Full Length Research Paper

Oxidative stress is considered a major cause of cellular injuries, including various clinical abnormalities. Here, the neuroprotective effect of extracts from the calyx of Diospyros kaki (DCE), peel of D. kaki (DPE), and flesh of D. kaki (DFE) of Diospyros kaki on oxidative stress-induced apoptosis in PC12 cells was investigated by an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) reduction assay. The results showed that H2O2 significantly decreased cell viability (50.9%), and DCE exerted the highest neuroprotective effect on H2O2-induced cytotoxicity. We identified that treatment with DCE of H2O2-stressed PC12 cells caused dose-dependent suppression of H2O2-induced leakage of lactate dehydrogenase (LDH) as released amount (11.1 to 30.4%); we further verified these findings by observing morphological features. H2O2 also induced severe apoptosis of the PC12 cells, as determined by Hoechst 33342 staining and flow cytometric analysis. DCE (100, 500 μg/ml) exerted a significantly high protective effect on PC12 cells against H2O2-induced cell injury, and the percentage of cells in the sub-G1 phase decreased to 3.7 and 4.6%. These results suggest that DCE can protect cells against H2O2-induced apoptosis, and might be a potential therapeutic agent for treating or preventing neurodegenerative diseases that are related to oxidative stress.


INTRODUCTION
Rice is the most vital food crop and a major food grain for more than a third of the world's population (Zhao et al., 2011).Its most important and essential plant nutrient is nitrogen and will increase the crop yield positively (Dastan et al., 2012).Chlorophyll, a green pigment present in plants, captures the sunlight that is used in photo synthesis (Swain and Jagtap, 2010).Rice plant needs a sufficient supply of nutrients from several sources for optimal growth.These nutrients are supplied by indigenous sources such as soil minerals, soil organic matter, rice straw, manure, and water (rain, irrigation), but the amount supplied is usually insufficient to achieve high and sustainable yields.However, the use of organic manures alone might not meet the plant requirement due to presence of relatively low levels of nutrients.Therefore, in order to make the soil well supplied with all the plant nutrients in the readily available form and to maintain good soil health, it is necessary to use organic manures in conjunction with inorganic fertilizers to obtain optimum yields (Rama Lakshmi et al., 2012).Many of our problems on declining productivity (increasing cost, declining yield) can be traced to imbalanced and inefficient use of nutrients.Improper nutrient management has resulted in the nutrient imbalances in *Corresponding author.E-mail: drkrishnakumar76@gmail.com.

S.No
Character/cultural operation Details 1.
Date of harvesting 05-10-2004  Technology Bulletin Series, 2002).Awareness about crop quality and soil health increased the attention of people towards organic farming (Sharma et al., 2008).Plant based tools for real time nitrogen management in rice through use of Leaf Color Chart (LCC) is becoming progressively popular in South and South East Asian rice farming.Leaf color chart (LCC) is made of high quality plastic material.It consists of six colour ranging from light yellowish green (NO1) to dark green (NO6) colour strips fabricated with veins resembling those of rice leaves (Nachimuthu et al., 2007).It has been observed that more than 60% of applied nitrogen is lost due to lack of harmonization between the nitrogen demand and nitrogen supply (Yadav et al., 2004) Farmers normally apply nitrogen fertilizer in fixed time advocated N split schedule (Pillai et al., 1993) in 1:2:1 or 2:1:1 ratio at basal, extreme tillering and panicle initiation phases respectively, without taking into account whether the plant really requires N at that time which may lead to loss or may not be found sufficient enough to harmonize nitrogen supply with actual crop nitrogen demand (Ladha et al., 2000).

Location
The experiment was conducted in low land series of wet lands of International Rice Research Institute (IRRI) Farm, Los Banos, Laguna, Philippines.

Crop and variety
Rice variety PSBRc 82 was raised during wet season (June to October 2004).The details of important cultural operations are shown in Table 1, such as sowing, transplanting and harvesting are as furnished below.

Treatment details
The field experiment was conducted in Randomized Block Design with 11 treatment combinations replicated thrice and treatment details are as follows (Table 2).

Layout of the field experiment
The field was divided into 44 plots of 15 m 2 size with adequate

Application of organic manures and fertilizers
There are three treatments involving various sources of organic manures such as fresh rice straw (FRS), rice straw compost (RSC) and poultry manure (PM) applied on equal N basis.These manures were analyzed for N, P and K contents and the quantity of organic manures used to supply 90 kg ha -1 were calculated and applied at ten days prior to transplanting.The treatment that received fertilizer N @ 90 kg ha -1 , P2O5 @ 40 kg ha -1 and K2O @ 60 kg ha -1 received the nutrients in the form of urea, super phosphate and muriate of potash.Half the dose of N and K2O and full dose of P2O5 were applied basally and the remaining half of the N and K2O were applied in 2 equal splits at tillering and panicle initiation stages.The absolute control treatment received no manures or fertilizers.

Amount of N added through treatments
The amount of nitrogen added through treatments are as shown in Table 3.

Biometrical observations
Five plants were selected at random and tagged for recording biometric observations.

Plant height
Plant height was recorded from ground level to the tip of the top most leaf or panicle and the mean height was worked out and expressed in centimeter.

Number of tillers and productive tillers m -2
Number of tillers was counted at tillering and flowering stages and productive tillers m -2 was counted at harvest stage.

Panicle length
Five panicles were taken from each plot and the length was Krishnakumar and Haefele 2061 measured from the scar of the panicle to the tip of the panicle and the mean panicle length was expressed in centimeter.

Number of grains per panicle
Five panicles were taken from each plot and the number of grains per panicles was counted and the mean number of grains of panicle was worked out.

Thousand grain weight
One thousand grains from each treatment were counted and the weight was recorded in gram.

Grain and straw yield
Grain and straw yields were recorded separately for each plot and expressed at 14% moisture level.

Available N
The available N status of soil was increased up to the tillering stage and there was a gradual decrease in N availability status at harvest stage (Tables 4 and 5).This might be due to the release of N from organic manures and simultaneous uptake by rice.Soil application of 50% N as RSC + 50% N as PM + RD P and K @ 90 kg N ha -1 gave significantly higher available N compared to control treatment.The per cent increase in 50% N as RSC + 50% N as PM + RD P and K @ 90 kg N ha -1 , 50% N through combined organics (RSC + PM) + 50% N as inorganic + RD P and K and 100% N FRS + RD P and K over recommended NPK fertilizer treatment (T 2 ) was 7.1, 7.0 and 6.3 respectively at tillering stage and 22.3, 19.2 and 9.3 respectively at post harvest stage of crop growth.This might be due to direct effect of decomposition and mineralization of nitrogen from various organic manures and indirectly due to rhizosphere effect in increasing the microbial activity and its impact on available N content in flooded soil.Ramesh (1998) reported that application of poultry manure increased the total as well as available N content of soil, besides increasing ammonical and nitrate N concentration in soil as compared to other organic sources.The available N content of soil decreased with stages of advancement of crop growth stages.
As could be expected, application of 140 kg N ha -1 in six splits (LCC 5) showed highest available N at different stages of plant growth compared to other LCC treatments.The higher N availability obtained due to the application of110 kg N ha -1 applied at different stages of crop growth was also reported by Michael and Behringer (1980).The lowest available N was observed in control treatment.Though the N management treatments viz., LCC 3, LCC 4 and LCC 5 did not show significant

Treatments
Available N (kg ha -1 ) Available P (kg ha -1 ) Available K (kg ha variation in soil available N during initial stages after transplanting.The LCC 5 had 3% increase in soil available N at post harvest stage.The probable reason might be the addition of higher quantity of N.

Available P
Significant difference in the available P status of the soil was observed during different stages of crop growth.The available P was observed to be higher at tillering stage and progressively decreased at the flowering and maturity stages of crop growth.The plant uptake of P was probably lower during the tillering stage and the amount of uptake increased due to increased biomass production towards maturity stage (Tables 4 and 5).The available P content of the soil was found to be significantly higher in the treatment that received 90 kg N ha -1 as 50% N through RSC + 50% N as PM + RD P and K than control treatment.The per cent increase in 50% N RSC + 50% N PM + RD P and K @ 90 kg N ha -1 and 50% N through combined organics (RSC + PM) + 50% N as inorganic + RD P and K over recommended NPK fertilizer treatment (T 2 ) was 45.7 and 30% respectively at active tillering stage and 31.3 and 28.0 respectively at post harvest stage of crop growth.Organic manures on decomposition in soil might release organic acid which would have solubilized the soil P as reported by Sripriya (1993).Among LCC treatments, LCC 4 and LCC 5 were found to give higher available P content of the soil.Johnkutty et al. (2000) also brought out similar results for increased P availability due to higher N applications.The recommended NPK fertilizer treatment gave lowest available P than organic manures treatment.

Available K
Significant variation in the available K content of the soil was observed due to the application of various sources of organic manures.The reduction in available K content from tillering to harvest stage might be due to the uptake of K by the crop and retention in clay complex (Tables 4  and 5).Application of N @ 90 kg ha -1 as 50% RSC + 50% N PM + RD P and K recorded the highest soil available K in all the stages of crop growth.The per cent increase in 50% N through RSC + 50% N as PM @ 90 kg N ha -1 , 50% N through combined organics (RSC + PM) + 50% N as inorganic fertilizer and 100% N as FRS over recommended NPK fertilizer treatment (T 2 ) was 20.6, 15.6 and 7.5 respectively at active tillering stage and 24.3, 20.7 and 20.6 respectively at post harvest stage of crop growth.The increased available K in this treatment might be due to the abundant release of K from poultry manure as well as its effects on release of soil K.Among all LCC treatments, LCC 4 showed highest available potassium.The recommended NPK fertilizer treatment showed lowest available K than organic manure treatment.
The LCC 4 (115 kg N ha -1 ) treatment showed higher available potassium at all the growth stages of rice crop which might be due to the synergetic effect of N on potassium.

Plant height
Soil application of various sources of organic manures @ 90 kg N ha -1 significantly influenced the plant height at different growth stages of the plant.Among various sources of organic manures, soil application of 90 kg N ha -1 in the form of 50% N as RSC + 50% N as PM + RD P and K showed significantly highest plant height than absolute control (Table 6).This might be due to higher available nutrients as a result of RSC and PM (Ramesh, 1998).Application of 90 kg N ha -1 in the form of 50% N as RSC + 50% N as PM + RD P and K showed significantly higher plant height of 5.4% more than the recommended dose of NPK fertilizer (T 2 ).Application of 140 kg N ha -1 in six splits under LCC 5 treatment increased the plant height which was attributed to higher amount of N addition.Positive correlation between plant height and N levels have been earlier reported by Balasubramanian et al. (2000).

Soil application of 90 kg N ha
-1 in the form of 50% N through RSC + 50% N as PM + RD P and K showed significantly higher number of tillers than absolute control treatment (Table 7).Increase in tiller production with the poultry manure application might be due to the dual benefits of improving the physical environment of rzhiosphere region and supplying plant nutrients.LCC 4 gave significantly highest number of tillers compared to LCC 3 treatment, but was on par with LCC 5.More number of early formed tillers under favourable N nutrition tends to bear more number of panicles.It may be due to higher availability and uptake of N, which is a substrate for synthesis of organic compounds, which constitute protoplasm and chlorophyll (Hatwar et al., 1992).This was the reason for the production of higher number of tillers in treatments LCC 4 and LCC 5 over the no fertilizer applied treatment.

Panicle number hill -1
Among the various treatments, application of 90 kg N ha -1 in the form of 50% N as RSC + 50% N as PM + RD P and K showed significantly higher number of panicles and also lengthier panicles (Table 8).Besides, higher nutritional status helped the production of panicle number and lengthier panicles too, with more number of filled grains and grain weight (Arumugaperumal, 2000).The treatments receiving higher N dose namely, LCC 4 and LCC 5 produced higher panicle number and lengthier panicle respectively, over no fertilizer applied treatment.Increased levels of N application in splits synchronized with the nutritional demand of rice at all the stages resulted in longer panicles.Application of 90 kg N ha -1 in the form of 50% N as RSC + 50% N as PM + RD P and K showed significantly higher number of panicles (23.1%) and 50% N combined organics (RSC + PM) + 50% N inorganic + RD P and K recorded significantly lengthier panicles (16.7%) than recommended dose of NPK fertilizer (T 2 ).The per cent increase number of panicles in treatments LCC 3 (90 kg N ha -1 ), LCC 4 (115 kg N ha -1 ) and LCC 5 (140 kg N ha -1 ) over was 9.1, 16.7 and 9.1 and LCC 5 (145 kg N ha -1 ) gave lengthier panicles than T 2 treatment.
Among various treatments, 100% N as FRS showed significantly higher filled grains panicle -1 and application of 90 kg N ha -1 in the form of 50% N as RSC + 50% N as PM + RD P and K showed lower unfilled grains panicle -1 (Table 9).Among LCC treatments, LCC 4 showed highest filled grains panicle -1 and lowest unfilled grains panicle -1 . This was due to the higher nutrient uptake and higher plant assimilation with adequate supply of photosynthates to grains, as explained by Morales et al. (2000).
Among organic manure treatments, 50% N as RSC + 50% N as PM + RD P and K (T 10 ) showed highest thousand grain weight and 140 kg N ha -1 (LCC 5) gave significantly higher thousand grain weight as compared to other LCC treatments.Asif et al. (1999) reported that the split application of nitrogen sustained the supply of N throughout the vegetative and reproductive development of plant, particularly during grain filling and ripening (Table 9).Nayak and Patra (2000) reported that the increased thousand grain weight due to N application at flowering could be attributed to the increased filled grains.The highest thousand grain weight was recorded in the 50% N as RSC + 50% N as PM + RD P and K and LCC 5 (140 kg N ha -1 ) treatments compared to recommended dose of NPK fertilizer treatment.

Yield of rice
The grain yield, straw yield and dry matter production of rice were significantly influenced by application of various sources of organic manures and other treatments.The results revealed that application of 90 kg N ha -1 in the form of 50% N through RSC + 50% N as PM + RD P and K recorded significantly higher grain and straw yield than other treatments (Table 10).High yield obtained for the application of rice straw compost and poultry manure may be attributed to the growth parameters and yield attributes viz., productive tillers, lengthier and more number of panicles besides thousand grain weight.The higher soil available nutrients as observed under this treatment would have contributed to more nutrient uptake ultimately resulting in high yield.
Among LCC treatments, LCC 4 gave highest grain yield, DMP and LCC 5 showed higher straw yield.The grain filling can be one prime reason for the yield differences.The LCC 4 had the highest filled grains and reduced unfilled grains.Due to this reason the increased grain yield was noted in LCC 4. The consumed level of ) had recorded the highest grain yield in LCC 4 which was much higher when compared to no fertilizer treatment.Stalin et al. (2000) also got the best yield of rice in the dose of 115 kg N ha -1 . Peng et al. (1993) ascribed the increased grain yield to the favourable effects of improved leaf N concentration, photosynthetic rate of flag leaves and increased filled grain percentage.A better response for N dose up to 130 kg N ha -1 in rice was reported by Stalin et al. (2000) who reported that the N doses have to be applied only after careful assessment of plant requirement through LCC and not by predetermined crop calender timings, because the need based application help in increased filledgrain per cent and thousand grain weight.Effect of different split application N-fertilizer was significantly on LCC values each of the time (Yoseftabar, 2013).Fertilizer N is applied whenever the leaves are less greenish than a threshold LCC value, which corresponds to a critical leaf N content (Singh et al., 2010).Application of nitrogen in split according to the crop needs was the reason for better rice growth parameter (Sathiya and Ramesh, 2009).
LCC is easy to use and is an inexpensive diagnostic tool for monitoring the relative greenness of rice leaf as an indicator for the plant N status and can be used as an alternative to chlorophyll meter (Sen et al., 2011).
The treatment receiving 90 kg N ha -1 in the form of 50% N through combined organics (RSC + PM) + 50% N as inorganic + RD P and K showed next best organic treatment and 100% N through FRS + RD of P and K gave lowest grain yield compared to other organic treatments.But 100% N as FRS + RD of P and K gave higher yield than absolute control as could be expected.Interestingly, it was on par with recommended NPK which may be ascribed due to the nutrient release from rice straw under lowland situations.Sodhi et al., (2009) did a study of continuous application of rice straw compost to soil properties.They apply rice straw compost either alone or in conjunction with inorganic fertilizers and studied the impacts on aggregate stability and distribution of carbon and nitrogen on a sandy loam soil.
The benefit gained by farmers is seen as the reduction of using chemical fertilizer based on the associate chemical components of the compost.According to (FAO, 2005), the recommended rate of nitrogen for rice is 95-145 kg/ha.If the straw compost contains 1.86% (Isroi, 2009) of nitrogen, about 26 kg of nitrogen is available from 1.4 ton of compost.Farmers can use this source of nitrogen and can save about one quarter of the nitrogen needs.For example, as mentioned earlier that 1 ton of rice straw compost is equivalent with 41.3 kg urea.Organic manures were often superior to inorganic fertilizer as they exhibit dual benefits of improving the physical environment of soil and also supply the plant nutrients, which might have contributed to the increased nutrient uptake and ultimately the higher grain yield.Application of 90 kg N ha -1 in the form of 50% N through RSC + 50% N as PM + RD P and K recorded significantly higher grain yield (15.6%) and straw yield (17.0%) over recommended dose of NPK fertilizer (T 2 ) treatment.The integrated nutrient management technique resulted in a positive influx of nutrients by increasing organic carbon content, available nitrogen, phosphorus, and potassium.It was found that the application of enriched poultry manure compost on equal N basis (2.3 t•ha −1 ) recorded higher yield attributes and grain yield of rice (4675 kg•ha −1 in 2007 and 4953 kg•ha −1 in 2008), which was however comparable with composted poultry manure (Sangeetha et al., 2013).

Conclusion
Application of nitrogen at 90 kg level as 50% through Rice straw compost + 50% nitrogen as poultry manure registered higher available N, P and K contents of soil during different growth stages as compared to the other treatment combinations.The growth and yield attributes were also found to be enhanced by the above treatment resulting in more grain and straw yield.With regard to N management, LCC 4 and 5 based N applications recorded higher grain yield as a result of higher soil available nutrients during the critical growth stages.

Table 3 .
Amount of Nitrogen added through treatments.

Table 4 .
Available NPK content of soil at tillering stage of PSBRc 82 rice as influenced by organic manures and LCC based N management.

Table 5 .
Available NPK content of soil at post harvest stage of PSBRc 82 rice as influenced by organic manures and LCC based N management.

Table 6 .
Height of PSBRc 82 rice as influenced by organic manures and LCC based N management.

Table 7 .
Number of tillers of PSBRc 82 rice as influenced by organic manures and LCC based N management.

Table 8 .
Panicle number and length of PSBRc 82 rice as influenced by organic manures and LCC based N management.

Table 9 .
Filled, unfilled grains and thousand grain weight of PSBRc 82 rice as influenced by organic manures and LCC based N management.

Table 10 .
Grain yield and straw yield of PSBRc 82 rice as influenced by organic manures and LCC based N management.