COMPARATIVE  STUDIES  BETWEEN  SEAWEEDS  AND COMMERCIAL  ALGAE  IN  ALLEVIATION  OF  HARMFUL  EFFECTS OF  DROUGHT  STRESS  OF  FABA  BEAN  (VICIA  FABA  L.) PLANTS

A. D. El-Gamal, M. A. Ismail, M. A. Amin and A. M. Sayed

Botany and Microbiology Department, Faculty of Science, Al-Azhar University,

Cairo, Egypt.

ABSTRACT:The effect of seaweed extract (SWE) obtained from two macroalgae species (Sargassum latifolium and Corallina elongate) and two commercial algae (Canada power and oligo x) on drought stress tolerance in faba bean (Viciafaba L.) plants was studied. Examination of growth parameters and some physiological and biochemical parameters showed that SWE extract and commercial algae under stress conditions enhanced shoot length and decreased root length, in most cases, at stages 1&2 in faba bean plants with comparison to stress conditions. All treatments, mostly, caused decreases in fresh and dry weight of faba bean plants under drought stress.  Maximal increases in shoot and root lengths were observed in stress case in presence sargassum extracts in comparison to drought stress. Number of leaves, flowers and yield parameters decreased in response to drought. Bio stimulant especially sargassum extract caused increasing in these parameters. In most cases, Chl.a, Chl.b, Chl. a+b and carotenoids of leaves of faba bean plants increased at stage 1 and decreased at stage 2 as a result of all treatments. Carbohydrate and protein contents of root, shoot and seed yield showed increases under stress of faba bean plants. Amylase and protease activities revealed different responses to all treatments. With respect to antioxidant enzymes, peroxidase activity of faba bean plants at both stages of growth increased in response to all treatments, with exceptions of stress + sargassum and stress + Canada power at stage1. In case of activities of super oxide dismutase and poly phenol oxidase showed decreases at both stages of growth with comparison to stress conditions, with exception of super oxide dismutase at stag 2 of faba bean plants. Total phenolic content was increased in faba bean plants under different treatments (with exception of treatment with stress + Canada power) with respect to stress conditions. Acidic growth hormones, IAA, GA3 and ABA exhibited increases in GA3 contents of faba bean plants as a result to all treatments as comparison to stress condition, however IAA and ABA contents decreased, with exception of treatment with stress + sargassum extract in case of ABA. The increased total phenolic content and the enhancement of antioxidant enzymatic activity by SWE and commercial algae in stressed faba bean plants may contribute to protection against peroxidation and reduce the severity of water deficit.

INTRODUCTION

The faba bean (Vicia faba), is known for its high protein concentration in its seeds. It ranks fourth among the most important legume crops in the world, after dry beans, dry peas and chickpeas. The crop is a stable food that provides adequate nutrition to many people in the Middle East (Ammar et al., 2017). Legumes are a major source of protein in human and animal nutrition and play a key role in crop rotations in most parts of the world. When it grows in rotation with other crops, under certain environmental conditions, they can improve soil fertility and reduce the incidence of weeds, diseases and pests (Mwanamwenge et al., 1998).

Agriculture is facing the dual challenges of increasing crop production and climate change. Rising temperature, drought, salinity, floods, desertification and weather extreme are adversely affecting agriculture especially in developing world IPCC (2007). Environmental factors are essential components which effect on quality and quantity of crop yield to a great extent. The introduction of resistance to salt, cold, and drought into crop plants have become a topic of major economic interest for agriculture. In the case of drought, scientists have been able now to uncover some of the extremely intricate mechanisms through which seed from orthodox plants acquires tolerance to desiccation during their final maturation period (Oliver et al., 2010). Drought triggers a wide variety of plant responses (Ajum et al., 2011).

Global climate change makes drought a serious threat to food security worldwide. Drought, as an abiotic stress, is multidimensional in nature, and it affects plants at various levels of their organization. Three main mechanisms reduce crop yield by soil water deficit: (1) reduced canopy absorption of photosynthetically active radiation, (2) decreased radiation-use efficiency and (3) reduced harvest index (Earl and Davis, 2003).  Therefore, use of foliar application of algae (algal extract and commercial algae) may have become a new trend to reduce the harmful effects of drought on some crops.

Drought stress has pronounced effects on the growth, phenology, water and nutrient relations, photosynthesis, assimilate partitioning, and respiration in the form of physiological, biochemical, and molecular responses (Usman, 2014).

Seaweeds are excellent source of vitamins A, B1, B12, C, D and A, riboflavin, niacin, pantothenic and folic acid. (Thirumaran et al., 2009) stated that recent researches proved that seaweed fertilizers are preferred not only due to their nitrogen, phosphorus and potash content but also because of the presence of trace elements and metabolite similar to plant growth regulators. Recently, seaweed extracts as liquid fertilizers has come in the market for the simple reason that they contain many growth promoting hormones like auxin, gibberellin, trace elements, vitamins, amino acids and micronutrients. (Strik et al., 2004) reported  that the seaweeds extracts are effective fertilizers in many crops.

The using of seaweed products improve seeds germination, seedlings development, increase plant tolerance to environmental stresses (Zhang and Ervin, 2008), and enhance plant growth and yield (Kumari et al., 2011). Liquid extracts obtained from seaweeds have gained importance as foliar sprays and soil drench for many crops including various grasses, cereals, flowers and vegetable species. Also, they apply to stimulate seedling germination and rooting. At present one of the most promising applications of seaweeds is their use as plant bio stimulants. For example, aqueous extracts of Sargassum johnstonii at concentration from 0.1 to 0.8% (w/v) that is equivalent 1–8 mg SW mL^-1 used as foliar spray and soil drench enhanced vegetative growth (plant height, shoot length, root length, and number of branches) and reproductive parameters (flower number, fruit number, and fresh weight) of tomato (Kumari et al., 2011).

Seaweed extracts are often regarded as soft or natural products that can influence crop growth and development (Norrie and Hiltz, 1999). A wide range of beneficial effects has been observed including increasing crop yield, nutrient uptake, resistance to frost and stress conditions, longer shelf life of fruit, improved seed germination, and reduced incidence of fungal and insect attack and reduced the effect of salinity stress on membrane permeability (Wang et al., 2005). The effect of crude seaweed extracts of three green seaweeds (Cladophora dalmatica, Enteromorpha intestinalis, Ulva lactuca) and the three red algae (Corallina mediterranea, Janiarubens, Pterocladia pinnate) from the Egyptian Mediterranean Sea coast were studied by (El-Sheekh and El-Saied, 2000) on seed germination, growth of seedlings, chlorophyll content and other metabolic activities of Vicia faba, They found that the crude extract of C. dalmatica showed maximal activity, and it increased seed germination, length of main root and shoot systems and the number of lateral roots. Also, all the crude extracts of seaweed increased protein content in root and shoot systems, total soluble sugars and chlorophyll content in leaves. The cytokinin content of the green algae was higher than that in red algae. Growth of seedlings of Vicia faba was stimulated but to different degrees.

Canada power is commercial product contain Ascophyllum nodosum as main source of biofertilizer. Ascophyllum nodosum is a large brown alga (up to 2m) of the Fucaceae family which is common on both sides of North Atlantic Ocean (Martin et al., 2015). Oligo-X is commercial product contain oligosaccharides 3% and alginic acid 5%. oligosaccharides are model compounds to represent domains from the larger, more complex polysaccharides (Kinnaert et al., 2017). Alginic acid obtained from brown algae; 61% mannuronic acid and 39% guluronic acid (Parker et al., 2015).

This study was conducted to investigate the influence of spray field application of seaweed extract (SWE) and commercial algae for mitigating harmful effects of drought stress on growth, yield and biochemical constituent of faba bean plants in order to select a suitable bio stimulant to this purpose.

MATERIALS AND METHODS

Plant material:

Seeds of faba bean (Vicia faba misr 1) plants were obtained from Agricultural Research Centre, Ministry of Agriculture, Giza, Egypt.

Methods of planting, treatments and collection of samples:

Sargassum latifolium (Turner) C. Agardii was collected from Hurgada Red Sea coast in June 2019 and Baltim, while  Corallina elongate J. Ellis was collected in May 2019 from shallow water beside the shore of Mediterranean Sea at AbouQuair coast in Egypt. Collecting algae were washed with fresh water then were dried in the oven at 60ºC for 5 hours, hand crushed and powdered with coffee-grinder, then heated in sterile distilled water in a ratio 1: 100 (w/v) at 60 ^ºC for 45 min. The extracts were filtered through a filter paper and stored at 4 ºC for further experimental studies.  Concentrations of extracts were prepared by diluting these extracts with distilled water (Mikhail et al., 2013). The algal extracts and commercial algae (Canada power and Oligo-x) were applied as a foliar treatment at the rate of 4 g powdered algae/L and 4ml/L of commercial algae , after 30 and 60 days from sowing.

Treatments and experimental design:

Uniform faba bean seeds were planted in natural loamy soil conditions in a plot (12 m width and 15 m length) containing 6 groups representing the following treatments: control (tap water every 7 days), drought stress (tap water every 14 days), drought stress in presence Corallina extract, drought stress in presence Sargassum extract, drought stress in presence Canada power as commercial algae and drought stress in presence Oligo-x as commercial algae The seeds were sown on one side of the ridge, with 10 cm apart between the hills. The in Botanical garden, Botany and Microbiology Dept., Fac. of Sci., Al- Azhar Univ., Nasr City, Cairo, Egypt, developed plants were irrigated whenever required. Concentrations of the used treatments were chosen according to a preliminary experiment in which they caused a maximum germination percentage. The plants were sprayed twice with the above-mentioned treatments, the first and second were added at 30 and 60 days of plant age respectively. The plant samples were collected for analysis when the plants were 37 (Stage I) and 67 (Stage II) days old. At the end of the growth season, analysis of the seeds yielded from the different treatments and the control were done.

Determination of Metabolites content of Faba bean:

Chlorophylls contents of were estimated using the method of (Vernon and Selly, 1966). Carotenoids contents of were estimated according to (Lichtenthaler, 1987). Soluble carbohydrates were measured according to the method of (Umbriet et al., 1969). Contents of soluble proteins were estimated according to the methods of (Lowery et al., 1951). A phenolic compound (mg/100 g of dry wt) was carried out according to that method described by (Daniel and George, 1972). Activities of amylases were determined using the method of (Afifi et al., 1986). Proteases activities were estimated using the method of (Ong and Gaucher, 1972). Peroxidase activity was assayed using the method of (Jaworek, et al., 1974). Superoxide dismutase activity was. determined by measuring the inhibition of the auto-oxidation of pyrogallol using a method described by (Marklund and Marklund, 1974). The activity of polyphenoloxidase enzyme was determined according to the method adopted by (Matta and Dimond, 1963). The method of extraction of endogenous acidic phytohormones extraction was essentially similar to that adopted by (Shindy and Smith, 1975) and described by (Hashem, 2006). 

Statistical Analysis:

Results were statistically analyzed by calculating the analysis of variance, in completely randomized design (Snedecor and Cochran, 1982).

RESULTS AND DISCUSSION

Morphological responses and yield parameters:

The present results in Table (1) revealed that algal extracts of (Sargassum latifolium and Corallina elongate) and two commercial algae (Canada power and oligo x) under stress conditions enhanced shoot length and decreased root length, in most cases, at stages I and II in faba bean plants with comparison to stress treatment. Maximum enhancement increases in shoot lengths were observed in presence sargassum extracts. Our results agree with those of (Bassal and Zahran, 2002) revealed that the blue green algae addition significantly increased flag leaf area, plant height of rice (Oryza sativa) plants. The importance of SWE in stress water effect can be correlated to improvement of glycine betaine content in treated plants. In several plant species, a positive correlation between leaf osmotic potential and glycine betaine, β-alanine betaine, and proline betaine has been observed (Rhodes and Hanson, 1993). These organic compounds are now known to also have osmoprotective effects in the cell (Ashraf and Harris 2004). Seaweed concentrate prepared from Ecklonia maxima (Kelpak) was found to increase the root length and root number of Pinus pinea seedlings (Atzmon et al., 1994), increased root and shoot growth in three species of Eucalyptus (Van Staden et al.,1995) and promoted root formation in a variety of plants (Crouch and Van Staden, 1991), which has been attributed to the relatively high concentrations of indoles present in the extract (Crouch et al., 1992).

The obtained results in Tables (2,3 and 4) showed decreases in fresh, dry weight, No. of leaves, flowers and branches of faba bean plants in response to drought stresscompared to the control plants. All treatments caused improvement of this parameters with comparison to stress treatment. The increases in some of growth parameters coincides with results of (Kumari et al., 2011) showed thataqueous extracts of Sargassum johnstonii at concentration from 0.1 to 0.8% (w/v) that is equivalent 1–8 mg SW mL^-1 used as foliar spray and soil drench enhanced  vegetative growth (plant  height,  shoot  length, root  length, and number of branches) and reproductive parameters (flower number, fruit number, and fresh weight) of tomato. The enhancement of growth parameters by spraying of Vicia faba plants with commercial or algal extracts may be due to the presence of cytokinins, minerals and many of nutrient in commercial or algal extracts. These ingredients are known to improve the growth and increase cell division and cell enlargement (Ahmed et al., 2014). This might explain the remarkable increase in shoot height plants even than their control counter parts. 

Our results in Table (5) showed that commercial algae and algal extracts caused remarkable improvement in yield parameters comparing to drought stress. These results in agreement to those obtained by  (Jayaraj et al., 2008); (Khan et al., 2009) and (Hernández-Herrera et al., 2014), who revealed that, in recent years, the use of bio stimulants, often based on natural extract such as from seaweeds, has been proposed as a sustainable Strategy for improving crop yields without adversely impacting on the environment.

Chemical constituents:

Results recorded in Tables (6 and 7) revealed that, in most cases, Chl.a, Chl.b, Chl. a+b and carotenoids contents of shoot of faba bean plants increased at stage I and decreased at stage II as a result of all treatments. Plants adapt to drought stress through synthesis of osmoprotectants (osmolytes or compatible solutes) which are low-molecular-weight and highly soluble compounds that are usually nontoxic even at high cytosolic concentrations (Tekle and Alemu, 2016).  Drought stress caused  a   significant   increase   in  total  soluble carbohydrates and protein contents of Vicia faba plants. These results are in harmony with those of (Mohamed and Akladious, 2014). This increases in soluble compounds can protect the cell under stress by balancing the osmotic strength of the cytosol with

Table 1: Effects of drought stress and bio stimulant (Canada Power, Oligo X, and Corallina elongata, Sargassum latifolium extracts) on shoot length and root length of bean plants .

Table 2: Effects of drought stress and bio stimulant (Canada Power, Oligo X, and Corallina elongata, Sargassum latifolium extracts) on fresh and dry weights of shoots of bean plants

Table 3: Effects of drought stress and bio stimulant (Canada Power, Oligo X, and Corallina elongata, Sargassum latifolium extracts) on Fresh and dry weights of roots of bean plants

Table 4: Effects of drought stress and bio stimulant (Canada Power , Oligo X,and Corallina elongata, Sargassum latifolium extracts) on  leaves, flowers and branches numbers  of bean plants

Table 5: Effects of drought stress and bio stimulant (Canada Power, Oligo X, and Corallina elongata, Sargassum latifolium extracts) on yield Parameters of bean plants.

that of the vacuole and the external environment (Anjum et al., 2011).  The importance of seaweed extract (SWE) in stress water effect can be correlated to improvement of glycine betaine content in treated plants. In several plant species, a positive correlation between leaf osmotic potential and glycine betaine, β-alanine betaine, and proline betaine has been observed (Rhodes and Hanson, 1993).

Data in the present study are similar to those of (Genard et al., 1991) reported that glycine betaine delays the loss of photosynthetic activity by inhibiting chlorophyll degradation during storage conditions in isolated chloroplasts the decrease in chlorophyll under drought stress is mainly due to the damage of chloroplasts by reactive oxygen species (Smirnoff, 1993). The seaweed extract applied as foliar spray enhanced the leaf chlorophyll level in plants (Blunden et al., 1996). The effect of water deficit was notably reduced by the foliar application of SWE. The benefit effect of algae extracts in protecting chlorophyll degradation may be attributed to betaine and betaine-like compounds present in seaweed (Khan et al., 2009). In plants, betaines serve as a compatible solute that alleviates osmotic stress induced by salinity and drought stress. Glycine betaine protects physiological processes such as photosynthesis and protein synthesis under drought conditions (Sulpice et al., 1998). On the other hand, it has been reported (Zhang and Ervin, 2008) that positive anti-stress effects of seaweed extracts may be related to cytokinin activity. Cytokinins mitigate stress-induced free radicals by direct scavenging and by preventing reactive oxygen species (ROS) formation by inhibiting xanthine oxidation (Fike et al. 2001).

Table 6: Effects of drought stress (in presence or absence of) and bio stimulant (Canada Power, Oligo X, and Cor. elongata, Sargassum latifolium extracts) on Chlorophyll a and b of bean plants at I and II stages.

Table 7: Effects of drought stress and bio stimulant (Canada Power, Oligo X, and Cor. elongata, Sargassum latifolium extracts) on total chlorophyll (a+ b) and Carotenoids of bean plants at I & II stages

As recorded in Table (8) the increase in the protein content at lower concentration of seaweed fertilizer (SLF) might be due to absorption of most of the necessary elements by the seedlings (Kannan and Tamilselvan, 1994; Anantharaj and Venkatesalu, 2001, 2002).

Furthermore, the obtained results in Table (9) indicated that the soluble carbohydrates content increased up to 20% concentration of SLF and the content decreased at higher concentrations. The same trend was observed in the H. musciformis with NPK application in blackgram (Tamilselvan and Kannan, 1994), V. catajung and D. bixorus (Anantharaj and Venkatesalu, 2001, 2002).

Data illustrated in Table (10) showed no apparent trend with respect to amylase and protease activities. The most significant increases in amylase activities were observed in case of treated faba bean plants with stress + Corallina at stage 1 and stress + Oligo-x at stage 2. The highest increases of protease activities were observed in case of treated faba bean plants with stress + Canada power at both stages of growth. The present results are in accordance with those obtained by (Sivasankari et al.,  2006) observed that the α-amylase activity was higher than the β- amylase activity. Th α- amylase and β-amylase activity increased at lower concentrations of both the treatments of seaweeds.

Table 8: Effects of drought stress and bio stimulant (Canada Power, Oligo X, and Corallina elongata, Sargassum latifolium extracts) on soluble proteins of bean (plants) Shoot and Root at stages I and II; Values means of three replicates.

Table 9: Effects of drought stress and bio stimulant (Canada Power, Oligo X,and Corallina elongata, Sargassum latifolium extracts) on soluble carbohydrates of bean plants (shoot and root) at I and II stages;Values means of 3 replicates.

Table 10: Effects of drought stress and bio stimulant (Canada Power, Oligo X, and Corallina elongata, Sargassum latifolium extracts) on Amylase and Protease activities (mg/g dry weight) on bean plants at stages I and II; Values are means of three replicates.

The demonstrated results in Table (11) showed that peroxidase activity of faba bean plants at both stages of growth increased in response to all treatments, with exceptions of stress + sargassum and stress + Canada power at stage1. In case of activities of superoxide dismutase and polyphenol oxidase showed decreases, mostly, in response to all treatments at both stages of growth as compared to stress conditions, with exception of increases in superoxide dismutase as a result of all treatments at stag 2 of faba bean plants. Our result may be explained by the effect of seaweed extract in reducing cell damage caused by reactive oxygen species (ROS) (Khan et al., 2009). Application of seaweed extract to turf grasses increased the activity of the antioxidant enzyme superoxide dismutase (SOD), which scavenges superoxide (Fike et al. 2001). Similarly, (Ayad, 1998) reported an increase in SOD of plants treated by seaweed extract. Many researchers have reported that seaweed extracts enhance the ascorbate peroxidase activities (Ayad, 1998), demonstrating the strong antioxidant properties of seaweeds which have been correlated to bioactive compounds (Meenakshi et al., 2009; O’Sullivan et al., 2011).

The obtained results in Tables (12&13) indicated that total phenolic content was increased in faba bean plants because of different treatments (with exception of treatment with stress + Canada power) with respect to stress conditions. Acidic growth hormones, IAA, GA3 and ABA exhibited increases in GA3 contents of faba bean plants as a result to all treatments as comparison to stress condition, however IAA and ABA contents decreased, with exception of increasing ABA contents as a result of treatment with stress + sargassum extract. Our results are similar to findings of (Nilsen and Orcutte, 1996) reported that, under drought, endogenous contents of auxins, gibberellins and cytokinin usually decrease, while those of abscisic acid and ethylene increase Nevertheless, phytohormones play vital roles in drought tolerance of plants. Auxins induce new root formation by breaking root apical dominance induced by cytokinins. As a prolific root system is vital for drought tolerance, auxins have an indirect but key role in this regard. Drought stress limits the production of endogenous auxins, usually when contents of abscisic acid and ethylene increase (Nilsen and Orcutte, 1996).

Table 11: Effects of drought stress and bio stimulant (Canada Power, Oligo X, and Corallina elongata, Sargassum latifolium extracts) on Peroxidase, Superoxidase dismutase and Polyphenol oxidase activities (µg/g dry weight) on bean plants at stage I and II; Values are means of three replicates.

Table 12: Effects of drought stress and bio stimulant (Canada Power, Oligo X, and Corallina elongata, Sargassum latifolium extracts) on total phenol (mg of gallic acid / 100 g fr. wt.), seed yield protein and carbohydrates (mg/gm dry wt.) of bean plants. Values are means of three replicates.

Table 13: Effects of drought stress and bio stimulant (Canada Power, Oligo X, and Cor. elongata, Sargassum latifolium extracts) on Phytohormones of bean plants Values are means of threereplicates.

Polyphenols may act as antioxidants to protect the plant against oxidative stress (Grace, 2005). Increase in total phenolic content by application of SWE in bean plans can be explained by enzyme activation. It was reported (André et al., 2009) that treatment with SWE caused significantly enhanced activities of phenylalanine ammonia lyase (PAL) the most important enzyme responsible for biosynthesis of polyphenols.

Conclusion

In the light of the present study, it seems reasonable to suggest that spraying of Vicia faba plants with commercial algae (Oligo x and Canada power) and algal extract can successfully ameliorate the deleterious effects of drought stress as well as enhance the plant  growth. Furthermore, it is worth noting that Sargassum extract were more effective than commercial algal in raising the plants’ tolerance to drought. Therefore, we would venture to recommend the use of spraying Vicia faba plants with Sargassum extract as a new natural and low-cost method for not only the alleviation of drought stress on plants but also for stimulating growth with no discernible adverse effects.

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