Enhancement effect of Hydroxyl Benzoic Acid (HBA) foliar application on growth performance of Triticum aestivum L. (Wheat) under induced chromium toxicity

Sami Ullah, Musawar Khan, Muhammad Idress, Sajjad Ali, Muhammad Nauman Khan, Muhammad Adnan and Fethi Ahmet Ozdemir 1. Department of Botany, UOP, KP, 25120-Pakistan 2. Department of Botany, BKUC, KP, 24420-Pakistan 3. Department of Chemistry, BKUC, KP, 24420-Pakistan 4. Department of Molecular Biology and Genetics, Faculty of Science and Art, Bingol University, 12000, Bingol-Turkey *Corresponding author’s email: sami_jan69@yahoo.com Citation Sami Ullah, Musawar Khan, Muhammad Idress, Sajjad Ali, Muhammad Nauman Khan, Muhammad Adnan and Fethi Ahmet Ozdemir. Enhancement effect of Hydroxyl Benzoic Acid (HBA) foliar application on growth performance of Triticum aestivum L. (Wheat) under induced chromium toxicity. Pure and Applied Biology. Vol. 9, Issue 4, pp2529-2542. http://dx.doi.org/10.19045/bspab.2020.90269


Introduction
Metals being essential component of the environment chiefly deposited in the soil due the over use of chemical fertilizers, pesticides and poor municipal waste disposal [1,2]. The term "heavy metals" refers to any metal element having high density and toxic in nature even at little concentration. Increasing heavy metal concentration in soil also increases the uptake of heavy metals by plants depending upon the type of soil, plant growth stages and species [3]. Heavy metals reduce nutrient uptake in plants and causing chaos in plants metabolic activities such as lowering chlorophyll content, mineral nutrients deficiency and finally chlorosis [4]. Chromium is amongst one of the prominent heavy metals whose concentration in the atmosphere is increasing at an alarming rate. Furthermore, degradation of chromium is not possible into harmless products, hence it retains in the environment for longer period of time [5]. Higher concentrations of Cr +3 is potentially harmful at germination and early seedling growth stages [6]. High levels of chromium cause inhibition of plant growth, chlorosis in young leaves, nutrient imbalance, wilting of tops, and root injury [7]. Heavy metals entering into plant tissues inhibit most of the physiological processes of metabolism including respiration, photosynthesis and nitrate assimilation [8]. In Pakistan the toxic heavy metals from industries are entering in food chain and ground water supplies; leading to serious ailments in local population [9]. The industrial effluents (untreated) are the main sources of heavy metal pollution [10]. Besides this, leather and tanning industries are the major sources of chromium and other potentially detrimental heavy metals in both water and soil (through leaching) thus polluting both to a great extent. Chromium releases in waste water from different industries such as food processing and match producing industries, due to which the vegetable fields irrigated with sewage around Peshawar and Faisalabad contained significantly higher concentration of chromium [11,12]. According to Ali et al., [13] chromium stress causes ultra-structural disorders in leaves, root ultra-structural modification, disruption and disappearance of nucleus. Higher concentration of Cr +6 in the plant root zone affects many physiological processes and inhibits plant growth [14]. Hexavalent chromium (Cr +6 ) causes a decrease in shoot length of wheat [15]. Common agricultural crops such as wheat, Indian mustard, sorghum and maize are affected by chromium, higher chromium concentration inhibited germination, plumule and radicle length [16].
Biochemical aspects like photosynthetic pigments (Chlorophyll a and Chlorophyll b), total protein and amino acids content decreased with increasing chromium concentration [17]. Different applied treatments of foliar spray increased all studied growth parameters as number of branches and leaves per plant, leaf area per plant and leaves dry weight as well [18]. Under the influence of 4-HBA superoxide dismutase and apoplastic forms of peroxidase in wheat coleoptiles were increased [19]. Chromium effluent is highly toxic to plant and is harmful to their growth and development [20]. Toxicity of chromium in plants also results in inhibition of enzymatic activities, impairment of photosynthesis, nutrient and oxidative imbalances, and mutagenesis [21]. High levels of chromium interfere with important metabolic processes causing toxicity and ultimately reduction in growth and yield. It leads to stunted growth decrease of rootshoot growth and total biomass, chlorosis and eventually plant death [22]. However many reports have focused on the toxic effects of Cr +3 and Cr +6 on plants, our knowledge about its toxicity is shallow, and the detailed mechanisms are not richly understood. Although due to our poor knowledge, we understood up to some extent that heavy metal chromium have negative effects on crop plants. To achieve the objective of studies, little efforts have been made to determine:  The effect of hydroxyl benzoic acid foliar application on growth performance of wheat.  The effect of hydroxyl benzoic acid foliar application on morphological characters of Triticum aestivum L.  The effect of hydroxyl benzoic acid application on agronomic characters of wheat and  To determine the Chromium toxicity of rhizospheric soil on growth of wheat.

Materials and methods Plant material and growing conditions
Seeds of two selected varieties of Triticum asetivum L. (wheat) including Ta-Habib and Saher were obtained from Cereal Crop Research Institute Persabaq Nowshehra (CCRI). The seeds were sterilized in 5% chlorox for 2 minutes then washed with 50% ethanol for 3 minutes. Seeds were washed with distilled water and sown in plastic pots filled with soil and sand in ratio of 3:1. Proper ventilation was maintained, weeds were eradicated periodically and pots were protected from harsh weather regimes. For each treatment 3 pots (replicates) were taken. The numbers of total pots was 30. Numbers of seeds per pot were 12. Ta-Habib was selected as variety-1 (V1) and Saher was variety-2 (V2).

Nutrient culture experiment
The seedling was grown in 2ppm and 4ppm of Cr +3 concentrations. These various concentrations of chromium were dilute to 100ml (used water) in volumetric flask. Application of heavy metal (Cr +3 ) concentrations to plants Required concentrations of heavy metal chromium are 2pp and 4ppm were applied to required replicates of plants in amount of 5ml was imposed withholding water supply for a period of 10 days. The first heavy metal treatment was started at 2ppm and 4ppm, the second and third was same as first. First treatment of heavy metal (Cr +3 ) concentrations (2ppm and 4ppm) were given to plants after 24 days of sowing. Second treatment of heavy metal (Cr +3 ) concentrations (2ppm and 4ppm) were given to require replicates of plants after 10 days of first treatment. Third treatment of heavy metal (Cr +3 ) concentrations (2ppm and 4ppm) were also given to plants after 10 days of the second treatment. Application of HBA foliar spray During experimental work HBA (Hydroxyl Benzoic Acid) foliar spray were prepared. 1ml of HBA taken by the help of puppet and were poured in 100ml of water (1ml/1liter). Then the prepared HBA spray was sprayed on required replicates by the help of spray pressure pump. First treatment of HBA spray was given to required replicates of plants on 24 days of sowing. Second treatment of HBA spray was given to required replicates of plants after 10 days of the first treatment. Third treatment of HBA spray was also given to required replicates of plants after 10 days of the second treatment.

Sampling
Sampling was done three times with intervals of 7, 14 and 21 days after the onset of chromium treatment. For each treatment 3 replicates were taken. The samples were stored and weighed till further analysis. Assessment and measurement of agronomic characters Leaf, shoot, root and soil fresh and dry weight in grams (g) were measured for all the replicates with the aid of electric balance.

Plant powder preparation
For further analysis plant samples were kept and dried at room temperature after drying the samples, grinded to fine powders then stored in moisture proof plastic bags for further analysis. Chemical analysis of rhizospheric soil Soil moisture content Soil (10 gram) was taken from uniform depth i.e. 6 inches from the surfaces of pots. Dry weight was determined after drying the soil in oven for 72 hrs at 70 ˚C till constant weight. The %age moisture content of soil is calculated by following formula: MC (%) = Fresh weight of soil (g)-Dry weight of soil (g) x100 Weight of dry soil (g)

Percent field capacity of rhizospheric soil
The field capacity (%) of rhizospheric soil can be calculated by following mathematical equation: FC (%) = Weight of wet soil (g)-Weight of dry soil (g) x100 Weight of dry soil (g)

Chromium analysis
The rhizospheric soil and plant powder was analyzed for chromium (Cr +3 ). Methodologies for the preparation of different reagents, stock solutions, working solution and standards solutions are given in appendix.
Chromium analysis of rhizospheric soil 0.25gm of rhizospheric soil extract and 10-20ml of distill water was taken in a test tube and analyzed for Cr +3 on atomic absorption Spectrophotometer. For the determination of chromium, stock solutions were made. 100 ppm stock solution of the Cr +3 were prepared by dissolving required amount of salts in distilled water. The availability of Cr +3 elements in the selected varieties of plants from Charsadda district was determined by Perchloric-acid digestion method [23].

Chromium analysis of selected plants of Charsadda district
For the determination of the abovementioned element, stock solutions were made. 100 ppm stock solution of the Cr +3 were prepared by dissolving required amount of salts in distilled water. The availability of Cr elements in the selected varieties of plants from Charsadda district was determined by Perchloric-acid digestion method [23]. For this whole plants powder were also 0.25 gm taken from each treatment. Dried samples (0.25g) were taken in 100 ml beaker and add 5 ml of mixed acid solution i.e., Perchloric acid, sulfuric acid and nitric acid having ratios of 1:0.1:5 respectively. Afterward the mixture was boiled in fume hood on hot plate till the digestion has been completed; indicated by white fumes coming out from the flasks. Moreover, few drops of distilled water were mixed and allowed to cool. The digested samples were transferred in 100 ml volumetric flasks and the volume was made up to 100ml by adding distilled water. Then filtered the extract with Whitman filter paper and filtrate were collected in labeled plastic bottles. Concentration of these elements in the entire samples was determined by Shimadzu AA-670 Atomic Absorption Spectrophotometer.

Results and discussion
Results in (Table 1) indicated that maximum percent moisture content, field capacity, percent germination has been reported in Ta-Habib and Saher has been recorded in treatment T5 (control) with 16.05% and 15.75% followed by treatment T2 (2ppm Cr +3 +HBA) with 13.40% and 12.70% after 7, 14 and 21 days respectively, indicating that Hydroxyl benzoic acid play a positive role in increasing of moisture content and field capacity, while chromium exhibiting negative effect on these parameters. Maximum shoot fresh weight ( Table 2) has been recorded in treatment T2 (2ppm Cr +3 +HBA) after 7 days with 0.238g followed by treatment T5 (control) with 0.238g in cultivar Ta-Habib while that of Saher has been recorded in treatment T5 (control) with shoot fresh weight 0.20g followed by T2 (2ppm Cr +3 +HBA) with 0.19g, while minimum shoot fresh has been recorded in T3 (4ppm Cr +3 ) with 0.181g and 0.16g respectively, result after 14 days of treatments shows that maximum shoot fresh weight has been recorded in the treatment T5 (control) with 0.40g and 0.42g followed by treatment T2 (2ppm Cr +3 +HBA) with 0.34g and 0.40g, while minimum shoot fresh weight has been recorded in the treatment T3 (4ppm Cr +3 ) with 0.18g and 0.35g respectively in Ta-Habib and Saher. Maximum shoot dry weight ( Table 2) has been recorded in treatment T5 (control) after 7 days in both wheat varieties (Ta-Habib and Saher) with 0.07g and 0.05g, while lowest shoot dry weight in both varieties has been recorded in treatment T3 (4ppm Cr +3 ) with 0.03 and 0.04g followed by T1 (2ppm Cr +3 ) with 0.061g and 0.04g respectively. Result after 14 days shows that maximum shoot dry weight in Ta-Habib and Saher has also been recorded in treatment T5 (control) with 0.13g and 0.13g followed by treatment T2 (2ppm Cr +3 ) with 0.12g and 0.13g, whereas lowest shoot dry weight after 14 days has been recorded in treatment T3 (4ppm Cr +3 ) with 0.06g and 0.09g respectively.
Result regarding to root length shows in (Table 3), result after 7 days shows that the maximum root length in both varieties (Ta-Habib and Saher) has been recorded in treatment T5 (control) with 5.60cm and 6.65cm, whereas the smallest values of root length has been reported in treatment T3 (4ppm Cr +3 ) with 2.75cm and 4.15cm respectively. Result showing greatest root length in Ta-Habib and Saher after 14 days in treatment T5 (control) with 6.30cm and 5.25cm followed by T2 (2ppm Cr +3 +HBA) with 6.0cm and 4.65cm, after 21 days greatest root length been recorded in T5 (control) with 5.80cm and 8.20cm followed by T2 (2ppm Cr +3 +HBA) with 5.65cm and 5.05cm respectively, while the smallest root length has been reported in both wheat varieties (Ta-Habib and Saher) in T3 (4ppm Cr +3 ) after 14 days with 3.70cm and 0.07cm, and after 21 days with 4.20cm and 3.80cm. Result shows in (Table 3) that the maximum root fresh weight in wheat (Triticum aestivum L.) varieties (Ta-Habib and Saher) has been recorded in treatment T5 (control) after 7 days with similar value 0.08g and 0.08g, after 14 days with root fresh weight, 0.44g and 0.10g and after 21 days with maximum root fresh weight 0.46g and 0.18g respectively. While minimum root fresh   the largest leaf width after 7 days of treatment in wheat variety Ta-Habib has been recorded in treatment T2 (2ppm Cr +3 +HBA) and T5 (control) with similar values 0.45cm and that of Saher also has been found in T2 (2ppm Cr +3 +HBA) followed by T5 (control) with 0.40 and 0.39cm, after 14 days in both varieties (Ta-Habib and Saher) leaf width recorded in T5 (control) with similar value 0.50cm, after 21 days in both varieties (Ta-Habib and Saher) leaf width has also been recorded in T5 (control) with similar value 0.75cm. While minimum leaf width has been recorded in the treatment T3 (4ppm Cr +3 ) in both wheat varieties (Ta-Habib and Saher) after 7 days with 0.30cm and 0.35cm, after 14 days with similar value 4.00cm and after 21 days with minimum leaf width 0.60cm and 0.64cm respectively.    (Table 6) after 7, 14 and 21 days of treatments that highest concentration of chromium in rhizospheric soil of Ta-Habib has been recorded in the treatment T4 (4ppm Cr +3 +HBA) with 2.17ppm, 2.09ppm and 2.66ppm followed by T3 (4ppm Cr +3 ) with 2.10ppm, 2.07ppm and 2.63ppm, whereas that of Saher has also found in T4 (4ppm Cr +3 +HBA) with chromium concentrations 2.12ppm, 2.13ppm and 2.55ppm followed by T3 (4ppm Cr +3 ) with 2.04ppm, 2.10ppm and 2.08ppm respectively . While minimum chromium concentration in rhizospheric soil in both wheat varieties (Ta-Habib and Saher) has been founded in treatment T5 (control) after 7 days with 1.97 and 1.82 ppm after 14 days with1.93ppm and 2.03ppm and after 21 days 2.48pp and 2.45ppm followed by T1 (2ppm Cr +3 ) after 7 days with 2.02ppm and 1.8ppm after 14 days with 2.01ppm and 2.03ppm, after 21 days with 2.02ppm and 2.07ppm respectively. Result in (Table 6) after 7, 14 and 21 days of treatments that highest concentration of chromium in whole plants both wheat (Triticum asetivum L.) varieties (Ta-Habib and Saher) recorded in treatment T4 (4ppm Cr +3 +HBA), after 7 days with 2.60 and 2.40ppm, after 14 days with 2.67 and 2.61ppm and after 21 days with 2.71 and 2.72ppm respectively, while minimum concentrations of chromium in whole plants has been recorded in treatment T5 (control) after 7 days with 2.43 and 2.29ppm, after 14 days with 2.48 and 2.48ppm and after 21 days with 2.59 and 2.64ppm followed by T1 (2ppm Cr +3 ) after 7 days with 2.47 and 2.29ppm, after 14 days with 2.49 and 2.50ppm and after 21 days with 2.60 and 2.65ppm respectively, showing that Hydroxyl benzoic acid can help to speedup accumulation of heavy metal chromium in plant. Experiment held for the present study to revealed the effect of hydroxyl benzoic acid foliar spray under various concentrations of chromium (Cr +3 ) on physiological and agronomic characteristics of Triticum aestivum L. (wheat) including two varieties Ta-Habib and Saher, for which three collections of agronomic & physicochemical characters with intervals of 7 days, 14 days and 21 days of chromium treatment were collected results revealed that the Hydroxyl benzoic acid has been increased the moisture content and percent field capacity while high concentrations of chromium reduced soil percent moisture content and field capacity in both wheat varieties (Ta-Habib and Sahar) significantly as shown in (Table 1). Shoot length has been reduced in the treatments which were treated with chromium in different concentrations i.e., 2ppm and 4ppm in all of the treatments, while hydroxyl benzoic acid foliar spray were found to improve the shoot characters even at high concentrations of chromium (4ppm) given in (Table 2). Similary Pati et al., [24] reported that the effect of chromium stress on plants exhibited a gradual decline in the growth, total chlorophyll content and protein content along with enhanced proline content with increasing levels of chromium. Shoot weights were also illustrated in (Table 2) showed significantly reduction along the concentration gradient in cultivar Ta-Habib and Saher. Highest values of shoot weight were recorded in T5 (untreated control) followed by spray treatments T2 and T4 in comparison to other treatments such as T1 and T3 in both varieties. Saif et al., [15] investigated that chromium on growth and biochemical parameters of Triticum aestivum, caused a significant decrease.
Experimental work revealed that the root length after 7 days, 14 days and 21 days was adversely affected by chromium in treatments T1 (2ppm Cr +3 ) and T3 (4ppm Cr +3 ) compared to spray treatments i.e., T2 (2ppm Cr +3 +HBA) and T4 (4ppm Cr +3 +HBA) while maximum root length in both varieties were recorded in T5 (untreated control) and minimum root length were reported in T3 (4ppm Cr +3 ) as described in (  The present study revealed that the leaf length has been decreased with chromium even at 2ppm concentration in all of the three treatments in both varieties (Ta-Habib and Saher) ( Table 4). Results showed that leaf weight also has been reduced with chromium, relatively the leaf weight has been lowered with chromium even at low concentration indicated that significantly chromium decreased the weight of leaf