 675 10 750 400 750 Sapota 1 200 200 300 2 200 200 300 3 200 200 300 4 200 200 300 5 200 200 300 6 1000 500 500 7 1000 500 500 8 1000 1000 1500 9 1000 1000 1500 10 1000 1000 1500 76 micronutrients, highest concentration was observed in Mn, followed by Fe, Zn and Cu. Surface soil recorded higher micronutrient concentration compared to sub- surface soil except for Mn. T he micr onutr ient concentr ation in soil was crucially inter pr eted considering the critical limit of soil availability of DTPA extractable Zn, Cu, Mn and Fe as 0.6, 0.2, 2 and 4.5 mg kg-1 respectively suggested by Lindsay and Norvel (1978) and Katyal(2018). The available Fe content in lime and sapota orchard ranged from 5.3 to 7.7 mg kg-1 and 3.4 to 8 mg kg-1 with mean value of 6.1 and 5.19 mg kg-1 respectively. However, sub surface mean values of DTPA Fe content in lime and sapota orchard was 5.4 and 4.59 mg kg-1 respectively (Table 3). Most of the soil sa mples showed Fe concentr a tion below the sufficiency range (6-8 mg kg-1) suggesting that Fe deficiency might arise in future in sapota plantation. Higher bicarbonate concentration of irrigation water used in fruit orchard could result in Fe deficiency. In the medium black soils of study site, Fe deficiency in lime plantations owing to increased concentration of bicarbonate ions in irrigation water was reported by Somasundaram et al. (2011). Similar report of Fe deficiency in pomegranate orchard was also reported by Gathala et al. (2004). Considering the critical concentr a tion of soil Mn (2 mg kg-1), D T PA extractable Mn concentration in both lime and sapota orchard were above sufficiency range. In lime and sapota orchard, DTPA-Mn of surface samples varied from 13.7 to 27.8 mg kg-1 and 8.4 to 15.2 mg kg-1 with mea n va lue of 20. 2 a nd 12. 11 mg kg -1 respectively. In sub surface soil Mn concentration varied from 12.7 to 24.6 and 6.57 to 16.03 mg kg-1 with a mea n va lue of 18. 8 a nd 11. 3 mg kg -1 respectively in lime and sapota orchard. In vertisol, high concentr a tions of both tota l a nd DT PA extractable Mn had been reported earlier by few authors (Singh et al., 2006; Kumar and Babel, 2011). However, Surwase et al.(2016) also found low status of Fe and Mn in silty clay loam soils under orange crop, although soils had optimum Zn and Cu. Available Zn concentration in lime orchard was higher than that of sapota orchar d. T he Zn content was low to marginal level in lime orchard. The DTPA extractable Zn concentration of lime and sapota orchard varied between 0.42 to 0.97 mg kg-1 and 0.17 to 0.74 mg kg-1 respectively in surface soil. Sub surface DTPA Zn concentration varied from 0.26 to 0.81 and 0.19 to 0.72 mg kg-1 respectively in lime and sapota orchard. Earlier study reported Zn deficiency in fruit orchard soils of south eastern Rajasthan (Kumar and Babel, 2011; Somasundaram et al. 2011). Among all the four micronutrients, Cu concentration was lowest in or cha r d soils. T he DT PA extr a cta ble Cu concentration varied between 0.082 to 0.51 mg kg-1 and 0.02 to 0.35 mg kg-1 in surface soils of lime and sapota plantations. Sub surface samples recorded lower Cu content varying from 0.05 to 0.33 mg kg-1 and.02 to 0.25 mg kg-1 in lime and sapota orchard.Soils of orchard have pH >7.5, Zn forms negatively charged ions called zincate ions (ZnO2 2-) which can reduce Zn availability in soils (Katyal, 2018). Except for Mn, Fe, Zn and Cu concentration were higher in surface compared to sub surface soil. Similar results were also reported by Surwase et al. (2016) who reported higher DTPA extractable micronutrients in surface soils of orange orchards due to higher soil organic carbon and biological activity in surface layer. Thus, balanced micronutrient fertilization is necessary to correct nutrient deficiency in soils of fruits crops for doubling farmer’s income. Micronutrient concentration in plant samples (leaves and petioles) Plant analysis is known as adiagnostic tool for managing mineral nutrition and the total nutrient concentrationin the leaf tissue provide an accurate production potential of fruit crop which mostly depends upon the supply and uptake of particular nutr ient (Sr iva sta va a nd Singh 2006). Lea f micronutrient concentration, like soil micronutrient content, showed wide variation (Table 4).The mean Fe content in leaves and petioles of lime trees were 196.8 and 161 mg kg-1 whereas, in sapota plantations it was 127 and 120 mg kg-1respectively. Leaf Fe concentr ations wa s higher tha n the normal Fe concentration in plant tissues. However, 12% plant samples were deficient in Fe and in case of petioles 8, 54 and 33% samples were deficient, sufficient and high in Fe concentration. Considering the optimum level of total Fe concentration in plant tissue (50-100 mg kg-1), more than 62% of samples were sufficient a nd 18% sa mples r ecor ded excess of Fe concentration. During field examination for sample collection, some trees showed interveinal chlorosis and necrotic symptoms were observed in leaves of both lime and sapota crop.(Fig. 2). Considering the normal Mn content in plant tissues (15 to 50 mg kg-1), most Rashmi et al. J. Hortl. Sci. Vol. 15(1) : 72-80, 2020 77 Micronutrient imbalance in lime and sapota J. Hortl. Sci. Vol. 15(1) : 72-80, 2020 Table 3. Micronutrient concentration and ranges (mg kg-1) in soils of lime and sapotaorchard Soil depth (cm) Fe Mn Cu Zn Lime Surface soil (0-15) Min 5.3 13.7 0.082 0.42 Max 7.7 27.8 0.51 0.97 Mean* 6.1±0.23 20.2±1.1 0.28±0.025 0.69±0.032 Sub surface (15-30) Min 3.8 12.7 0.05 0.26 Max 7.2 24.6 0.33 0.81 Mean* 5.4±0.30 18.8±0.81 0.18±0.02 0.53±0.031 Sapota Surface soil (0-15) Min 3.4 8.4 0.02 0.17 Max 8.0 15.2 0.35 0.74 Mean* 5.19 ± 0.33 12.11± 0.5 0.19 ± 0.03 0.38±0.05 Sub surface (15-30) Min 2.8 6.57 0.02 0.19 Max 6.5 16.03 0.25 0.72 Mean* 4.59 ± 0.29 11.3 ± 0.72 0.10 ± 0.02 0.42 ± 0.05 *Mean of 15 samples, ± Standard error of mean Table 4. Leaf and petiole micronutrient concentration and ranges (mg kg-1) in lime andsapotaplantations Fe Mn Cu Zn mg kg-1 Leaf Petiole Leaf Petiole Leaf Petiole Leaf Petiole Lime Range 45 – 61.2 – 18.6 – 5.64 – 45 – 63 – 46.3 – 68 – 470.3 313.5 84.9 55.3 1588 941 650.8 473.2 Mean* 196.8 ± 161.1 ± 42.87 ± 18.6 ± 869 ± 526 ± 411 ± 293 ± 22.7 20.1 3.58 2.2 89.6 57.9 39.8 21.3 Sapota Range 89.41 – 33.52 – 22.2 – 11.31 – 4.35 – 2 – 0.62 – 0.54 – 231.24 284.51 97.61 69.26 19.78 18.48 48.12 36 Mean* 127.66 ± 119.8 ± 48.84 ± 33.46 ± 8.25 ± 9 ± 16.66 ± 13.01 ± 6.56 12.23 3.98 2.92 0.77 0.89 2.25 2.1 *Mean of 30 samples, ± Standard error of mean 78 Rashmi et al. J. Hortl. Sci. Vol. 15(1) : 72-80, 2020 Fig. 2. Iron deficiency in lime (a) and sapota (b) plants Fig. 1. Collection of representative plant samples from sapotafruit orchard of the leaf samples showed deficient to sufficient status. The average Mn concentration in lime varied between 43 and 19 mg kg-1 and in sapota was 48 and 33 mg kg-1 respectively for leaves and petioles samples (Table 4). Leaf samples registered 64% sufficientand 36% excess concentra tionof Mn, wher ea s in petiole sa mples 8% sa mples wer e deficient. Excessive Mnconcentration in plant tissues can alter various processes such as enzyme activity, absorption, translocation and utilization of other mineral elements (Ca, Mg, Fe and P), causing oxidative stress (Ducicand Polle, 2005; Lei et al., 2007). Mean Cu concentration in lime leaf samples varied between 869 and 526 mg kg-1 in leaf and petiole sample. In contrast, lower Cu concentration values of leaf samples were recorded in sapota plants. Copper concentration of sapota leaf samples varied from 4.35 to 19.78 mg kg- 1 with a mean value of 8.25 mg kg -1. T he Cu concentration of petiole samples varied from 2 to 18.48 mg kg-1 with a mean value of 9 mg kg-1 (Table 4). The Cu concentration range in plant samples vary from 5 to 16 mg kg-1. Based on the normal range of 79 Micronutrient imbalance in lime and sapota J. Hortl. Sci. Vol. 15(1) : 72-80, 2020 Cu (100 mg kg-1) in plants, lime plants samples showed excessive total Cu content. This was mainly attributed to the spray of Cu based fungicide to control fungal disease in orchard. Some plants with young leaves showed chlorosis symptoms due to Cu toxicity. However, in sapota crop, 13 and 21% of leaf and petiole samples were recorded as deficient and 79% wer e sufficient in Cu. However, Cu deficiency symptoms (dieback of apical buds) in sapota were observed during plant sampling in some sapota trees. Some common symptoms included pr ema tur e defoliation and die back of twigs occurred. The tip of the twigs developed multiple buds which died soon. Zinc concentration of lime crop for leaf and petiole varied from 46.7 to 650.8 mg kg-1 and 68 to 473.3 mg kg-1 respectively with a mean value of 411 and 293 mg kg-1. In sapota crop, the Zn concentration of leaf and petiole samples varied between 0.62 - 48.12 and 0.54- 36.0 mg kg-1 with a mean value of 16.7 and 13.0 mg kg-1 respectively (Table 3). Wide difference between Fe content in sapota and lime was observed in the study. Based upon the Zn concentration (<20 mg kg-1), more than 73% samples were sufficient in Zn content in lime orchard. However, in sapota crop 50% of leaf samples were deficient where as 42% recorded optimum to highand 8% had excess Zn status.In petioles, 67 and 33% samples recorded deficiency and sufficiency of Zn respectively in sapota plants. High Zn concentration in lemon orchard was also reported by Somasundaram et al. (2011) where more than 88% leaf samples recorded higher Zn content. They suggested accumulation of excess of Cu in leaf resulted in greater accumulation of Zn to maintain nutrient balance. Soil and foliar method of fertilizer application is utilized for sapota crop.Foliar application of micronutrients is considered as quickest means to correct nutrient deficiency in fruit trees. In sapota crop, Fe, Mn, Zn and Cu deficiency can be corrected by foliar spray ferrous sulfate (0.2 to 0.4%), manganese sulphate (0.3%), zinc sulphate (0.2 to 0.5%) and copper sulphate (0.1%) (Satyagopalet al., 2015).Copper based fungicide (copper oxychloride with 3g l-1 of water) sprays will be helpful in correcting the Cu deficiency of sapota. Possibility of micronutrient response to its application in crops could be as high as 90% for very low, 60 to 90% for ‘low’ and 30 to 60% for ‘optimum’ levels of extr a cta ble micronutrients (Cooper and Abi-Ghanem, 2017). 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