The Interactive Effect of Sewage Sludge Application with Phytohormones IAA or SA on Three Broad Bean Cultivars

The present work has been performed to study the growth and metabolic activities of three broad bean cultivars (Giza 843, Sakha 1, and Giza 716) which are shown to have different response to sewage sludge applications and to determine the effects of phytohormones IAA or SA. Along with identifying the mechanisms of broad bean response and the role of growth regulators (200 ppm of IAA or 10 mM of SA) in counteract sewage sludge effects is ex-amined. The situation can be recorded that growth parameters fresh, dry matter and water content of pods were significantly increased as increasing SSL in cv. Giza 843 and in cv. Sakha 1 up to 70% level. However, these parameters were markedly decreased as increasing SSL in cv. Giza 716. Length and number of pods were unchanged in cv. Sakha 1, increased in cv. Giza 843 while, they were decreased in cv. Giza 716 with increasing SSL. Phytohormonal application stimulated growth parameters in pods of three tested cultivars Giza 843, Sakha 1 and Giza 716 with sewage sludge treatments. Soluble sugar and soluble protein contents were decreased as increasing SSL in both cv. Sakha 1 and cv. Giza 716. In cv. Giza 843, soluble sugar was significantly increased while soluble protein was decreased as increasing SSL. Proline content was unchanged in cv. Sakha 1, increased in cv. Giza 716 while, it was decreased in cv. Giza 843. Metabolites (soluble sugar, soluble protein and proline) showed variations in three broad bean cultivars to IAA or SA application plus sewage sludge treatments. In cv. Giza 716, SS treatment increased Zn, Ni, PB and Cu. In cv. Giza 843, SS treatment decreased Zn, Pb, and Cu while increased Ni. In cv. Sakha 1, SS treatment decreased Zn, Ni, Pb and Cu. Phytohormonal treatments showed an increasing or decreasing effect in heavy metals in three tested cultivars.


Introduction
Agriculture actions in the El-Minia distinct of central Egypt hinge on water removal from the River Nile along secondary canals. Maximum of cultured land are watered by saturating methods that next generate an additional of subsoil water. A result of the constant rise in the water quantity, soil strength and salt concentrations are affected, which clues to problems of water logging. For these reasons, drilling of the El-Moheet drain is directed to decrease the water level, mainly on the western bank of the Nile Valley. This drain receives sewage, industrial, and agronomic unwanted waters from other negligible drains. The main danger is the straight linking between this drain and the River Nile through a subsidiary Etsa drain. However, contamination is probable for groundwater in the region around the drain as exposed by Abdel-Dayem, (2011) [1]. The region between nearby the drains was sensibly chosen for polluted soil [2] [3] [4].
Sewage sludge is waste organic wide and biogenic compounds especially nitrogen and phosphorus which are essential for plant growth [5] [6] [7]. Conversely it also contains heavy metals in addition to those classified as poisonous (Cd, Cr, Cu, Hg, Ni, Pb and Z) [8] [9]. Provisional on the concentration and exposure time, an assumed metal can carriage environmental and fitness risks which are unified with its bulk to bioaccumulation the food chain [10]. The highest community origins of heavy metals in sewage sludge are national and manufacturing wastewaters and deterioration of sewage systems, as well as surface run from recognized areas or streets [11] [12]. Zeid and Abou El Ghate (2007) [13] showed that seed germination and growth of bean seedlings were fortified by irrigation with sewage water. Growth encouragement was attended with increase pigment production, carbohydrate, nucleic acids, and nitrogenous compounds, except amino acids N was reduced. Increased enzymes activity (amylase, invertase and protease) with sludge treatments may be due to the presence of specific ions which action as activator for these enzymes. Additioally, the effect of sewage sludge on nutrient uptake and growth parameters of larch seedlings (Larix decidua), minerals and heavy metals and scattering were studied by Bourioug (2014) [14]. The extreme foremost metals in sludge were Zn and Cu. Sludge differences in hormone and stress prompting activities of municipal wastewater in straight activated sludge wastewater use plant water [15]. Correspondingly, Klimas et al. (2016) [16] stated that presentation of products as composts covering phytohormones in plants. Growth corrected of signals system improved nutrient uptake, improved stress and decease resistance and de- Thus the present work was carried out to illustrate the effect of sewage sludge and interactive with phytohormones (IAA or SA) on growth, metabolites and heavy metals on three broad bean cultivars.

Experimental Sites and Sewage Sludge Treatments
Broad bean plant is important economic crop plant and considers the first plant food for Egyptian people because it contains highly benefit protein and other essential elements for man healthy. In Egypt, faba beans is the most common fast food item in the Egyptian diet, eaten by rich and poor alike. Egyptians eat faba beans in various ways. The prepared beans, called fulmedames, are then eaten with bread. Faba bean is an excellent source of protein (20% -25%), calcium (0.15%), phosphorus (0.50%), lysine (1.5%) and methionine-cysteine (0.5%) in dry weight. It is also an excellent source of complex carbohydrates, dietary fiber, choline, lecithin, minerals and secondary metabolites (phenolic and levo-dihydroxy-phenylalanine (L-DOPA), which is the precursor of the neurotransmitter dopamine and naturally found in seedlings, green pods and beans [5] (1:1) for 3 minutes, followed by several washings with sterile distilled water.
Ten seeds were sown per each pot. Each pot contained 3.6 kg of garden clay soil.
All pots were irrigated with tap water for four weeks until full germination. After one month the pots were irrigated with the following Moheet drain water of the desired concentrations: 0.0%, 20%, 50%, 70% and 100% in each tested pots with the same concentrations of sewage sludge treatments and were classified into three groups.

Sewage Treatment and Combined with IAA
From three of previous groups which treated with different sewage concentrations (0%, 20%, 50%, 70% and 100%), one group was sprayed by 200 ppm of IAA, the other group was sprayed with 10 mM of SA. In order to maintain the osmotic potential, the soil moisture content was kept near the field capacity using tap water. The seedlings were left to grow in natural conditions under these conditions for 150 days. At the end of the experimental period (5 months) yields of the pods were determined.

Laboratory Analysis for Metabolites
To determine the dry matter yields of pods, they were dried in an oven at 80˚C.
Successive weighting was carried out until the constant dry weight of each sample was reached. The soluble sugars were determined by the method of anthrone sulphoric acid which was stated by Fales (1951) [19]. The soluble proteins were determined according to the method adopted by Lowery et al. (1951) [20] and proline by Bates et al. (1973) [21].

Statistical Analysis
The triplicate sets of the experimental data for the different tested parameters were subjected to the one way analysis of variances (ANOVA) test in accordance with the experimental design using the SPSS program, version 13.0 and the means were compared using the least significant differences, L. S. D. at P levels of 0.05% (Steel, 1960) [22].

Growth Parameters as Affected by Sewage Sludge and Phytohormones Applications
Fresh, dry matter and water content of pods in cv. 843 were significantly in-  Figure 1). Additionally, length and number of pods were became unchanged with increasing SSL treatments ( Figure 2(a), Figure 2(b)). Increasing sewage sludge level application stimulated the production of fresh, dry matter and water content in pods of cv. Sakha 1 up to 70% SSL, after that, a sudden inhibition effect was induced (Table 1 & Figure 1). The percent of increase was 183.2%, 199.0% and 169.6% at 70% SSL compared with control plants (Table 1 & Figure 1). Correspondingly, length and number of pods in cv. Sakha 1 were increased with increasing SSL reached a maximum percent value at 70% SSL with percent 303% and 181.2% and then, a surprising reduction was induced at 100% SSL ( Figure 2(a), Figure 2(b)). Fresh, dry matter and water content of pods in cv. 716 were significantly decreased as increasing SSL application with percent 32.5%, 42.3%, and 29.4% at 100% level as compared with unsludge plants (Table 1). Additionally, length and number of pods were also reduced as elevating SSL treatment with percent 21.9% and 60.2% at 100% level ( Figure 2(a), Figure   2(b)). Treatment cultivars Giza 843 and Sakha 1 with either IAA or SA induced a stimulation effect on fresh, dry matter, water content, number and length of pods especially at higher sewage sludge treatments (Table 1, Figure 1 & Figure 2(a), Figure 2(b)). This effect was more pronounced in Sakha 1 cultivar than cv. Giza 843. Phytohotmonal application was generally enhanced the production of fresh, dry matter, water content, length and number of pods in cv. Giza 716 at 50% to 100% sludge application when compared with non-hormonal plants treatments (Table 1, Figure 2(a), Figure 2(b)). This stimulation trend was more pronounced at IAA than SA application and at higher SSL. American Journal of Plant Sciences  The mean difference is significant at the 0.05% level.

Metabolites as Affected by Sewage Sludge and Phytohormones Applications
Soluble sugar and soluble protein contents were decreased as increasing SSL in both cv. Sakha 1 and cv. Giza 716 (Table 2). However, in cv. Giza 843 soluble sugar was significantly increased, soluble protein was markedly decreased as increasing SSL (Table 2)    The mean difference is significant at the 0.05% level.

Micronutrient as Sewage Sludge and Phytohormones Applications
In cv. Giza 843, sewage sludge application with 100% level illustrated no marked change in Zn while a slight increase in Ni content was detected (Figure 3(a)). However, a huge reduction was observed in the accumulation of both Pb and Cu which reached to over a half value in relation with their control. No marked change in the accumulation of Zn and Ni was observed in plants grown with SS plus IAA treatment (Figure 3(a)). On the other hand, Pb and Cu ranked against each other, while Pb was markedly decreased, Cu was markedly increased reached 0.861 mg/g. d. m. in relation with its control 0.339 mg/g d. m. Plants of cv. Giza 843 grown under SS plus SA exhibited a significant reduction in the contents of Zn, Pb and Cu. Except Ni showed a smooth increase in relation with control plants (Figure 3(a)). Sludge application decreased the contents of Cu, Pb, Ni and Zn in pods of Sakha 1 (Figure 3(b)).  (Figure 3(b)). Sludge    (Figure 3(c)).  [28] was illustrated that generally adding sewage sludge to soil enhanced soil texture raised organic matter contents, total sugar and proteins. Wyriwicka et al.

Discussion
(2019), [35] showed that cucumber plants grown in sewage sludge rise soluble protein. Application of SS to forest plantations could potentially increase tree growth and wood making and improve several soil features [36]. Abdel Latef et al. (2015) [29] stated that sewage sludge applications effect on biochemical anal-ysis (sugar, protein, free amino acids, and proline and antioxidant enzymes activity) of maize plants. Dar et al. (2018) [26] illustrated that sugar and protein of Brassica juncea improved as increasing sludge submissions. Proline has different roles in plant metabolism, became unchanged in cv. Sakha 1 while a reduction was recorded in cv. Giza 843. This indicated that proline has not any significant role in cv. Sakha 1. While in cv. Giza 843, proline reduction was contributed in increasing its tolerant to sewage sludge application, here proline cannot consider as a sign of stress. On the other side proline tended to increase in cultivar cv.