Mitigation of the adverse effects of soil salinity in rice using exogenous proline and organic manure

Salinity causes cellular damage and limits crop productivity. Accumulation of organic compound is one of the major adaptive mechanisms for salinity tolerance in plants. The main objective of the present study was to mitigate the adverse effects of soil salinity in rice through organic amendments like proline and organic manure. The field experiments were conducted at the farmer’s field of Batiaghata upazilla under Khulna district with aman rice. The soil was silty clay loam having pH 7.2, EC 6.6 dS/m, CEC 26 meq/100 g soil and organic matter 0.84%. Rice variety BR 23 was used as a test crop. There were 15 treatment combinations with different doses of proline and organic manures. Recommended doses of N, P, K, S and Zn fertilizers were applied to all the experimental plots. Proline was applied as foliar spray at seedling and/or vegetative stages. Organic manures were added to soils during final land preparation. The experiments were laid out in a randomized complete block design with three replications. Significant reductions in growth and yield of rice were observed under saline conditions. Application of both proline and organic manures significantly increased growth, yield contributing characters, and grain and straw yields of rice under salinity conditions. There were no significant variations in growth and yield of rice due to use of different doses of proline and organic manure. Increased nutrient uptake and K + /Na + ratio were observed in rice due to proline as well as organic manure application under saline conditions. The present study suggests that exogenous proline or organic manure confers tolerance to salinity in rice by increasing K + /Na + ratio and nutrient uptake.


Introduction
World agriculture is facing a lot of challenges like producing 70% more food for an additional 2.3 billion people by 2050 while at the same time fighting with poverty and hunger, consuming scarce natural resources more efficiently and adapting to climate change (FAO 2009 ).The lower productivity in most of the cases is attributed to various abiotic stresses.It has been projected that more than 50% of yield reduction is the direct result of abiotic stresses (Rodriguez et al. 2005).Rice (Oryza sativa L.) is a staple food for more than half of the world's population.So it is imperative to increase rice production in different rice growing ecosystems to feed the increasing world population (Khush, 2005).Soil salinity is a major concern to agriculture all over the world because it affects almost all plant functions.More than 6% of the world's land and one third of the world's irrigated land are significantly affected by soil salinity (FAO, 2008).Agriculture is the most important sector of Bangladesh's economy.Out of 2.86 million hectares of coastal and offshore lands, about 1.06 million hectares are affected by varying degrees of salinity (SRDI, 2010).Rice is mainly grown in the saline areas but the yield is very low due to lack of salt-tolerant high yielding variety and inappropriate management practices.Salinity imposes both ionic toxicity and osmotic stress to plants.Salt stress disturbs cytoplasmic K + /Na + homeostasis, causing an increase in Na + to K + ratio in the cytosol (Zhu, 2003).Plants have evolved a variety of adaptive mechanisms to respond to environmental stress including salt stress.One of the main adaptive mechanisms to salt stress in plants is the accumulation of compatible solute like proline.Increased levels of proline accumulated in plants correlate with improved salt tolerance (Okuma et al., 2004;Asharf and Foolad, 2007).Salt stress induces the accumulation of reactive oxygen species (ROS) in plant cells.The excess production of ROS is toxic to plants and causes oxidative damage to cellular constituents, leading to cell death (Banu et al., 2009(Banu et al., , 2010)).Proline has been shown to scavenge free radicals and ROS.Up-regulation of the components of antioxidant defense system offered by proline protects plant against NaCl-induced oxidative damage.In cultured tobacco cells, proline suppresses cell death and improves salt tolerance by increasing the activity of enzymes and/or expression of genes involved in the antioxidant defense systems (Hoque et al., 2007a, b;2008;Banu et al., 2009Banu et al., , 2010)).Soil salinization is a major process of land degradation that decreases soil fertility and crop productivity.There is a report that coastal regions of Bangladesh are quite lower in soil fertility (Haque, 2006).The best means of maintaining soil fertility, productivity and salt tolerance could be through addition of organic manures.Soil amendments with organic manure reduce the toxic effects of salinity in various plants (Idrees et al., 2004;Abou El-Magd, 2008;Leithy et al., 2010;Raafat and Tharwat, 2011).The role of organic compounds like proline and organic manures in the mitigation of salt stress in plants is yet to be elucidated in context of Bangladesh.The present study is crucial for the improvement of salinity tolerance and economic crop production in the coastal areas of southern Bangladesh.Therefore, the present study was undertaken to investigate the effect of exogenous application of proline and organic manure on the growth and yield as well as nutrient uptake of rice under saline condition.

Field experimentation
The field experiments were carried out at the farmer's field of Batiaghata upazilla under Khulna district to investigate the improvement of soil salinity tolerance in rice by organic manure (farmyard manure and poultry manure) and proline application.The soil of Batiaghata upazilla was extremely saline due to higher EC value (6.4 dS/m) and due to this salinity rice production shows significant reduction in yield.The experiment was conducted here so that the performance of exogenous proline and organic manure application could be assessed under field condition.The experiment was laid out in a randomized complete block design (RCBD) with three replications.Rice cultivar BR23 was used as a test crop in this study.The total numbers of plots used in this study were 45.The unit plot size was 4m×2.5m and the plots were separated from each other by ails (1m).Treatments were randomly distributed within the block.

Management practices and proline application
Recommended doses of Urea, TSP, MP, gypsum and zinc sulphate were applied as the source of N, P, K, S and Zn (BARC, 2012).Recommended doses of fertilizer were applied to all experimental plots during final land preparation.Urea was applied in three split doses; first dose was applied after ten days of transplanting, second dose was applied at active tillering stage, and third dose was applied at panicle initiation stage.Weeding and other management practices were performed as and when necessary.Birtako 10g (2g dissolved in 10 L water) was applied as plant protection measures when insect, pests and diseases were appeared.Proline solutions were sprayed over plant leaves with the help of a sprayer.For making 25 mM, 50 mM and 100 mM proline solutions, 2.88 g, 5.76 g and 11.52 g of proline powder were dissolved in 1000 ml of water and 1 ml of Tween-20 was properly mixed with it respectively.Tween-20 was used as a sticky substance which helps proline solution's droplet, maintaining a close contact with plant leaves.

Crop harvesting and data collection
Maturity of crop was determined when about 90% grains became golden yellow.The crop was harvested on 29 December, 2014 at 120 days after transplanting.Grain and straw yield as well as plant growth parameters (Plant height, number of effective tillers per hill, panicle length and 1000 grains weight) were recorded after harvesting.

Chemical analysis of the plant samples
Grain and straw samples of maize were analyzed for N, P, K, S and Na contents following standard method as described by Khanam et al. (2001).

Statistical Analysis
Data were analyzed statistically using analysis of variance (ANOVA) to examine the treatment effects.The mean differences were adjudged by Duncan's Multiple Range Test (DMRT) (Gomez and Gomez, 1984) and ranking was indicated by letters.

Growth and yield components
Soil salinity caused a significant decrease in different growth parameters e.g.plant height, number of effective tillers per hill, panicle length and 1000 grains weight as well as grain and straw yield of rice.Table 1 show that addition of FYM and PM to soil significantly increased different growth parameters and yield of rice cultivar (BR23) under saline conditions.It was found that application of 8 t ha -1 PM achieved the highest plant height among all the treatments while lowest in control.The number of effective tillers per hill, panicle length and number of grains per panicle of BR23 was also found the lowest in control (no manure) and the highest was found when the soil was amended with FYM (10 t ha -1 ).Lakhdar et al. 2008 also found that soil salinity can be reduced by organic matter amendment in soil and growth performance of crops was better with organic matter addition in soil under saline condition.Tejada et al. 2006 also found that organic matter application reduced salinity and increased the growth and yield components of rice.Leithy et al. 2010 Zaki et al. (2009) found that organic manure increased all the vegetative growth parameters and vegetative yield under saline conditions.Amanullah (2008) showed that among the reclamation practices of coastal saline soils, FYM recorded better growth, yield parameters, yield and nutrient uptake by rice crop.From the second experiment, proline application also significantly increased different plant parameters and grain and straw yield of BR23 (Table 2).It was found that 100 mM proline application at seedling and vegetative stage achieved the highest plant height among all the treatments.The number of effective tillers per hill, thousand grains weight and panicle length was the highest when the plants were treated with 50 mM proline at both seeding and vegetative stages.Deivanai et al. (2011) conducted an experiment on rice to ameliorate the adverse effect of salinity and found that different growth components significantly decreased due to salinity but proline application increased the growth and yield components.Dogan (2011) also found that salinity decreased growth parameters in soybean and proline application ameliorates the adverse effect of salinity.

Grain and straw yields
Soil salinity caused significant reduction in grain and straw yields of rice.It is evident from the present experiment that addition of organic manure and exogenous application of proline increased both grain and straw yields of rice.From Figure 1, it was found that the highest grain yield of BR23 was found for application of PM 8 t ha -1 while FYM 10 t ha -1 and PM 4 t ha -1 showed same grain yield while the lowest grain yield was produced in control.The highest straw yield was found in PM 8 t ha -1 and the lowest was in control.The similar observations were also found by Lakhdar et al. 2008 andTejada et al. 2006 who also concluded that soil salinity problem could be ameliorated by the addition of organic matter and yield of crops increased due to addition of organic manure and this was probably due to higher uptake of nutrients by plants.Sharma (1997) reported that application of FYM alone or in combination with inorganic amendments increased the grain yield of wheat and rice in salt affected soils.Haq et al. (2001) reported that combined application of gypsum and FYM produced the highest rice yield in a saline-sodic soil.Proline application also significantly increased both grain and straw yields of rice (Figure 2).Application of 50 mM proline at seedling and vegetative stages produced the highest grain and straw yields of BR23 and the lowest was found in control.Islam et al. (2011) on hybrid rice also reported that grain yields decreased by the salinity.Ali et al. (2004) also found the similar result on grain yield of rice.Miah et al. (1992) on two rice varieties also found that salinity decreased straw yield of rice.Talat et al. (2013) observed that the exogenous application of proline significantly ameliorated the harmful effects of salt stress on wheat.Where, T 0 = Control (no manure), T 1 = FYM (5 t ha -1 ), T 2 = FYM (10 t ha -1 ), T 3 = PM (4 t ha -1 ), T 4 = PM (8 t ha -1 ).Where, T 0 = Control (no proline), T 1 = 25 mM proline at seedling stage, T 2 = 25 mM proline at vegetative stage, T 3 = 25 mM proline at seedling and vegetative stages, T 4 = 50 mM proline at seedling stage, T 5 = 50 mM proline at vegetative stage, T 6 = 50 mM proline at seedling and vegetative stages, T 7 = 100 mM proline at seedling stage, T 8 = 100 mM proline at vegetative stage, T 9 = 100 mM proline at seedling and vegetative stages.Where, T 0 = Control (no manure), T 1 = FYM (5 t ha -1 ), T 2 = FYM (10 t ha -1 ), T 3 = PM (4 t ha -1 ), T 4 = PM (8 t ha -1 ).Where, T 0 = Control (no proline), T 1 = 25 mM proline at seedling stage, T 2 = 25 mM proline at vegetative stage, T 3 = 25 mM proline at seedling and vegetative stages, T 4 = 50 mM proline at seedling stage, T 5 = 50 mM proline at vegetative stage, T 6 = 50 mM proline at seedling and vegetative stages, T 7 = 100 mM proline at seedling stage, T 8 = 100 mM proline at vegetative stage, T 9 = 100 mM proline at seedling and vegetative stages.

Total nutrient uptake
We investigated whether organic manure and exogenous application of proline influenced nutrient uptake by rice under salt stress.To investigate the nutrient uptake by rice, we measured nutrient content in grain and straw of rice.Salt stress caused reductions in nutrient (NPS) uptake by grain and straw of rice.On the other hand, application of organic manure (FYM and PM) and exogenous proline increased nutrient (NPS) uptake by rice under saline conditions.Addition of organic manure resulted in an increase in nutrient uptake by rice under saline condition.From Table 3, it was found that total N (88.54 kg/ha), P (20.58 kg/ha) and S (19.17 kg/ha) uptake was the highest when soil was amended with 8 t ha -1 poultry manure while the lowest was found from untreated control and it was N (75.80 kg/ha), P (15.89 kg/ha) and S (15.47 kg/ha).There are evidences that organic manures reduce the adverse effects of various stresses on plants by affecting the uptake and accumulation of inorganic nutrients (Zaki et al. 2009. Abou El-Magd et al. 2008).Exogenous application of proline also showed higher uptake of nutrients under saline condition (Table 4).The highest total N (90.45 kg/ha), P (21.95 kg/ha) and S (19.07 kg/ha) uptake was found when the plants were treated with 100 mM proline at both seedling and vegetative stages and the lowest nutrient (NPS) uptake was found in untreated control plants.Hamed et al. 1994 on maize also reported that total nutrient uptake was decreased due to salinity and increased due to exogenous application of proline.Momayezi et al. (2010) also found that salt stress decreased the total nutrient uptake by rice.Kumar and Sharma (1989) reported that nutrient uptake in mungbean was reduced due to salinity and it can be increased by exogenous application of proline.

K + /Na + ratio
Potassium (K) and sodium (Na) are most important element for the plant to survive in salt stress.Salt tolerance is directly associated with K contents because of its involvement in osmotic regulation and competition with Na.Plant salt tolerance requires not only adaptation to Na + toxicity but also the acquisition of abundant K + whose uptake by the plant cell is affected by high external Na + concentrations.Soil salinity significantly decreased K + /Na + ratio in both grain and straw samples of rice.Addition of organic manure (FYM and PM) and foliar application of proline resulted in a significant increase in K + /Na + ratio in rice.Organic manure application significantly increased K + /Na + ratio in grain and straw of BR23 under stress condition.All rates of organic manure application increased the K + /Na + ratio in grain and straw.In grain the highest K + /Na + ratio was found by both in PM (4 t ha -1 ) and PM (8t ha -1 ) and lowest in control.In straw K + /Na + ratio was observed highest at PM 4t ha -1 and lowest was observed in control (Table 3).Shazia et al. (2004) also found that salinity decreased K + /Na + ratio and it could be increased by organic matter amendment in soil.All the doses of proline increased the K + /Na + ratio in grain and the same trends were found in straw (Table 4).The higher K + /Na + ratio in grain was found when the plants were treated with 50 mM proline application at vegetative stage and 100 mM application at vegetative stage.A slight variation in straw K + /Na + ratio was observed among the doses of proline application.Proline application at different doses showed insignificant difference in K + /Na + ratio of rice straw samples but significant difference was observed between proline treated and untreated plants.The highest K + /Na + ratio of rice straw was found when the plants were treated with 100 mM proline at seedling and vegetative stages.There are evidences that exogenously supplied proline reduced Na + accumulation under salt stress and increases K + /Na + ratio (Zhu, 2003).Ahmed et al. (2011) also conducted an experiment on young olive and also found that exogenous application of proline increases K + /Na + ratio significantly.Haq et al. (2009) on seven rice varieties, Nounjan et al. (2012) on Thai aromatic rice and Miah et al. (1992) on two rice varieties at different salinity level also found that salinity decreased K + /Na + ratio and proline application increases the K + /Na + ratio in rice.

Conclusions
Salt stress caused a significant decrease in growth and yield of rice.Foliar application of proline and organic manures both FYM and PM significantly improved growth and yield of rice by mitigating the inhibitory effects of salinity stress.Application of poultry manure (8 t ha -1 ) or farm yard manure (10 t ha -1 ) showed higher yield of rice.Poultry manure showed higher yield performance than FYM application.Besides, 100 mM proline application at both seedling and vegetative stages showed better yield performance compared to other treatments.In addition to field research work, better understanding about physiological and bio-molecular mechanisms of proline will be helpful for plant breeders to develop stress-tolerant crop varieties.On the other hand, organic manures like FYM and PM are more available and less expensive.

Figure 1 .
Figure 1.Effect of organic manure on the grain and straw yields of BR23 under saline condition.

Figure 2 .
Figure 2. Effect of proline on the grain and straw yields of BR23 under saline condition.
on peanut, Abou El-Magd et al. 2008 on sweet funnel and Raafat et al. 2011 on rice have shown that organic amendments increased plant growth at salt stress.

Table 4 . Effect of proline on nutrient uptake and K + /Na + ratio of BR-23 rice under salinity conditions.
Same letter in a column represents insignificant difference at p< 0.05.