The Integrated Application of Phosphorous and Zinc Affects the Physiological Status, Yield and Quality of Canola Grown in Phosphorus-suffered Deficiency Saline Soil

Despite the soil could contain high amount of phosphorus (P), salinity reduce its availability for crop plants. Hence, farmers should practice several tactics to ameliorate P deficiency in soils. The current study aimed to assess the importance of zinc (Zn) supply for mitigating the deficiency of P for canola grown in saline soil. The effects of three Zn rates (0, 150 and 300 mg L−1, Zn0, Zn150 and Zn300, respectively) under three P rates (0, 36 and 72 kg P2O5 ha−1, P0, P36, and P72, respectively) on physiological status, yield and quality of canola were measured. Treatments were arranged in the strip plot design based on completely randomized blocks with three replicates. Findings exhibited that P36 recorded the highest values of membrane stability index in the 2nd season, while statistically leveled P72 for relative water content and chlorophyll fluorescence in both seasons. Zn300 exhibited potent effect on all canola physiological traits in both seasons. In both seasons, P36 × Zn300, P72 × Zn150 and P72 × Zn300 showed the maximum chlorophyll fluorescence and performance index values. Plots treated with P72 achieved 70.0% increase in canola seed yield, greater than the untreated ones. Seed yield obtained with Zn300 were higher than Zn0 and Zn150 by1.30 and 1.10 times in 2019/20 season and 1.23 and 1.05 times in 2020/21 season. The highest oil % was recorded with P0 × Zn150 and P72 × Zn0 in the 1st season and with P72 × Zn150 in the 2nd season.


Introduction
Globally, canola or rapeseed (Brassica napus L.) is ordered as the third oil crop after oil palm and soybean for oil production with ~76.0 million tons, obtained from ~35.0 million hectares (FAO 2019).Since its oil is fortified by a high amount of oleic acid as a main unsaturated fatty acid, canola seeds are typified by high quality and quantity of oil (Zhou et al. 2019;Mamnabi et al. 2020).According to the cultivated genotype, canola oil comprises 7% saturated fatty acids, 66% monounsaturated fatty acids, and 27% polyunsaturated fatty acids (Safavi Fard et al. 2018).The quality of fatty acid in oilseed crops is mainly depends on the environment and genotype (Enjalbert et al. 2013;Safavi Fard et al. 2018).
Production and cultivation of agricultural crops are hugely influenced by numerous eco-stresses involving drought, salinity, extreme temperatures, and nutrient deficiency (Rady et al. 2020;Saudy et al. 2020a;Abd El-Mageed et al. 2021;El-Metwally and Saudy 2021a;Abou Tahoun et al. 2022;El-Bially et al. 2022a).It has been documented that such stresses adversely affect crop growth and development, hence the productivity and quality (Mubarak et al. 2021).
Soil salinity is one of the most distinctive abiotic stresses causes major reductions in cultivatable lands and crop yield and quality (Salem et al. 2021;Abd El-Mageed et al. 2022;Al-Elwany et al. 2022;Shaaban et al. 2022).It is forecasted K that approximately 50% of all agricultural lands will be impacted by salinity by 2050 (Shrivastava and Kumar 2015).Consequently, owing to soil salinity, more than US$12 billion global losses yearly due to reduced crop productivity is expected (Jägermeyr and Frieler 2018).Thus, it is important to recognize the crop responses to salinity stress to reduce the economic loss and save food security.At the plant cellular level, salinization causes excessive accumulation of salt ions in soil resulting in toxicity and osmotic effects of the soil around the roots of the plant.Due to high osmotic potential in soil rhizosphere, the ability of the crop plants to absorb soil water is reduced (Machado and Serralheiro 2017).Furthermore, salt stress including osmotic and ionic stress interposes with cellular functions of plants owing to activating production of reactive oxygen species (ROS), which result in oxidative damage in various cellular complexes (Gupta and Huang 2014) with altering vital metabolic processes (Liang et al. 2018).Also, lipid peroxidation rated increased with salinity (Hernández 2019;Yu et al. 2020), hence higher membrane permeability and loss of ions from the cells occur (Gupta and Huang 2014).Accordingly, severe agricultural concerns can be emerged due to the limited plant productivity associated with soil salinity (Isayenkov and Maathuis 2019).To ameliorate the adverse impacts of salinity on crop plants, several tactics should be adopted.
Macronutrients play a crucial role in plant growth and development and productivity (Abd-Elrahman et al. 2022;Elgala et al. 2022;Saudy and El-Metwally 2022).Of these, phosphorus (P) is an essential element for enhancing yield and quality (Saudy and El-Metwally 2019;Saudy et al. 2020b).However, P availability in soil is substantially influenced by soil condition, resulting in low P accumulation in the economic product of the crops (Salem et al. 2022).The exposure of P to fixation in soils represents a critical issue.In this regard, conversion of about 80% of P fertilizers soil application was observed to be in unavailable form (Walpola and Yoon 2012).For instance, in calcareous or normal soils and acidic soils, P converted into an insoluble complex (Satyaprakash et al. 2017;Kumar et al. 2018), causing P deficiency (Saudy et al. 2022c).Lack of P affects the normal plant growth and brings about premature death of older leaves (Niu et al. 2013).
Being it is an activator of phosphoenolpyruvate carboxylase and ribulose diphosphate carboxylase and oxygenase, zinc (Zn) increased photosynthesis rate and photo-assimilates translocation and proteins synthesis (Olama et al. 2014;Ebrahimian et al. 2017;Manaf et al. 2019;Afsahi et al. 2020).Hence, protein and oil content of oilseed crops were stimulated by Zn supply (Weisany et al. 2014;Shahsavari and Dadrasnia 2016).The biosynthesis of enzymes such as carbonic anhydrase, alcohol dehydrogenase, and superoxide dismutase (Cakmak 2000) as well as indole-3-acetic acid (Fang et al. 2008) cannot be completed without Zn.A physiological stress was reported in plants subjected to Zn deficiency (Vojodi Mehrabani et al. 2018).While, under normal and stressed conditions Zn exhibited significant improvements in crop growth and yield (El-Metwally and Saudy 2021b; Saudy et al. 2021bSaudy et al. , 2022a)).
Despite the availability of P and Zn in most cultivated soils is low, such issue could be addressed by fertilization (Montalvo et al. 2016;Saudy et al. 2020b).We hypnotized that combined application of P and Zn could adjust the nutrient balance in canola plants grown in saline soil.Therefore, physiological status and productivity of canola as influenced by P plus Zn were assessed under soil salinity conditions.

Experimental Treatments and Crop Husbandry
In our experiment, three P fertilizer rates, i.e., 0 (P0), 36 (P36), and 72 (P72) kg P2O5 ha -1 were supplied in the form of superphosphate (15.5% P2O5), which is commonly used as P fertilizer.The P fertilizer rates were incorporated into the soil prior to planting.Plants exogenously sprayed with three concentrations of Zn in the form of ZnSO4 namely, 0 (Zn0; tap water as a control), 150 (Zn150), and 300 (Zn300) mgL -1 .The experiment was in a two-factor strip plot design based on completely randomized blocks with three replicates, where the horizontal factor included the P fertilization rates and the vertical factor included foliar-applied Zn levels.The Zn concentrations were added as foliar application twice at 45 and 60 days from planting (DFP).Foliar Zn application was applied by a fan nozzle K on a hand sprayer to lessen solution drift.To guarantee optimal Zn absorption into canola plant leaves, 0.1%, v/v of the non-ionic surfactant Tween ® 20 was added to the foliar-sprayed Zn solution.Healthy seeds of Brassica napus L. cultivar Serw4 were obtained from the Crop Research Institute, Agricultural Research Center, Egypt.Seeds were sown by hand on the 15th and 10th of November at 5 kg seed ha -1 in hills spaced by 0.2 m on one side of 0.6 m distanced rows.Each individual plot was 4 m in length × 3 m in width forming 12 m 2 net area with five rows.The thinning process was carried out at the 4-leaf stage (25 DFP) to maintain the strongest and healthful two canola plants per hill.The surface irrigation system was used, and canola plants needed six irrigations through the growing season according to the daily reference crop evapotranspiration, totaling 3066 m 3 ha -1 .During soil tillage and plant growth, the recommended nitrogen (N) and potassium (K) fertilizers in form of ammonium sulphate and potassium sulphate with rates of 108 kg ha -1 and 58 kg ha -1 , respectively were applied.The total amount of N fertilizer was top-dressed in three split doses at 21, 35, and 50 DFP, while the K fertilizer, in two equal applications, was added directly during the soil tillage and after thinning process, respectively.

Physiological Traits
Membrane stability index (MSI%) and relative water content (RWC%) were measured according to Premachandra et al. (1990) and Hayat et al. (2007), respectively.Chlorophyll fluorescence and photosynthetic performance index as a convenient tool to assess photosynthetic efficiency, was determined according to Maxwell and Johnson (2000) and Clark et al. (2000) by Handy PEA, Hansatech Instruments (Ltd, Kings Lynn, UK).

Seed Yield and Protein and Oil Content
During the two canola cropping seasons, measurements of seed yield and its attributes and seed quality were recorded at harvest (162 DFP).Five guarded canola plants were randomly chosen and carefully harvested from each plot to count the siliques number plant -1 .Canola yield was determined by manually harvesting all the plants of three inner rows from each plot.These harvested plants were field sundried for three days to reduce plant moisture to the greatest extent possible before oilseed separation by threshing.Canola seed yield and weight of 1000 seeds were recorded based on 12% seed moisture content.Canola seed representative subsamples (~200 g from each plot) were further purified to eliminate impurities or damaged seed for seed oil and protein determination using a Zeltex ZX-50 portable seed analyzer (Zeltex Inc., Hagerstown, Maryland, USA).

Statistical Analysis
Analysis of variance using InfoStat statistical software (Di Rienzo et al. 2013) was performed to determine the impacts of phosphorus and zinc levels and their interaction on canola performance according to the strip-plot design based on completely randomized blocks.Wherever, the Ftest showed significant (p ≤ 0.05) differences among mean values, the differences among treatments were compared using Duncan's test (Steel and Torrie 1980).

Physiology of Canola
Data in Table 1 illustrated that under saline soil condition, without P or Zn application, reductions in membrane stability index, relative water content, chlorophyll fluorescence and performance index were recorded in both growing seasons of 2019/20 and 2020/21.On the contrary, P supply enhanced all canola physiological parameters.Herein, high rate of P (P72) showed the maximum values of membrane stability index in the 1st season as well as relative water content, chlorophyll fluorescence and performance index in both seasons.P36 recorded the highest values of membrane stability index in the 2nd season, while statistically leveled P72 for relative water content and chlorophyll fluorescence in both seasons.
Spraying of Zn at a rate of 300 mg L -1 (Zn300) exhibited potent effect on all canola physiological traits in both seasons.However, the differences between 300 mg L -1 and 150 mg L -1 Zn did not reach the level of significance (p ≥ 0.05) for membrane stability index in the 2nd season, chlorophyll fluorescence, in the 1st season as well as relative water content and performance index in both seasons.

Canola Yield Traits
Siliques no.plant -1 , seed index and seed yield of canola significantly (p ≤ 0.05) influenced by P and Zn and their interaction in both seasons (Table 2).The main effects of for each P and Zn clarified the progressive increase in all yield traits with increase the application rate.Accordingly, as averages of the two seasons, plots treated with P72 achieved increases of 35.6, 14.8 and 70.0% in siliques no.plant -1 , seed index and seed yield, respectively, greater than the untreated ones.Zn300 possessed the maximum increases in all yield traits however, significantly equaled Zn150 for siliques no.plant -1 , in the 1st season and seed index n both seasons.Seed yield of Zn300 were higher than Zn0 and Zn150 by1.30 and 1.10 times in 2019/20 season and 1.23 and 1.05 times in 2020/21 season.
Concerning the interaction, seed index and seed yield of canola significantly responded to P × Zn, while siliques no.plant -1 did not affect (Table 2).All combinations between P and Zn showed similar improvements in seed index and seed yield in both seasons, except for P0 × Zn0 and P0 × Zn150, which recorded lower values.

Canola Protein and Oil
The responses of seed oil and protein content as affected by individual effects of P and Zn are illustrated in Fig. 1.P36 gave the maximum value of seed protein % in 2020/21 season.While, P72 showed the highest increase in seed oil % in both seasons statistically leveled with P36 in 2019/20 season.
Addition of Zn300 caused the highest seed protein content in both seasons, without significant differences with Zn150 in the 2nd season.Also, Zn150 showed the maximum oil content in both seasons, significantly leveled with Zn300 in the 2nd season.
Remarkable effects of the interaction between P and Zn on seed and protein content of canola were obtained K

Discussion
There is no doubt that the crop plants that subjected to environmental stresses cannot grow and develop normally (El-Bially et al. 2018, 2022b;El-Metwally et al. 2022).
Under abiotic stresses such as salinity, drought and heat, disturbance in the physiological status is realized (Semida et al. 2015;Saudy et al. 2021a;Abd El-Mageed et al. 2020;Makhlouf et al. 2022).Consequently, crop productivity and quality adversely influenced (El-Metwally et al. 2021;Saudy et al. 2022b).Specifically, salinity has multiple adverse effects start in the soil and extend to plant metabolism.Despite the large-scale supply of P fertilizer could increase the total quantity of P in arable lands, large amount of P is fixed in the saline soil, which is difficult to move to the crop rhizosphere.Hence, P utilization rate did not exceed 25% (Perassi and Borgnino 2014).Soil environment was affected by salinity causing nutrient lack and affected the content of available phosphorus in soil (Xie et al. 2022).In salt-affected soils (salinity or alkalinity impacts) the applied phosphorus to the soil transforms into insoluble form of phosphate with low availability (Bruland and DeMent 2009).Saline soil is typified by low nutrient ion activity, involving P and Zn, due to intemperate ratios of Na + /Ca 2+ , Na + /K + , Ca 2+ /Mg 2+ , and Cl -/NO 3-in the soil solution, which affected the plant growth and nutrient uptake (Grattan and Grieve 1992;Bidalia et al. 2019).In sodium chloride medium, significant reduction in nitrogen, potassium and zinc was obtained (Murat et al. 2007).Additionally, high osmotic pressure and increase Na + and Cl - inflow into root cells, generated by salinity, create shortage in the vital nutrient uptake, causing ionic imbalance in plant cells (Wang et al. 2017).Accordingly, salinity generates another abiotic stress expressed in P deficiency.Due to such stress, membrane stability index, relative water content, chlorophyll fluorescence and performance index of canola were deteriorated under salinity.Unlike, providing the saline soil with P improved the physiological state of canola.
On the other site, by adjusting the permeability of cell membrane zinc quenches excessive Na uptake under salinity (Aktaş et al. 2006).Also, zinc decreased Na accretion and improved K/Na ratio in plants under salinity (Saleh et al. 2009;Nadeem et al. 2020).Therefore, canola cell membranes exhibited high permeability, hence leakage of some compounds from the roots under Zn deficiency.While, Zn supply improved the physiological state of canola expressed in enhancing membrane stability index, relative water content, chlorophyll fluorescence and performance index.Consequently, yield traits and seed quality were increased.
Since phosphorus level in soil is one of the significant factors affecting the zinc mobility and uptake (Wei et al. 2007), foliar application of Zn was more efficient for enhancing canola physiology and yield under P supply.Also, Zn has a crucial act in indole acetic acid biosynthesis, beginning of the primordia of the reproductive organs and metabolic reactions (Brown et al. 1993;Rehman et al. 2012), hence canola yield traits were improved.

Conclusion
Since salinity has disturbed the physiological processes of the plant, there have been quantitative and qualitative losses in the canola yield.According to the findings of the current study, the integration between P and Zn helped canola plants grown in saline soil to produce high yield with good quality oil and protein.It is worthily to note that supplying canola grown in saline soils with Zn can compensate the deficiency in P and vice versa.Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Fig. 1
Fig.1Seed protein and oil content of canola plants grown in saline soil as affected by phosphorus and zinc in 2019/20 and 2020/21 seasons.P0, P36 and P72: 0, 36 and 72 kg P2O5 ha -1 , respectively; Zn0, Zn150 and Zn300: 0, 150 and 300 Zn mg L -1 , respectively.Values are the mean of 3 replicates ± standard errors.Means not sharing the common letters for each factor in each bar differ significantly at p ≤ 0.05

Fig. 2
Fig.2Seed protein and oil content of canola plants grown in saline soil as affected by the interaction of phosphorus (P) and zinc (Zn) in 2019/20 and 2020/21 seasons.P0, P36 and P72: 0, 36 and 72 kg P2O5 ha -1 , respectively; Zn0, Zn150 and Zn300: 0, 150 and 300 Zn mg L -1 , respectively.Values are the mean of 3 replicates ± standard errors.Means not sharing the common letters for each factor in each bar differ significantly at p ≤ 0.05

Funding
Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).Conflict of interest A. Shaaban, T.A.A. El-Mageed, W.R.A. El-Momen, H.S. Saudy and O.A.A.I.Al-Elwany declare that they have no competing interests.

Table 1
Physiological attributes of canola plants grown in saline soil as affected by phosphorus, zinc and their interaction in 2019/20 and 2020/21

Table 2
Yield attributes of canola plants grown in saline soil as affected by phosphorus, zinc and their interaction in 2019/20 and 2020/21 seasons