Improving the physiological indices and biological yield of different Atriplex species with organic fertilizers in different irrigation regimes

Abstract Because of water crises, the cultivation of forage plants is greatly important in arid and semi-arid regions of the world. In order to study the effect of different organic fertilizers and irrigation regimes on the growth and biochemical responses of different Atriplex species, this experiment was performed as a factorial split with three replications in the city of Bandar Abbas in 2017-2018. The main factor included three irrigation regimes (irrigation at 80, 60, and 40% of field capacity) and the sub-factor were different sources of fertilizer (poultry manure and sheep manure about 5 tons per hectare, and chemical fertilizer) and different species of Atriplex (A. canascens, A. leucoclada, and A. lelentiformis). Based on the results, increasing drought stress increased sodium (Na) and proline of the leaf and decreased the concentration of nitrogen, phosphorus, potassium, and soluble carbohydrates. In irrigation of 40% of field capacity, the highest amount of leaf nitrogen was observed in sheep manure (1.47%), representing a significant increase compared to the chemical fertilizer. In irrigation of 80% of field capacity, the highest amount of leaf phosphorus (0.222%) was observed in chemical fertilizer. The highest levels of catalase and peroxides were obtained in poultry and sheep manure. Also, in the irrigation of 40% of field capacity, poultry manure and then sheep manure could show higher levels of soluble protein in the leaf, respectively. In general, this study showed the successful effects of organic fertilizers on increasing nutrient uptake and physiological traits and improving the biological yield of Atriplex.


Introduction
Atriplex belongs to the family of Chenopodiaceae.It has various annual and perennial species including Atriplex canescens, Atriplex lentiformis, and Atriplex leucoclada (Sadeghi and Delaviz 2016).Atriplex is tolerant to salinity and drought and can be used in forage production for livestock when there is no access to optimal soil and water resources (Mata-Gonz� alez et al. 2017).Atriplex can be effective in stabilizing production in arid and semi-arid regions of the world where other crops cannot grow and produce.Therefore, it has gained special importance in arid and semi-arid regions of the world due to the rapid growth and high forage production (Osmond, Bj€ orkman, and Anderson 2012).However, the management of forage plants and identification of plants with the ability of quality forage is a perspective for forage production in these areas (Masters, Benes, and Norman 2007).
The essential nutrients to plants are usually supplied through chemical fertilizers applications.However, large amounts of nutrients in chemical fertilizers become insoluble after entering the soil and unreachable for plants.It has been reported that the efficiency of nutrient utilization by crops in different environmental conditions is less than 50% (Baligar and Fageria 2015).In calcareous soils of arid and semi-arid regions, the presence of high calcium carbonate, high pH, and low organic matter reduces the efficiency of nutrient uptake (Attarzadeh et al. 2019;Parvizniaa et al. 2021).In addition to the use of chemical fertilizers, using of organic fertilizers in order to provide the essential nutrients to plants has been taken into account by growers.The results of different studies show that the application of organic and biofertilizers can increase plant access to nutrients and consequently improve plant growth and yield (Mousavi, Bahmanyar, and Pirdashti 2013;Sheikhi, Ronaghi, and Mousavi 2015;Mousavi et al. 2017;Sheikhi et al. 2018;Mousavi et al. 2018;Cheraghi et al. 2022).Furthermore, the use of organic fertilizers increases plant tolerance to environmental stresses such as drought stress (Bibi and Ilyas 2019).Organic compounds do not have unilateral effects, as they help to provide nutrients and improve the chemical and physical properties of the soil (Mousavi, Srivastava, and Cheraghi 2022).Khalid, Arshad, and Zahir (2006) stated that the use of organic fertilizers can be one of the important approaches to sustainable agriculture and they have further effect on alleviating environmental stresses compared to chemical fertilizers (Wang et al. 2018).
Drought stress, as an important environmental constraint, is a critical inhibitor of crop production in many arid and semi-arid regions of the world (Basirat et al. 2019;Cheraghi, Mousavi, and Zarebanadkouki 2023).It causes changes in the vegetative and biochemical traits of plants and consequently reduces the plant yield (Loreto and Centritto 2008;Attarzadeh et al. 2020;Basirat and Mousavi 2022;Cheraghi, Motesharezadeh, et al. 2023).Numerous factors cause plant tolerance to drought stress and change in the physiological and biochemical mechanisms of plants (Ilyas et al. 2020).The development of environmentally friendly agriculture in arid and semi-arid regions is of great importance (Niu et al. 2019).One of these solutions is to select plants with high production efficiency.Therefore, identifying and introducing plant species tolerant and compatible with the conditions of these areas are particularly important (Ghaffarian et al. 2020).
Soils in arid and semi-arid regions of the world are unable to meet the nutritional needs of plants due to a lack of organic matter and biological activities.On the other hand, due to the environmental problems caused by using chemical fertilizers, organic and biological compounds can play an important role as a solution to increase the development of sustainable agriculture and crop yield.As a matter of fact, the present study is an applied research and its results will confidently use by growers and stakeholders in these regions.Therefore, the present study aimed to use poultry and sheep manure to improve physiological parameters and yield of forage of Atriplex in different irrigation regimes.

Experimental design and treatments
This experiment was performed as a factorial split with three replications in 2017 and 2018 in the city of Bandar Abbas, Iran, at 56 � and 35"E and 27 � and 21"N and an altitude of 16 m above sea level.In Bandar Abbas, the summers are long, sweltering, oppressive, and arid; the winters are short, comfortable, and dry; and it is mostly clear year round.Over the course of the year, the temperature typically varies from 54 � F to 100 � F and is rarely below 48 � F or above 105 � F. The main factor was three irrigation regimes (including irrigation at 80, 60, and 40% of field capacity) and the sub-factor was different sources of fertilizer at three levels (including poultry manure about 5 tons per hectare, sheep manure about 5 tons per hectare, and chemical fertilizer based on soil requirements) and different species of Atriplex (including A. canascens, A. leucoclada, and A. lelentiformis).
Before the experiment, the soil was sampled from a depth of 0-30 cm and the physical and chemical properties of the soil were determined (Table 1).Also, the chemical properties of the organic fertilizers were measured (Table 2).
The seeds were planted in a suitable environment and then transferred to the main farm on July 10, 2017, and the growth period lasted about six months.In each experimental unit, 12 seedlings of the Atriplex plant were planted with a row space of 80 cm and a distance of 40 cm between two plants.The distances between the main and sub-plots and the distances between the blocks were 2, 1, and 2 m, respectively.After preparing the land for planting, organic and chemical fertilizers were applied based on the treatments used.Hence, poultry and sheep manure were used sparsely for the organic fertilizer treatments in each experimental plot and were mixed with the soil.Moreover, in the chemical fertilizer treatment, the urea fertilizer was used as a nitrogen source required by the plant and applied in the amount of 150 kg ha −1 in three equal parts.To supply phosphorus, about 150 kg of triple superphosphate was used per hectare before planting.In order to supply the potash required by the plant, before planting, 100 kg of potassium sulfate per hectare was uniformly put under the planting lines.During the growth period, weeding was performed manually on the plots.In order to irrigation, a furrow technique was used.
After planting the seedlings, all plots were planted for 14 days based on control irrigation treatment (irrigation at 80% of field capacity) for proper seedlings growing in the field.Then, the irrigation treatments were started.Irrigation treatments were applied based on the percentage of crop capacity in the depth of root development.Also, in order to determine the soil moisture levels and irrigation regimes a pressure plate device was used.

Measurements
In order to measure the nutrient content of the leaf, the collected samples were thoroughly washed with distilled water and dried in an oven at 70 � C for 48 h.Then, 1 gr of the dried sample was ashed in an oven at 500 � C. The concentration of sodium was read by a photometer flame device (Jenway 7, German model).Finally, the read numbers were adjusted using the graphs obtained from standard samples (Patterson, Macrae, and Ferguson 1984).The leaf nitrogen was measured based on titration after distillation by Kajeldal model V40.The leaf phosphorus was measured in an extract of plant ash dissolved in 2 N hydrochloric acid.In the obtained extract, phosphorus concentration was measured by molybdate-vanadate reagent using a colorimeter with Vis 2100 spectrophotometer at 420 nm.Potassium concentration was measured by flame diffusion method using 620 G photometer flame (Attarzadeh et al. 2019).The amount of soluble carbohydrates was determined via the Nelson method (Nelson 1944).To measure proline, the method of Bates, Waldren, and Tear (1973) was used.Catalase activity was measured by Cakmak and Horst method (Cakmak and Horst 1991).Peroxidase was measured based on Ghanati et al.'s method (Ghanati, Morita, and Yokota 2002).To measure soluble proteins of the leaf, the method of Bradford (1976) was used.Also, the plant height and dry weight of aerial parts were measured by using standard methods (Mousavi et al. 2017).

Statistical analysis
The statistical analysis of the data was performed by the computer program of SAS.Before data analyses, the normality test was done by using Minitab software and the Kolmogorov-Smirnov test (Fitrianto and Chin 2016).The results of the normality test showed that P-Value was greater than 0.05 which confirms the normality of the data (Ataei, Baharlouei, and Ataabadi 2022).Then, after analysis of variance (ANOVA) of the data (St and Wold 1989), the mean separation was done using Duncan's test at a 5% probability level.The figures were created by EXCEL.

Concentration of leaf nutrients
Based on the results of analysis of variance (ANOVA), the effect of drought stress, fertilizer, and harvest was significant on the concentration of sodium in the leaf; moreover, this trait was affected by the interaction of harvest, drought stress, and fertilizer.Increasing the drought stress increased the leaf sodium so that the highest leaf sodium was observed in irrigation of 40% of field capacity (Table 3).In the first and second harvests, in irrigation of 80% of field capacity, the lowest concentration of sodium was observed in sheep manure treatment representing a significant decrease compared to the chemical fertilizer (Table 3).Moreover, by increasing drought stress to 40 and 60% of field capacity, poultry manure could reduce the sodium concentration of leaf at a lower level than sheep manure.Therefore, the organic fertilizers reduced the sodium uptake of Atriplex leaves to some extent compared to the chemical fertilizers.The effect of drought stress, fertilizer, and harvest on the nitrogen content of the Atriplex leaf was significant.Moreover, this trait was affected by the interaction of drought stress and fertilizer.Increasing the drought stress decreased the uptake of nitrogen in the leaf (Table 4).The highest amount of leaf nitrogen was obtained in irrigation of 80% of field capacity.Also, the amount of leaf nitrogen in irrigation of 60 and 80% of field capacity in the chemical fertilizer showed no significant difference compared to the poultry and sheep manure.In irrigation of 40% of field capacity, the highest amount of leaf nitrogen was observed in the treatment of sheep manure (1.47%) representing a significant increase compared to the chemical fertilizer (Table 4).Also, the nitrogen concentration of the leaf was 1.66%, in the second harvest showing a significant increase compared to the first harvest (1.30%) (Table 5).
The effect of drought stress, fertilizer, and harvest on the phosphorus content of Atriplex leaf was significant and was affected by the interaction of drought stress and fertilizer.In irrigation of 80% of field capacity, the highest amount of leaf phosphorus (0.222%) was observed in chemical fertilizer treatment representing a significant difference compared to the poultry and sheep manure (Table 4).On the other hand, the amount of leaf phosphorus in irrigation of 60 and 40% of field capacity in the chemical fertilizer regime showed no significant difference compared to the poultry and sheep manure.Other results indicate that increasing drought stress reduces the uptake of leaf phosphorus (Table 4).Also, the concentration of leaf phosphorus in the second harvest was 0.193% showing a significant increase compared to the first harvest (0.170%) (Table 5).
Based on the results of ANOVA, the effect of drought stress, fertilizer, and harvest on Atriplex leaf potassium was significant and it was affected by the interaction of harvest, drought stress, and fertilizer.Increasing the drought stress decreased the leaf potassium so that the lowest amount of leaf potassium was observed in irrigation of 40% of field capacity (Table 3).In the first and second harvests, in irrigation of 80% of field capacity, the highest amount of leaf potassium was observed in chemical fertilizer treatment (Table 3).Moreover, in the drought stress of 60% of field capacity, there was no significant difference between the levels of chemical fertilizers compared to organic fertilizers.On the other hand, in the second harvest, in the treatment of 40% of field capacity, the potassium content of Atriplex leaf in chemical fertilizer treatment was 1.43%, which was lower than the organic fertilizers (1.51%).The results showed that the highest accumulation of sodium ions in the Atriplex leaf occurred in irrigation of 40% of field capacity.

Soluble carbohydrates and leaf proline
The ANOVA showed that the effect of drought stress and fertilizer on soluble carbohydrates of Atriplex leaves was significant and also was affected by the interaction of drought stress and fertilizer.The decrease of soluble carbohydrates in the Atriplex leaf was observed by increasing the drought stress (Table 4).The highest leaf soluble carbohydrates were obtained in irrigation of 80% of field capacity.On the other hand, irrigation of 40% of field capacity significantly reduced the content of soluble carbohydrates in the leaves.The content of soluble carbohydrates in leaves in irrigation of 80 and 60% of field capacity in chemical fertilizer treatment showed no significant difference compared to the poultry and sheep manure.In irrigation of 40% of field capacity, the content of soluble carbohydrates of leaf in the poultry and sheep manure treatments showed an increase of 8% and 7%, respectively compared to the chemical fertilizer (Table 4).
Considering the results of ANOVA, the effect of drought stress, fertilizer, and harvest on the proline content of the Atriplex leaf was significant.It was affected by the interaction of harvest, drought stress, and fertilizer.An increase in leaf proline was observed with increasing the drought stress so that the highest leaf proline was observed in irrigation of 40% of field capacity (Table 3).In the first and second harvests in the irrigation of 80% of field capacity, the highest amount of leaf proline was observed in sheep manure treatment (Table 3).In drought stress of 60% of field capacity, in the first and second harvests, the highest amount of leaf proline content was observed in poultry manure treatment representing an increase of 9% and 11%, respectively compared to the chemical fertilizer.Also, in the treatment of 40% of field capacity, the amount of leaf proline in the chemical fertilizer was lower than in poultry and sheep manure treatments.

Antioxidant enzymes of catalase and peroxidase
Considering the results of ANOVA, the effect of drought stress and the fertilizer treatments on the activity of antioxidant enzymes of catalase and peroxidase was significant.This trait was not affected by the interaction of experimental factors.Increasing the drought stress increased the activity of catalase so that the increase in catalase in irrigation of 40% of field capacity was observed compared to 80 and 60% of field capacity by 27 and 15%, respectively (Table 6).On the other hand, the highest activity of catalase was obtained with 7.12 and 6.99 mmol mg −1 min −1 of protein in poultry and sheep manure treatments (Table 7).
Increasing the activity of peroxidase similar to catalase was achieved by increasing drought stress so that peroxidase enzyme in irrigation of 40% of the field capacity represented a significant increase compared to the irrigation of 80 and 60% of field capacity (Table 6).On the other hand, the highest amount of peroxidase was observed in sheep manure treatment, which showed a 5% increase compared to the chemical fertilizer, however, it was not significantly different from the poultry manure (Table 7).

Soluble protein of leaf
The ANOVA showed that the effect of drought and fertilizer treatments on the soluble protein content of Atriplex leaves was significant.This trait was affected by the interaction of drought stress and fertilizer.Increasing the drought stress decreased the amount of soluble protein in the leaves (Table 4).The highest amount of leaf soluble protein was obtained in irrigation of 80% of field capacity.However, increasing the drought stress at the irrigation level of 40% of field capacity caused a significant decrease in the soluble protein content of leaves.On the other hand, in irrigation of 80% of field capacity, the amount of protein in chemical fertilizer shows an increase of 8% and 14% respectively compared to poultry and sheep manure treatments.In irrigation of 60% of field capacity, no significant difference was observed between different levels of fertilizer (Table 4).Furthermore, in irrigation of 40% of field capacity, poultry manure and then sheep manure could show higher levels of soluble protein in leaves.Increasing the drought stress led to a decrease in soluble proteins of Atriplex leaf while using organic fertilizer moderated the adverse effects of drought stress on the amount of soluble proteins in leaves.

Plant height and biological yield
Based on the ANOVA, the effect of drought stress, fertilizer, and harvest on the plant height of Atriplex was significant.Such a trait was affected by the interaction of drought stress and fertilizer.Increasing the drought stress decreased the plant height of Atriplex (Table 4).In irrigation of 80 and 60% of field capacity, no significant difference was observed between different levels of fertilizer.On the other hand, at 40% of field capacity, plant height in poultry and sheep manure treatments showed an increase of 28% and 38% compared to the chemical fertilizer (Table 4).Moreover, the plant height in the second harvest was 63.36 cm representing a significant increase compared to the first harvest (51.89 cm) (Table 5).
The ANOVA also showed that the effect of drought stress on the biological yield of Atriplex was significant, also this trait was affected by the interaction of drought stress and fertilizer.The decreased yield of Atriplex was observed by increasing the drought stress (Table 4).No significant difference in the biological yield in irrigation of 80 and 60% of field capacity was observed between different levels of fertilizer.On the other hand, in irrigation of 40% of field capacity, biological yield in poultry and sheep manure showed an increase of 4% and 8% compared to the chemical fertilizers (Table 4).

Concentration of leaf nutrients
Drought stress can affect nutrient content by impairing the absorption and transport of nutrients within plant vessels or by the accumulation of ions possibly disrupting the metabolism (Dotaniya and Meena 2015).In support of this claim, the results of this study showed that increasing the drought stress increased the leaf sodium.However, excessive uptake of sodium ions within the cell will be problematic (Cheeseman John 2013), for example, based on the results of this study, leaf sodium has a significant negative correlation with the concentration of nitrogen and phosphorus (Table 8).In some plants under salinity stress, the negative effects of salt on plant metabolism were minimized by the accumulation of sodium ions in the vacuole and other salts such as potassium in the cytoplasm (Flowers Timothy, Rana, and Colmer 2015).Therefore, due to the sodium toxicity, physiological characteristics will be impaired and plant yield will be reduced.On the other hand, the use of organic fertilizers under drought stress conditions reduced sodium uptake and increased the uptake of nitrogen and phosphorus from soil (Sheikhi, Ronaghi, and Mousavi 2015).Thus, it can be concluded that organic fertilizers increased the content of nutrients compared to chemical fertilizers by reducing soil salinity.Moreover, using organic compounds enhanced nutrient uptake by improving soil physicochemical properties and the gradual availability of nutrients (Mousavi, Srivastava, and Cheraghi 2022).However, the use of organic fertilizers, especially in drought stress conditions, improves the growth environment and better absorption of nutrients such as nitrogen and potassium for plants, which refers to the positive effect of organic fertilizers on biochemical responses of the plant which consequently improves plant growth and nutrition.

Soluble carbohydrates and leaf proline
Drought stress decreased soluble carbohydrates in the leaves of Atriplex but increased the content of proline.Although researchers have reported that increasing environmental stresses reduces the soluble sugar (carbohydrates) content of plant leaves, contradictory results have been obtained in some studies (Seki et al. 2007).It has been reported that some plants in high environmental stress conditions increase the content of compatible osmolytes such as proline to maintain cell structure, regulate osmosis, and tolerate stress (Hameed et al. 2012).Proline plays an important role in regulating osmosis and reducing the severity of adverse effects of drought stress.In general, the accumulation of compatible osmolytes such as proline is important in reducing damage to the cell by free radicals, protecting and stabilizing enzymes, and stabilizing membrane structures.In this regard, proline content showed a positive and significant correlation with leaf Na, however, soluble carbohydrates showed a negative and significant correlation with leaf Na (Table 8).On the other hand, the accumulation of proline and soluble sugar (carbohydrates) in Atriplex leaves was affected by using organic fertilizers.Increasing soluble sugar (carbohydrates) and proline content of leaves, as a type of biochemical response of the plant, may provide better conditions for water absorption under drought stress (Molinari et al. 2007).

Antioxidant enzymes of catalase and peroxidase
The antioxidant systems are activated under stress conditions in order to control and defend the plant under stress conditions (Blokhina, Virolainen, and Fagerstedt 2003).In general, the highest level of antioxidant enzyme activity may be caused by increasing cell damage under severe environmental stresses like drought stress (Attarzadeh et al. 2019).The present study showed that NS, � and �� : Not-significant and significant at 5% and 1% error probability, respectively.
using poultry and sheep organic fertilizers moderates the adverse effects of drought stress.The higher activity of antioxidant enzymes of catalase and peroxidase due to the application of organic fertilizers confirms this claim.In other words, using organic and biofertilizers in a plant's response to drought stress is associated with the regulation of oxidative reactions and the induction of antioxidant defenses (Karimkhani et al. 2021).Also, the activity of antioxidant enzymes of catalase and peroxidase showed a positive and significant correlation with leaf Na (Table 8), which this is referred to the fact that antioxidant enzymes such as catalase and peroxidase act as a defense barrier against plant stresses (Pyngrope, Bhoomika, and Dubey 2012).

Soluble protein of leaf
It seems that the amount of protein decreases with increasing the activity of protein-degrading enzymes under stress (Zahoor et al. 2017).Oxidative stress as a result of drought stress leads to the production of oxygen-free radicals with a high affinity to proteins.Oxygen-free radicals lead to the destruction of cellular proteins and thus reduce the amount of leaf-soluble proteins (Abbasi et al. 2014).The decreased protein content may also be due to the reduction of activity of enzymes of nitrate reductase and glutamine synthetase due to the lack of nitrogen availability for the plant.Confirming this claim, the results obtained from this study show that leaf protein has a positive and significant correlation with the concentration of nitrogen and phosphorus.Nitrogen deficiency has led to a reduction in soluble leaf proteins.However, under the application of organic fertilizer, the amount of soluble protein in the leaves increased by increasing the availability of available nitrogen.

Plant height and biological yield
Drought stress in irrigation of 40% of field capacity impaired the nutrient uptake and measured physiological traits in Atriplex.Hence, the reduction of biomass yield of these plants in severe drought stress was not unexpected.Furthermore, increasing the plant height and biological yield in organic fertilizer systems increased the growth of the Atriplex plant by providing a more appropriate level of uptake for nutrients.In this regard, plant height and biological yield showed a positive and significant correlation with the concentration of all nutrients.The reduced levels of water and nutrients can affect the physiological characteristics of plants, leading to the production of reactive oxygen species possibly causing cell damage (Zgallai, Steppe, and Lemeur 2005).On the other hand, nutrients can maintain higher carbon dioxide and photosynthesis under stress, which can be considered as an effective strategy to improve plant growth in drought stress (Attarzadeh et al. 2019).It has been reported that organic fertilizers increase photosynthetic production by improving nutrient uptake, which in turn improves plant growth (Mousavi, Bahmanyar, and Pirdashti 2011;Chatzistathis et al. 2020;Cheraghi et al. 2022).It has also been suggested that organic and biofertilizers affect plant growth and development through the production of plant-stimulating hormones and increase biological yield (Al-Amri 2021; Srivastava et al. 2021).

Conclusion
The results of this study showed that with increasing drought stress the status of soluble nutrients, carbohydrates, and protein contents of Atriplex were adversely affected.Also, the activity of the antioxidant enzymes of catalase, peroxidase, and leaf proline was increased affected by increasing the drought stress.Use of organic fertilizers (i.e.poultry and sheep manure) moderated the adverse effects of drought stress by increasing compatible osmolytes such as soluble sugar and proline, and the antioxidant enzymes of catalase and peroxidase.In general, the results of this study showed the successful effects of organic fertilizers on increasing nutrient uptake, and improving the physiological traits and the biological yield of Atriplex.

Table 1 .
The physical and chemical properties of the studied soil.

Table 2 .
Some important properties of the organic fertilizers used in this experiment.

Table 3 .
Means comparison of the interaction effects of harvest time, irrigation regimes and fertilizer on leaf Na, K and proline of Atriplex.
Means followed by the same letters in each column are not significantly different by Duncan test at 5% probability level.

Table 4 .
Means comparison of the interaction effects of irrigation regimes and fertilizer on leaf N and leaf P, soluble carbohydrate, protein, plant height and biological yield of Atriplex.Means followed by the same letters in each column are not significantly different by Duncan test at 5% probability level.

Table 5 .
Effect of harvest time on leaf N, leaf P and plant height of Atriplex.Means in each column followed by the same letters have no significant difference on the basis of Duncan's multiple range test at 5% error probability.

Table 6 .
Effect of irrigation regimes on catalase and peroxidase of Atriplex.

Table 7 .
Effect of fertilizer on catalase and peroxidase of Atriplex.
Means in each column followed by the same letters have no significant difference on the basis of Duncan's multiple range test at 5% error probability.

Table 8 .
Pearson correlation coefficients between measured traits.