天美传媒

Industrialization and urbanization have led to factors causing food crisis along with increased human population adding concerns for world’s food security being affected by severe climatic condition, Chemical fertilizers and drought conditions. Population pressure has farther amplified the need of increased agricultural productivity across the globe. Cereal crops especially wheat, barley and rice have become the necessity of time to be improved for yield and yield attributing characters but these crops face the major of problems of drought and heat stresses in semi-arid soil conditions of Pakistan that reduce their productivity. Barley (Hordeum vulgare L.) crop is affected by drought stress which can simply be said shortage of water but has disastrous effect on morphology, physiology, molecular and biochemical processes of the crop that reduces plant growth. Drought stress can occur at any stage depending upon local environmental conditions and also on genotypic variability and resistance of the varieties sown. Drought tolerance can be estimated by determining the particular traits of plant at certain growth stages. Several biotic along with abiotic stresses like salinity, variable temperatures, water logging and water deficit etc. reduce cereal crop productivity [1].

Drought serves as major climatic factor which persists under zero rainfall remains for a long term in a specific area causing water deficit to a crop and it is the major scarcity which causes restricted crop rowth around the globe. Drought stress being abiotic stress threatens the crop productivity and imparts detrimental effects owing to Reactive Oxygen Species (ROS) and free radicles initiated by cellular biomolecules resulting into cellular necrosis. Plant also stimulates Superoxide Dismutase (SOD), Ascorbate Peroxidase (APX) and catalases to resist damages of ROS. Certain chemical substances like plant growth stimulators and osmoprotectants are given to plants for enhancing resistance against biotic and abiotic stresses. Many macronutrients like potassium hold a decisive function in growth and development of cereals by activating certain enzymes. Building blocks of proteins and hence enzymes i.e., the amino acids are crucial for cellular growth and stimulate osmotic ion concentration due to internal osmoregulation. Organic and inorganic fertilizers influence physiological as well as biochemical processes throughout plant development for continuation of osmoregulation, cytosolic pH, neutralization of charged proteins, and stimulation of enzymes, phloem loading and nutrient use efficiency of crop [2].

Applied nutrients variability maintains adequate cellular efficiency during drought stresses and improves ionic retention in the leaf mesophylls. These nutrients (organic or inorganic) when applied as foliar application enhance physiological process and plant growth also that improves the plant capability to restrain grain-filling stages under water deficit conditions nevertheless, a little work has been done in the past concerning effects and alleviation of drought stress on barley plants. Biochar; an organic source of nutrients, Chitosan; a polysaccharide was observed to mitigate water deficiency in barley by Sanchez-Reinoso et al., and improved nutrient uptake along with increased chlorophyll concentration and improved plant growth were observed respectively under drought stress. Many inorganic nutrients having geochemical origin when applied with these organic fertilizers were observed to reduce water deficit imparted defects in plant growth and hence improved crop growth and productivity. Several strategies had been adopted by researchers to make plant adopted to survive water deficiency such as reduced leaf size to minimize water loss by improved stomatal conductance as it influences transpiration rate but it on the other hand affects photosynthetic rate also. But using drought tolerant and more susceptible consents using organic and inorganic inputs to barley associate drought tolerance and improve carbon metabolism, scavenging of ROS, membrane and protein stability [3].

To combat drought stimulated drawbacks leading to reduced plant growth of barley, a screening was organized to investigate the outcomes of certain organic as well as inorganic fertilizers to negate negative impacts of water deficiency which cannot only be due to low rainfall but also due to unavailability of water by poorly irrigated soil. As a result, the goal of this research trial was to find the optimal treatment combination for alleviating drought stress in barley. It is believed that using NPK and NPB together rather than separately to improve barley growth characteristics under drought stress would be a prime approach. The main reason behind is to validate which amendments are truly impressive in relieving the issues of drought stress on barley. It is hypothesized that combining nitrogen fixing and phosphorus solubilizing bacteria NPB and NPK fertilizer to improve barley characteristics would be more effective than using them separately in drought impacts. However, there is need to fulfil the gap of studies where more physiological characters of barley should be focused to be improved in future work especially membrane ionic balance and improved drought tolerant proteins in plants that needs high levels of investigation on field level considering certain other climatic conditions validating this study as an efficient improvement for drought compatibility [4].

Experimental location and design

Barley was implanted in botanical experimental area of Faisalabad (30°-31.5° N and 73°-74° E, 184 m above the sea level) in pots. This experiment was managed in controlled environment in a greenhouse during the year 2018-19. Experimental design was Randomized Complete Block Design (RCBD) and each treatment was replicated thrice [5].

Soil characteristics

Soil from 7-10 inches depth was obtained from experimental locality of university. Soil was than sterilized through solarization, using thin plastic layer covering. Solar waves from sun helped soil attaining temperature to deplete the microbes, weeds and pests. The was then desiccated and sieved through a 2 mm sieve. Soil texture was clay loam. Some soil chemical composition included EC 7.78 dSm^-1, organic matter 1.38%, pH 7.3, available N 0.032 ppm, available P 5.93 ppm, available K 32.3 ppm [6].

Seed collection and sterilization

Newly developed befitting barley genotype was selected as it survives better in several abiotic stress conditions. Seeds were sterilized with 95% ethanol, washed with 70% sodium hypochlorite mixture followed by cleaning with distilled water thrice.

Treatments

The treatments (three sets of pots with 75% FC, 50% FC and 35% FC irrigation regimes) were:

• Control (untreated, no fertilizer).
• NPB (Nitrogen and phosphorous Bio-fertilizers (nitrogen fixing and phosphorus solubilizing bacteria)).
• NPK (N-P-K fertilizer (0.51-0.45-0.38 N-P-K g) by urea (46% N), sulphate of potash (50% KO) and diammonium phosphate (46% PO, 18% N)). • NPK+NPB (a combined application of NPB and NPK).

Seed sowing, fertilizer applications and establishment of drought stress

10 seeds were sown in every pot that had 12 kg of soil (30 cm height × 25 cm diameter) with average distance of 5-6 cm apart. After their complete emergence, the seedlings were thinned to 6 plants in every pot. For NPK treatment, soil was fertilized with N-P-K fertilizer (0.51-0.45-0.38 N-P-K g) with urea (46% N), sulphate of potash (50% K₂O) and diammonium phosphate (46% P₂O₅, 18% N) at the start of the experiment [7].

For NPB treatment establishment, an aerobic, free-living, soil nitrogen fixing and phosphorous solubilizing bacterial strain of Pseudomonas aeruginosa; isolated by the method of serial dilution, was used. At 37°C temperature, successive dilutions up to 10-7 were disseminated on Luria-Bertani agar plates and inoculated overnight. A spectrophotometer was used to record optical density at 600 nm, which was used to determine bacterial growth. Prior to 15 minutes of sowing, seeds were soaked with 20% gum arabic which serves as binding agent and then submerged in a bacterial culture of 108 cfu ml⁻¹ strength. To carefully control irrigation regimes and induction of drought environment, pots were transferred to canopies after 20 DAS. Well-irrigated environment was managed at 70 FC (70% field capacity) and drought stress was sustained at 45 FC (45% field capacity). Plants were left under variable water regimes prior to harvesting [8].

The pots were placed in canopies after 20 DAS to carefully manage irrigation regimes and to impose drought stress. Severe drought stress was sustained at 35% FC (35% field capacity), mild drought stress was inducted at 50% FC (50% field capacity) while well-irrigated conditions were held at 70% FC (70% field capacity). Until harvesting, the plants were kept at these specified varying water regimes. The drought stress was imposed at initial growth (seedling) stage because we wanted to check the performance of barley plants at premature level of growth at given fertilizer treatments [9].

Harvesting and recording of growth attributes

First harvest was taken after 40 DAS. At the experimental site, root and shoot fresh weights and lengths were recorded at the spot after harvest. For fresh analysis, samples were kept in biomedical refrigerator at -30°C. Three samples from every treatment were oven dehydrated for 3 days at 65°C to measure root and shoot dry weights and acid digestion ionic content analysis [10].

Photosynthetic pigments

Dere et al., prescribed a quantification method the chlorophyll content in barley leaves. 0.2 g of randomly picked leaves were mixed in 10 ml of 96% methanol for one minute, then filtered and centrifuged for 10 minutes at 2500 rpm. Supernatant was extracted and acquired to quantify chlorophyll concentration at wavelengths of 666 (chlorophyll a), 653 (chlorophyll b), and 470 nm (total carotenoids) with help of spectrophotometer (Model SM1200; Randolph, NJ, USA). The carotenoids were evaluated from fresh leaf samples by Arnon method.

Leaf biochemical and mineral content analysis

Each replicate's fully developed leaves were collected, enclosed in aluminum foil, submerged in liquid nitrogen, and placed in plastic bags. For subsequent examination, these materials were kept at -80°C. A spectrophotometer was used to perform the following biochemical assay (Model SM1200; Randolph, NJ, USA).

50 mg dehydrated leaves were mixed in 10 mL 80% ethanol and filtered, then re-extracted in 10 mL ethanol with a final volume of 20 mL to assess osmolytes as sugars and non-enzymatic antioxidants. This solution was used to test for soluble sugars, proteins, proline, and ascorbic acid contents. Na ions were evaluated using acid digested plant samples by Atomic Absorption Spectrum (AAS; Shimadzu instruments, Inc., Spectra AA-220, Kyoto, Japan) [11].

CAT content assay

0.2 g of thawed fresh leaf sample was squeezed to catalase activity. The plant sample was mixed in 1 mL of 0.05 M buffer Tris-HCl (pH=7.5). At 4°C, the resulting solution was centrifuged for 20 minutes at 13,000 rpm. Supernatant was utilized to calculate the activity of CAT enzymes using the Kar and Mishra method.

Evaluation of Malondialdehyde (MDA), electrolyte leakage and H₂O₂ contents

The lipid peroxidation extent of chloroplasts was assessed by following Heath and Packer method, that included quantification of MDA concentrations by thiobarbituric acid reaction.

MDA (μmol g^-1)=6.45 (Abs.532-Abs.600)-0.56 × Abs.450

The Mukherjee and Choudhari method was used to calculate the H2O2 contents. In 10 mL cold acetone, 0.1 g of leaf sample was excavated and centrifuged at 10,000 rpm. The solution was then mixed to with 5 mL concentrated ammonium solution and 4 mL titanium reagent. After centrifugation at 10,000 rpm for 5 minutes, the resulting precipitate was poured in 10 mL of 2N H₂SO₄. The leftover was centrifuged once more to eliminate any particles. The optical density at 415 nm was measured contrary to blank with help of spectrophotometer (UV-2550; Shimadzu, Kyoto, Japan).

Statistical analysis

Each treatment was tri-replicated and statistical analysis of data set was done by two-way ANOVA in a completely randomized block design comprising of split-plot arrangement. A mean separation and a variance analysis with a 5% level of significance were the treatment means (p ≤ 0.05). Where required, a logarithmic data transformation was employed to create a distribution prior to analysis close to normal, where necessary [12].

Length, fresh and dry weights of roots and shoot

A significant outcome of applied treatments was recorded on root and shoot fresh weights, dry weights and lengths (Figure 1) under control (75% FC), mild drought stress (50% FC) and severe drought stress (35% FC) conditions. NPK+NPB was remained significantly efficient in increasing root fresh weight and shoot fresh weight at both 50% FC and 35% FC over control treated (75% FC), barley plants. Additionally, MLE 2 differed significantly for the increase in root and shoot dry weight during the imposed drought stress (50% FC 35% FC) than control (75% FC). Treatments NPB, NPK and NPK+NPB were significantly varying for root and shoot length in comparison with control under drought stress. A prominent difference was noted in application of both fertilizers and in their combination in relation to control for all studied parameters at all irrigation treatments i.e., 75% FC, 50% FC and 35% FC. Notably the application of NPK+NPB boosted up plant growth attributes that signifies the ameliorating efficiency of given amendments in drought tolerance potential of barley [13].

 

Figure 1: Influence of solitary and integrated applications of NPB and NPK on diversified growth attributes of barley plants under 75% FC, 50% FC and 35% FC irrigation levels. Bars indicate mean values of three replicates. Error bars depict SE (standard error). Various letters on bars determine significant difference at p ≤ 0.05; LSD. (75% FC=well-irrigated; 50% FC=mild drought stress and 35% FC=severe drought stress conditions; NPB=Nitrogen and phosphorous Biofertilizers (nitrogen fixing and phosphorus solubilizing bacteria); NPK=N-P-K fertilizer (0.51-0.45-0.38 N-P-K g)).

Photosynthetic pigments

Photosynthetic pigments like chlorophyll a-b, total chlorophyll and carotenoids were predominantly influenced when disclosed to drought environment in comparison with well-irrigated ones (75% FC) (Figure 2). alternative treatments significantly enhanced plant photosynthetic pigments in comparison with control. NPK+NPB treatment was prevailed proficiently in elevating chlorophyll a-b at both 50% FC and 35% FC irrigation regimes over controlled barley plants (75% FC). Furthermore, NPK+NPB also differed significantly for upgradation of overall chlorophyll components during drought. A significant difference was recorded in NPB and NPK treated plants for all attributes under consideration at all irrigation levels. Our results affirm the worth of selected interventions in boosting the efficacy of water absorption and usage by growing plants [14].

 

Figure 2: Influence of solitary and integrated applications of NPB and NPK on photosynthetic pigments of barley plants under 75% FC, 50% FC and 35% FC irrigation levels. Bars indicate mean values of three replications. Error bars depict SE (standard error). Various letters on bars reveal significant difference at p ≤ 0.05; LSD. (75% FC=well irrigated; 50% FC=mild drought stress and 35% FC=severe drought stress conditions; NPB=Nitrogen and phosphorous Biofertilizers (nitrogen fixing and phosphorus solubilizing bacteria); NPK=N-P-K fertilizer (0.51-0.45-0.38 N-P-K g)).

Net photosynthesis, osmolytes, oxidative stress indicators and sodium contents

All alterations had positive significant progress in ameliorating drought effects in barley. Net photosynthesis, Total Soluble Sugars (TSS), Total Soluble Proteins (TSP), H₂O₂ (hydrogen peroxide), Malondialdehyde (MDA), proline, Ascorbic Acid (AsA), catalase and sodium contents of barley plants were remarkably impacted during drought stress compared to well-irrigated plants (75% FC) (Table 1). Both solitary and mixed applications of NPB and NPK significantly enhanced plant net photosynthesis, TSS and TSP over the control. NPK+NPB treatment was remained efficient in decreasing H₂O₂, MDA, AsA, proline and catalase contents at both drought levels of 50% FC 35% FC over 75% FC. Moreover, the combined effect NPK +NPB differed significantly better than sole applications of NPB and NPK at all irrigation regimes [15].

Ionic Sodium (Na) contents raised significantly in plants which were irrigated at 35% FC compared with plants irrigated with water to reach 50% FC and 75% FC (Table 1). Na contents were increased both in root and shoot of barley plants in drought stress. The given amendments had positive significant effects in ameliorating sodium toxicity effects in barley with NPK+NPB as the most effective one.

All figures were mean of three replications ± SD. various labels represent significantly variable alphabets by LSD test. (75% FC=well irrigated; 50% FC=mild drought stress and 35% FC=severe drought stress conditions; NPB=Nitrogen and phosphorous bio-fertilizers (nitrogen fixing and phosphorus solubilizing bacteria); NPK=N-P-K fertilizer (0.51-0.45-0.38 N-P-K g); TSS=Total Soluble Sugars; TSP: Total Soluble Proteins; H2O2: Hydrogen Peroxide; MDA: Malondialdehyde; AsA: Ascorbic acid; R. Na: Root Sodium contents and S. Na: Shoot Sodium contents).

Table 1: Net photosynthesis, osmolytes, oxidative stress indicators and sodium contents of barley grown in well-irrigated and drought stress conditions.

Pessarson correlation

Pearson correlation indicated that water stress impacted on plants was significantly far in comparison with shoot and root length, fresh and dry weights, photosynthetic pigments and osmolyte contents. Parameters like i.e., flavonoids, phenolics, proline, flavonoids, malondialdehyde, hydrogen peroxide, catalase and Na constituents were found remarkably positive in correlation with water stress (Figure 3). Drought stress increased the antioxidants while retarded the growth attributes and photosynthetic pigments and ionic ingredients of barley [16].

 

Figure 3: Pearson correlation of barley attributes in different water regime conditions.

Tolerance to drought stress in barley can be enhanced by supplementing it with chemical and bio-fertilizers to mitigate oxidative stress by enhancing plant antioxidant defense mechanisms. The provision of organic or biofertilizers as a worldwide drive against expensive fertilizers is studied by various investigators. The primary impacts of irrigation and fertilizer treatments were significant on all examined variables. Furthermore, the combination between irrigation and fertilizer treatments had a substantial impact on fresh and dry matter yield, plant length, photosynthetic pigments, osmolytes and several oxidative stress indicators [17].

Many diverse processes are involved in the plant stimulants that promote abiotic stress resilience to alleviate stress-induced yield decline, the majority of which involve phytohormones and antioxidants. The presence of growth stimulants, either indigenous or exogenously given, increased nitrogen absorption and an up-regulated antioxidative defense system that is linked to the induction or increase in tolerance during stress. By minimizing electrolyte leakage and maintaining membrane stability, plant growth regulators are associated to delayed photo-inhibition provoked by extrinsic stress factors and an increase in plant sterols [18].

Plant bio-stimulants, such as bacteria, may promote carbon-nitrogen metabolism, resulting in a boost in photosynthetic efficiency. These bioactive stimulate a number of cell-signaling pathways that benefit plant development, phytohormone production and yield factors. Sugars accumulated by these stimulants are thought to increase plant vegetative growth characteristics, providing more energy to stressed plants. Our research is in validation to these facts. In drought stress and well watered conditions, rhizobacteria enhanced photosynthetic pigment levels, lengths, fresh weights, and dry weights of both roots and shoots [19].

Rhizobacteria increase atmospheric nitrogen fixation capability of roots, mineral absorption and siderophores formation. The bacteria reduce several plant-growth restricting environmental hazards indirectly either by inducing stress tolerance or by producing antagonistic substances in plants and enhance vegetative growth. Inoculation of seeds by bacteria increased nitrate contents in barley observed in all given treatments with NPK+NPB as most effective one. Zulfiqar et al., noted enrichment in soil nutrients presence and advancement in fruits quality resulted by bio-stimulants in stress conditions. Development in crop nutritional condition and vigor is owing to efficient soil microbial dynamics and either due to the direct nutrient absorption, accumulation of amino acids and proteins by applied products or by existence of binding agents that increase nutrients emulsification in soil [20].

Previous studies support our findings in demonstrating that drought stress results in an elevation in Reactive Oxygen Species (ROS), which activates plant enzymatic and non-enzymatic antioxidant operations to maintain cellular redox balance. Present study determines that all considered growth, biochemical, ionic and antioxidant components of barley plants were enhanced by utilizing either of two devised amendments. NPK and NPB reduced all oxidants and oxidative stress indicators. Particularly, alternative amendments might offend each other’s impact up to some level in their mixed applications, as their solitary usage had declined physiological, biochemical and ionic attributes. Thus, fundamental effects NPK and NPB were more embellished thus resulting in drought tolerance in barley. The steep slope of plant growth and osmolytes increment in organic fertilizer treatment could be due to a higher mineralization rate under drought stress, that provides greater accessible nitrogen for the plant to absorb, resulting in a higher nitrogen concentration in the barley plants. Because of the greater oxidation rate, the mineralization rate might be boosted under drought stress circumstances.

But Organic fertilizers offer less nitrogen to the crop than chemical fertilizers due to lesser N intake or instability of N delivery with plant needs, resulting in narrow grain N in organic treatments. These findings correspond to proceedings by Rengel et al., Woese et al., Zhang et al., and Ryan et al.,. Integrated fertilizers i.e., chemical fertilizers are more reliable in providing more nutrients than organic fertilizers. Application of chemical fertilizer increase plant biomass as more N is available in soil and chemical fertilizer have better effect on root efficiency of N absorption. These findings are accompanied by Brezink's et al. findings.

Plant nutrient concentrations, according to Ryan et al., generally reflect soil exchangeable minerals and pH. As a result, it's reasonable to conclude that using chemical fertilizers in the soil reduced soil pH, which enhanced nutrient availability to plants. When the pH of the soil falls to roughly 7 or below due to drought stress, the presence of nutrients in soil and plant tissue increases. As a consequence, NPB application inclines to lower soil pH, which might comprehend the outcomes achieved in our experiment [21].

The provision of both nitrogen fixing and phosphorus solubilizing bacteria along with N-P-K fertilizer either in solitary or integrated treatments can diminish nutrient and drought stress issues. Although, composite application of NPK and NPB (NPK+NPB) can prominently improve barley growth attribute, photosynthetic pigment contents and nutrients uptake during drought stress. Integrated augmentation of NPK and NPB had more significant positive results in comparison to sole treatments. NPK+NPB was also competent in declining oxidative stress indicators and enzymatic antioxidants of barley and may a good promising approach in mitigating drought stress. Additional investigations at field levels are required while taking in account the impact of other environmental limitations to competitively prove our findings better.

The authors declare that there is no conflict of interest to publish this article.

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