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Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1247-1265 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 7 Number 07 (2018) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2018.707.150 The Selective Vulnerability of Rice Root System Architecture to Organic and Inorganic Nitrogen Jayeshkumar A. Bhabhor2, Kirti Bardhan1*, Dhiraj P. Patel3, Ajay V. Narwade2 and Harshad N. Chatrola1 1 Department of Basic Sciences and Humanities, 3Department of Natural Resource Management, ASPEE College of Horticulture and Forestry, Navsari Agricultural University, Navsari (Gujarat) 396450 India 2 Department of Genetics and Plant Breeding, N. M. College of Agriculture, Navsari Agricultural University, Navsari (Gujarat) 396450 India *Corresponding author ABSTRACT Keywords Root system architecture, Root plasticity, Rice, Organic and inorganic nitrogen Article Info Accepted: 08 June 2018 Available Online: 10 July 2018 N fertilizers and high yielding varieties were major drivers of the enormous increase in rice productivity during the past 50 years. Despite increasing food production higher nitrogen use also contributed in environmental pollution and Increasing consciousness of conservation of environment and mitigation of climate change brought a major shift in cultivation practices of major crops towards organic agriculture. An important issue regarding the acceptance of organic agriculture is the question of productivity. In addition to readily available ammonium and nitrate ions, the soil of organic agriculture can contain a wide range of organic nitrogen compounds such as peptides, proteins, free amino acids, amino sugars and nitrogen heterocyclic compounds. The root system architecture (RSA) features are of utmost importance for increasing nitrogen use efficiency of future climateresilient varieties. From a fundamental point of view, the influence of nitrogen on root development is still poorly understood. Modulating root architecture is a strategy that aims at developing crops that capture nutrients more efficiently and are thus suitable for sustainable agriculture with fewer fertilizer inputs. Our experiment aimed to study the responses of IR-28 root system architecture to the availability of different forms of nitrogen, including organic at seedling stage, so that we can understand up to what extent our current rice varieties, which are exclusively breed for intensive agriculture, are suitable for organic agriculture. From the experimental results it was indicated that root parameters viz. primary root length was significantly reduced due to the availability of nitrogen in nitrate and ammonia while increased with organic N. Main root angle was significantly increased with increasing N concentration and it became more steeper under the deficiency of N. Straightness of main root does not affected by the availability of N while the sources of nitrogen had significant effect and maximum straightness observed in organic N treated plant while minimum in ammonium treated. Lateral root number was increased with increasing nitrogen as nitrate and ammonia up to 100% as compared to organic N. Mean lateral root length was significantly affected by the sufficiency of nitrogen, while does not fluctuate with various forms of nitrogen. Sum of lateral root length was higher in nitrogen sufficiency. Lateral root density was significantly responding to availability and non-availability of nitrogen and significant reduction was observed in 0% N condition. Total root system size significantly influenced by sources and levels of nitrogen. Moreover, amongst root traits, total root system size was found least phenotypic plastic while lateral root density was ranked as highest phenotypic plastic trait due to nitrogen. The results indicate that combined nitrogen nutrition through nitrate and ammonia is most suitable for root system and seedling growth of rice as compared to the sole sources. The results of these study support the view that we need to breed varieties suited for organic agriculture and varieties such as IR-28, which is select and breed for high nitrogen input intensive agriculture, may not be efficient for organic nitrogen uptake and/or assimilation. 1247 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1247-1265 Introduction Nitrogen (N) is a paramount element for crop productivity since it is a core component of many plant structures and for their metabolic processes. It is quantitatively the most important nutrient for plant development. Limited N availability has severe consequences for plant metabolism and growth resulting in lower biomass and yield of storage compounds (Epstein and Bloom, 2005). The ‘green revolution’ of the 1960s and 1970s helped agriculture to meet the food demands of a rapidly growing global population, through the development of dwarfed cereal cultivars, with high nitrogen responsiveness and better irrigation facilities. Past cereal production, including rice is mainly due to high application of N fertilizers together with the development of high yielding varieties. Approximately 85 to 90 Million Metric tons (MMt) of nitrogenous fertilizers are added to the soil worldwide annually up from 1.3 MMt in 1930 and 10.2 MMt in 1960 (Frink et al., 1999) and this is predicted to increase up to 240 MMt by the year 2050 (Tilman et al., 2002). Nitrogen fertilization has been used for decades to increase crop yield with relatively low efficiency since a considerable fraction (up to two-third) of N input accumulates as runoffs (Frink et al., 1999), which dramatically affect the N cycle and associated processes (Vitousek et al., 1997 and galloway et al., 2008). The Nitrogen Use Efficiency (NUE) which may be defined as the yield obtained per unit of available N in the soil has declined sharply with increasing application of nitrogenous fertilizer. In most intensive agricultural production systems, over 50% and up to 75% of the N applied to the field is not used by the plant and is lost by leaching into the soil (Raun and Johnson, 1999) and nitrogen use efficiency is only 33% (Abrol et al., 2007). The NUE of cereals, including rice, may be low because of modern breeding methods where selection trials are routinely carried out with sufficient to excess N provision often based on previous best practices and lines selected which respond to the applied N levels in the soil (Kamprath et al., 1982). N fertilization introduced N into the environment largely that resulted in significantly negative environment consequences (Brown, 2011). Nitrogen lost from agricultural system will entered to groundwater, lakes, estuaries and coastal water where the reactive nitrogen can participate and induces death of aquatic life as well as harms human and animal population as its negatively influence on drinking water availability. The production and extensive application of N fertilizer also contributed in major environmental problems due to soil leaching and greenhouse gas emission that play a large role in ozone depletion and global warming (Donner and Kucharik, 2008). In addition to these negative environmental effects, synthetic nitrogen fertilizer is typically the single highest input cost for many crops, since commercial fertilizer production (via Haber Bosch method) is energy intensive process. Agriculture sector activities (mainly nitrogen fertilizer use) are the main contributor of global anthropogenic N2O emission (ca. 58%), soil deterioration and nutrients imbalance (Wuebbles, 2009). Nitrous oxide (N2O) is the third most abundant greenhouse gas (GHG) with only carbon dioxide (CO2) and Methane (CH4) being most prevalent (Montzka et al., 2011) and is a 300 times more potent GHG than CO2 (Johnson et al., 2007). India share 49% N2O emission in 2005 (Out of 267 Gg, where Gg=1000000 kg) compared to 40% in 1985 (144 Gg) (Garg et al., 2012). In order to reduce eutrophication and the costly component of crop production, there is an immediate need to reduce N fertilizer inputs. The concept of sustainable agriculture leads to conservation of natural resources with the 1248 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1247-1265 mitigation of climate change and increasing the productivity tend to move towards organic agriculture. It is based on minimizing the use of external inputs through use of on-farm resources efficiently compared to intensive agriculture and thus the use of synthetic fertilizers is avoided. Awareness of organic food for health conscious people steadily increases. There is an annual average growth rate of 20-25% (Ramesh et al., 2005) increase in organic products demand. Worldwide over 130 countries produce certified organic products in commercial quantities (KortbechOlesen, 2000). An important issue to the acceptance of organic agriculture is found in the question of its low productivity. At present carrying capacity of organic agriculture only for 3-4 billion, well below the present world population (≈ 7 billion) and that is projected up to 9 billion for 2050 (Connor, 2008). To meet food requirements of this future population FAO estimates that food production will have to increase by 70 %. In organic agriculture, crop productivity is mainly limited due to nitrogen availability which is not easily controllable (Owen and Jones, 2001). The N availability dependent on mineralization of crop residues and farm yard manures applied on the farm. In early crop growth stages when demand is low, N is lost while in later stages the demand from the plant is often much greater than the supply from mineralization and matching N need and mineralization one of the major limiting factors in organic agriculture system. One of the basic principles of soil fertility management in organic agriculture is that crop nutrition depends on biologically derived nutrient. Organic residues of low nutrient content (FYM, vermicompost, green manure etc.) added to the soil surface or incorporated into soil undergo decomposition by soil microbes. In addition to the readily available ammonium and nitrate ions, soil of organic agriculture can contain a wide range of organic nitrogen compounds such as peptides, proteins, free amino acids, amino sugars and nitrogen heterocyclic compounds (Mader et al., 2002; Jones et al., 2002). The organic nitrogen fraction typically comprises 0.1 to 0.5 % of total soil N (Barber, 1984). NO3- and NH4+ ions are usually considered to be the main N forms in soil solution taken up by crop plants and to a lesser extent as proteins, peptides or amino acids. Some agricultural crop species have been shown to absorb organic N (Okamoto and Okada, 2004), for example, in rice, the N uptake rate increases with organic N supply rather than nitrate application (Yamagata and Ae, 1996). Studies on plant grown in solution culture or upon excised roots have also demonstrated that uptake of organic N can occur at a rate comparable to or in excess of N uptakes from inorganic N sources (Chapin et al., 1993; Raab et al., 1999). Under organic agriculture crop productivity mainly limited due to nitrogen availability (Mader et al., 2002) and additionally literature suggested that we need to breed differently for organic agriculture to increase their efficiency for nitrogen utilization (Sharma and Bardhan, 2017, Sharma et al., 2017). Moreover acquisition of the various N forms is regulated not only by their chemical nature and spatial availability in the soil, but also by root system architecture, transport system in the plasma membrane of root cells and mechanism that regulate the activity of N transport systems and root growth, depending on plant requirements. From a fundamental point of view, the influence of nitrogen on root development is still poorly understood. Modifying root architecture is a strategy that aims at developing crops that capture nutrients more efficiently and are thus suitable for sustainable agriculture with fewer fertilizer inputs. The present investigation aims to study 1249 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1247-1265 the responses of rice root system architecture to availability of different forms of nitrogen at seedling stage. However such experiment cannot be peprformed in the soils because of spatial and temporal variability of nitrogen in the soil, thus we choose to conduct our experiment in solid growing media under laboratory condition. Materials and Methods The study was conducted at the plant tissue culture laboratory, N.M. College of Agriculture, Navsari Agricultural University, Navsari, Gujarat, India (20.9467° N, 72.9520° E) in September-October 2017. The rice seeds of IR-28 were provided by Regional Rice Research Station, Navsari Agricultural University, Vyara. In the study, polystyrene made optically clear and gamma irradiated pre-sterilized square petri dishes (125 x 125 x20 mm3) having 139 cm2 of growing area were used. Growing condition The seeds were surface sterilized by immersing in 0.1 percent mercuric chloride for 10 minute. Then seeds were washed thoroughly with deionized water and before placing in nutrient media, the seeds were soaked for 24 hours in deionized water. Three rice seeds were placed in one petri dish (Fig. 1). The cultured dishes were placed in vertical orientation in the racks of culture room of the laboratory. The culture room was maintained at 25oC ±1oC, 60-70% RH with providing 12 hours of photoperiod at about 2000 lux by cool white fluorescent tube light. Growing media Yoshida’s solution (Yoshida et al., 1976) was used as a growing media and each petri dish was poured with 100 ml volume. The culture media was substituted with different sources of nitrogen on the basis of molar mass and the pH was maintained at 5.7 after mixing all the stock solution. Before pouring the media into the square dish the media was autoclaved at 121ºC for 2 hrs.8 g/L Phytagel was added for the solidification of culture media. Nitrogen treatment application The experiment consist of fourteen treatments viz., three levels of nitrogen sources (nitrate, ammonia and organic nitrogen) and their combination with four levels of concentrations (50, 75, 100 and 125% N) besides two controls (control – I, 100 % N as in Yoshida solution and control- II, 0% N, Yoshida solution). According to the experimental treatments, all the individual stock solution prepared and calculations of different nitrogen sources and its level were calculated by using total nitrogen concentration in the standard Yoshida solution. In standard Yoshida solution, nitrogen was supplied by NH4NO3 (91.4 g/lit). As per our experimental treatments, the nitrogen was substituted by using different sources and desired concentration level were maintained on the basis of molecular mass (Table 1 to Table 3). NH4NO3, KNO3, (NH4)2SO4 and amino acid mixture (Glycine, L- Glutamic acid and L-Aspartic acid) were used for substitution of nitrogen from the Yoshida solution. Root system analysis Total 126 petri dishes were used for the experiment. The experiment was laid down in complete randomized design with three repetitions. Total 126 images of root system were captured by digital camera (Sony Cybershot) on 21 day after emergence (3 images per 1250 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1247-1265 treatment per repetition). These images were processed as per the guidelines of the EzRhizo software (Armengaud et al., 2009) and the data obtained was converted to mean value for each parameter (Fig. 2). Table.1 Substitution of nitrate as sole N source (g/lit) Chemicals KNO3 CaNO3 CaCl2 HCl 50% 82.85 gm 38.10 gm 70.69 gm 9.88 ml 75% 82.85gm 105.45gm 39.04gm 27.36 ml 100% 82.85 gm 172.89 gm 7.33 gm 44.86 ml 125% 82.85 gm 236.44 gm -48.91 ml Table.2 Substitution of NH4+ as sole N source (g/lit) Chemicals NH4H2PO4 (NH4)2SO4 NH4Cl 50% 29.71 gm 58.36 gm -- 75% 29.71 gm 96.02 gm -- 100% 29.71 gm 133.74 gm -- 125% 29.71 gm 133.74 gm 30.55 gm Table.3 Substitution of organic nitrogen as sole N source (g/lit) Amino acid 50% 28.56 gm Glycine L- glutamic acid 55.98 gm L- Aspartic acid 50.65 gm 75% 38.53 gm 83.93 gm 75.92 gm 100 % 57.13 gm 111.97 gm 101.29 gm 125 % 71.44 gm 140.01 gm 126.67 gm difference among treatment found significant. Results and Discussion Other observations Nitrogen content in shoot and root samples were observed by weight digestion method along with shoot and root dry weight per seedling at 21 days after emergence. Statistical analysis All mean values were subjected for statistical analysis for control vs rest design of factorial concept (Panse and Sukahtme, 1978) of the experiment and analysis was performed by department of agricultural statistics, N. M. College of Agriculture, NAU, Navsari. The significance of difference was tested by ‘F” test at five per cent level. The critical differences was calculated whenever the Needless to say, thus the ability of plants to quickly and efficiently modulating its root architecture may determine its comparative success and productivity in nitrogen limiting environments. Under such condition plants activate foraging responses that induced morphological changes and modulated root system architecture, besides physiological and metabolic changes. Both nutritional status of the plant and the external nutrient availability can induce changes in overall root morphology (Giehl et al., 2014). Root system architecture (RSA) defined as the spatial configuration of root system is the fundamental aspect in plant productivity 1251 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1247-1265 (Lynch, 1995). The ability of a plant to modify the RSA based on nutritional status of the surrounding, so called plasticity is currently the most accepted target trait for nitrogen use efficiency. The results of primary root length (Fig. 3A) indicated significant increase in root length due to nitrogen unavailability. The primary root length of rice seedling was found significantly influenced by nitrogenous forms and their different levels. Results indicated that in control-I (100 % N, Yoshida solution) significantly minimum root length was observed while control-II (0% N, Yoshida solution) recorded significantly maximum root length. Deficiency in N results in a shift in dry matter in favor of root growth. (Ericsson 1995) and thus higher root growth was observed in control- II. Similarly in nitrogen deficiency, increase in primary root growth was observed by other workers (Linkohr et al., 2002, Lopez-Bucio et al., 2003). Various forms of nitrogen also influenced primary root length and minimum root length was observed with nitrate form which was statistically at par with ammonical form and control- I. Root proliferation and overall plant growth are usually greater with mixture of NH4+ and NO3- than with either form alone (Wang and Below 1992, Saravitz et al., 1994, Schortemeyer and Feil, 1996) as indicated in 100 % N Yoshida solution (control-I). Rice is known as efficient for NH4+ (Wang et al., 1993) as well as exceptionally efficient in absorbing NO3- also (Duan et al., 2006). Maximum primary root length was recorded for organic nitrogen nutrition, which also exhibited maximum carbon allocation and up to 33 % of shoot biomass reduction was recorded as compared to control- I (100 % N Yoshida solution) (Fig. 5). This suggesting nitrogen starvation in IR-28 seedlings under organic nitrogenous nutrition. Root length under various levels also supported the above discussed fact that nitrogen deficiency increases the root length and maximum and minimum root length were recorded with 50 % and 125 % levels of nitrogen, respectively. However, at 125 % of nitrogen, decreased in root length was observed with nitrate and ammonical form but not in organic form. Reduction in ammonical nitrogen may be attributed to its toxicity. Although rice is known as NH4+ tolerant species (Wang et al., 1993) can be negatively affected by elevated NH4+ levels (Balkos et al., 2010). Excessive NH4+ is also known to inhibit the growth of most crop species (Roosta and Schjoerring 2008). Infact stunted root growth is considered as principal symptom of ion toxicity (Gerendas et al., 1997; Britto and Kronzucker, 2002, Balkos et al., 2010). However, in the present study, at 125 % level of nitrogen, nitrate nitrogen found more toxic than ammonical nitrogen. In several crop species, genetic variation in axial root growth angle is associated with rooting depth as in common bean and maize shallow growth angles enhances top soil foraging and acquisition of top soil resources such as phosphorus (Zhu et al., 2005; Lynch 2011). In common bean, wheat, sorghum and rice, steep growth angles enhances subsoil foraging and water acquisition under terminal draught (Ho et al., 2005; Manschadi et al., 2008; Uga et al., 2011; Mace et al., 2012). In the present study, main root angle (O) (Fig. 4G) tend to steeper in nitrogen deficit treatments and maximum angle was recorded for control-I while significantly narrow angle was recorded in control-II. Nitrate and ammonical nitrogen had similar effect on root angle while organic nitrogen treatment significantly decreases the main root angle. Previous reports suggesting no influence of nitrogen on root angle (Forde and Lorenzo, 2001), However, Trachasel et al., (2013) reported decreased brace and crown root angle under low N condition in maize. In the present study, straightness of main root (Fig. 4H) was 1252 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1247-1265 not significantly influence by nitrogen availability, though form of nitrogen significantly influencing straightness of main root and in organic form, it was maximum as compared to ammonium and nitrate. This might be due to the fact that in organic nitrogenous treatment, seedling experiencing nitrogen limitation as supported by all other traits and thus root angle decrease more in organic and increases straightness as compared to ammonia and nitrate. lateral root primordia and thus lateral root emergence is hampered. In present study, significantly lower lateral root development was observed in 50% nitrogen supply. Though, it seems that in present study, rice seedlings were not experiencing severe nitrogen deficiency as reported elsewhere that in severe deficiency, total lateral root length was decreased and completely absent of lateral root formation (Krouk et al., 2010, Gruber et al., 2013). Lateral root number (Fig. 3B) of rice significantly varied in different forms of nitrogen nutrition and significantly maximum lateral root numbers were observed under nitrate nutrition while lowest number recorded with organic form of nitrogen. It has been shown that, nitrate stimulates lateral root growth by regulating auxin activity and it increases auxin accumulation in primary root tips (Vidal et al., 2010). There are strong connections reported between auxin and nitrate signaling, which could cooperatively regulate lateral root development (Zhang et al., 1999; Gutierrez et al., 2007, Tian et al., 2008; Krouk et al., 2010; Mounier et al., 2014). Moreover, lateral root length and sum of lateral root length was decreased at higher dose of nitrogen nutrition (Fig. 3 C and 3 D). It may be due to besides auxin signaling, high nitrogen may affect auxin concentration also. For instance, when external nitrate concentration was greater, the elongation of lateral root in maize was inhibited, which was due to reduction of auxin translocation from shoot to root in phloem (Tian et al., 2008). Therefore, a reduced auxin level in root resulted in the inhibition of lateral root growth. Moreover higher concentration of nitrate also inhibited lateral root growth through ABA signaling (Signora et al., 2001, Vidal et al., 2010). Lateral root density of IR 28 seedling (Fig. 4E) was found more in nitrate as compared to ammonical and organic nitrogen nutrition. As in the present study, along with lateral root number, sum of total lateral root length (Fig. 3D) was also recorded maximum in nitrate nitrogen. However, lateral root number, mean lateral root length and sum of lateral root length were significantly higher recorded in control-I (100% N Yoshida solution), where both nitrate and ammonical nitrogen were available. It seems to have complementary effects of availability of nitrate and ammonia on lateral root development because ammonium stimulates branching whereasnitrate stimulates lateral root elongation (Remans et al., 2006; Lima et al., 2010). Moreover, nitrogen deficiency shown to inhibit lateral root emergence as Krouk et al., (2010) suggested that severe nitrogen limitation caused less auxin accumulation in However, at higher concentration of supply, in all forms of nitrogen, lateral root density was decreased. It may be attributed to root plasticity where plant roots adopt an economic saving model with lateral root inhibition to reserve energy and carbon skeleton for other usage. Similar to our results, where at 100% of nitrogen nutrition, greater lateral root density, lateral root number and sum of lateral root length observed, in barley also nutrient rich ‘patch’ elicit in lateral root initiation and elongation (Drew, 1975). 1253 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1247-1265 Table.4 Shoot dry weight (mg/plant) of rice seedlings as influenced by various nitrogenous treatments Level of nitrogen Forms of nitrogen NO3- 50% 75% 100% 125% Mean Control-I (100% N Full Yoshida solution) mean Control-II (0% N Yoshida solution -N) mean 20.2 24.1 27.5 18.9 22.6 NH4+ Mean ANOVA Organic 20.5 22.5 23.6 19.3 21.5 18.0 18.0 18.8 17.6 18.1 19.6 21.5 23.3 18.6 20.73 28.1 17.5 Treatment Nitrogen forms (N) Level of nitrogen (L) NxL 0 % N vs Rest 100% vs 0% Nitrogen CV % S.Em± CD at 5% 0.55 0.28 0.32 0.55 0.41 0.55 1.60 0.80 0.93 1.60 1.18 1.60 4.55 Table.5 Root dry weight (mg/plant) of rice seedlings as influenced by various nitrogenous treatments Level of nitrogen Forms of nitrogen NO3- 50% 75% 100% 125% Mean Control-I (100% N full Yoshida solution) mean Control-II (0% N Yoshida solution N) mean 14.2 14.7 17.5 13.2 14.9 NH4+ Mean ANOVA Organic 14.0 15.2 18.2 13.5 15.3 13.3 13.7 14.0 13.7 13.7 13.8 14.6 16.6 13.5 14.6 18.7 13.5 Treatment Nitrogen forms (N) Level of nitrogen (L) NxL 0 % N vs Rest 100% vs 0% Nitrogen CV % 1254 S.Em± CD at 5% 0.45 0.22 0.26 0.45 0.33 0.45 1.30 0.65 0.75 1.30 0.96 1.30 5.24 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1247-1265 Table.6 Nitrogen content in shoot (mg.g-1) of rice seedlings as influenced by various nitrogenous treatments Level of nitrogen Forms of nitrogen 50% 75% 100% 125% Mean Control-I (100% N Full Yoshida solution) mean Control-II (0% N Yoshida solution -N) mean Mean NO3- NH4+ Organic 1.92 2.15 2.94 1.33 2.09 1.49 1.63 2.21 1.46 1.70 1.29 1.25 1.15 1.09 1.20 1.57 1.68 2.10 1.29 1.66 3.06 1.07 ANOVA Treatment Nitrogen forms (N) Level of nitrogen (L) NxL 0 % N vs Rest 100 % vs 0% Nitrogen CV % S.Em± CD at 5% 0.03 0.01 0.01 0.03 0.02 0.03 0.07 0.04 0.04 0.07 0.05 0.07 2.57 Table.7 Nitrogen content in root (mg. g-1) of rice seedlings as influenced by various nitrogenous treatments Level of nitrogen 50% 75% 100% 125% Mean Control-I (100% N full Yoshida solution) mean Control-II (0% N Yoshida solution N) mean Forms of nitrogen NO3- NH4+ Organic 0.76 0.91 1.36 0.81 0.96 0.99 1.04 1.14 0.75 0.98 0.70 0.73 1.01 0.96 0.85 Mean 0.81 0.89 1.17 0.84 0.93 1.48 0.52 ANOVA Treatment Nitrogen forms (N) Level of nitrogen (L) NxL 0 % N vs Rest 100% vs 0% Nitrogen CV % 1255 S.Em± CD at 5% 0.02 0.01 0.01 0.02 0.02 0.02 0.06 0.03 0.04 0.06 0.05 0.06 3.91 Int.J.Curr.Microbiol.App.Sci (2018) 7(7): 1247-1265 Figure.1 Root System Architecture of IR-28 at 21 days after emergence, under varied nitrogen sources and availabilities 1256