Differential Responses of Common Bean, ( Phaseolus vulgaris L.) to the Interactive Effects of Ascorbic Acid and Trichoderma harzianum under Salinity Stress

Recent ecological perturbations are presumed to be minimized by the application of biofertilizers as a safe alternative to chemical fertilizers. The current study aims to use biofertilizers ( Trichoderma harzianum ) and non-enzymatic antioxidants (ascorbic acid) in combination with NaCl on growth and some metabolic activities as well as esterase enzyme in Phaseolus vulgaris cv Bronko. Results showed that NaCl inhibited both fresh and dry weights, shoot and root lengths. Treatment of plants with either ascorbic acid or Trichoderma enhanced these parameters. All pigments (Chl.a, Chl.b, Chl.a+b and carotenoids) were enhanced at lower and higher salinity levels. Plants treated with either ascorbic acid or Trichoderma promoted carotenoids at moderate and higher levels of salinity. Soluble proteins, soluble carbohydrates and amino acids were increased in shoots and decreased in roots with increasing salinity, treatment of plants with either ascorbic acid or Trichoderma reduced these components in shoots. Treatment of plants with either ascorbic acid or Trichoderma decreased MDA and significantly increased proline in both shoots and roots. Salinity increased the DPPH% activity in both shoots and roots, and treatment with either ascorbic acid or Trichoderma significantly enhanced the scavenging activity of DPPH in both organs. Plants treated with either ascorbic acid or Trichoderma exhibited a reduction in phenols and increment in flavonoids, especially with Trichoderma. The assessment of esterase isozyme profiles on 7.5% polyacrylamide gel revealed the presence of 6 isoforms in response to water presoaking and treatments with either ascorbic acid or Trichoderma under different concentrations

Recent ecological perturbations are presumed to be minimized by the application of biofertilizers as a safe alternative to chemical fertilizers. The current study aims to use biofertilizers (Trichoderma harzianum) and non-enzymatic antioxidants (ascorbic acid) in combination with NaCl on growth and some metabolic activities as well as esterase enzyme in Phaseolus vulgaris cv Bronko. Results showed that NaCl inhibited both fresh and dry weights, shoot and root lengths. Treatment of plants with either ascorbic acid or Trichoderma enhanced these parameters. All pigments (Chl.a, Chl.b, Chl.a+b and carotenoids) were enhanced at lower and higher salinity levels. Plants treated with either ascorbic acid or Trichoderma promoted carotenoids at moderate and higher levels of salinity. Soluble proteins, soluble carbohydrates and amino acids were increased in shoots and decreased in roots with increasing salinity, treatment of plants with either ascorbic acid or Trichoderma reduced these components in shoots. Treatment of plants with either ascorbic acid or Trichoderma decreased MDA and significantly increased proline in both shoots and roots. Salinity increased the DPPH% activity in both shoots and roots, and treatment with either ascorbic acid or Trichoderma significantly enhanced the scavenging activity of DPPH in both organs. Plants treated with either ascorbic acid or Trichoderma exhibited a reduction in phenols and increment in flavonoids, especially with Trichoderma. The assessment of esterase isozyme profiles on 7.5% polyacrylamide gel revealed the presence of 6 isoforms in response to water presoaking and treatments with either ascorbic acid or Trichoderma under different concentrations of NaCl with different density bands.
al., 2017). Salinity stress-induced reduction in growth parameters undergoes osmotic regulation and affected many biochemical processes such as photosynthetic pigments and protein content (Kapoor andSrivastava, 2010, Rady et al., 2019). Proline is a compatible solute that accumulated in salt-tolerant plants under stress conditions and acts as an osmoprotectant (Munns and Tester 2008). Salt stress causes an imbalance in cellular ions, releasing ion toxicity which leads to ROS overproduction. This causes damage to DNA, lipids, and proteins, concurrently with chlorophyll degradation and membrane lipid peroxidation, decreasing membrane fluidity and selectivity (Rady et al., 2018). To overcome these ROS effects, plants have developed various antioxidant defense systems including non-enzymatic (e.g., proline, AsA, carotenoids, etc.) and enzymatic (e.g. SOD, CAT, POD, etc.) antioxidants (Hodges et al., 1997). Plants with high levels of constitutive and/or induced antioxidants have been reported to have greater capacity tolerance to oxidative damage. It has been suggested that the extent of oxidative cellular damage in plants exposed to abiotic stress is controlled by the capacity of their antioxidant systems (Rady et al., 2018).
Biofertilizers such as plant growth-promoting rhizobacteria (PGPR), Trichoderma played a very important role in yield improvement. The use of PGPR and Trichoderma harzianum biofertilizers may be useful in developing strategies to facilitate plant growth in saline soils (Shaharoona et al., 2007, Abd El-Baki and Mostafa, 2014, Zhang, et al., 2019, Niu et al., 2020and Negi et al., 2021. Plant beneficial fungi of the genus Trichoderma have been successfully used worldwide both to control fungal diseases and to promote plant growth. Trichoderma spp. represent a fundamental component of the rhizosphere microbiome because these fungi help plants to overcome numerous environmental constraints by stimulating defense responses including the secretion of antimicrobial reactive oxygen species (ROS), the production of secondary metabolites (Shoresh et al., 2010) and improving fitness and development (Hermosa et al., 2013). These abilities have supported the application of Trichoderma strains as biocontrol agents or plant biostimulants in agriculture.
The effect of ascorbate on plant growth has been extensively studied by Niakan et al. (2012). Ascorbate is an important antioxidant in plants and much literature showed that it has an essential role in several physiological processes in plants, including growth, differentiation, and metabolism Foyer, 1998 andHoremans et al., 2000). Ascorbate functions as a reductant for many free radicals because of its ability to donate electrons in a number of enzymatic and non-enzymatic reactions (Gill & Tuteja, 2010). Ascorbate enhances plant tolerance to environmental stressors such as saline stress (Khan et al., 2011;Ejaz et al., 2012, Abbasi andFaghani, 2015;Cai et al., 2016). Among the most important Fabaceae crops, the common bean (Phaseolus vulgaris L.) is a legume food that produces a significant amount of seeds rich in the protein content needed for human nutrition (Rady et al., 2013). The common bean plant is classified as salt-sensitive, suffering yield losses at soil salinity of less than 2 dS m −1 (Pessarakli, 1999).
The understanding of physiological responses of common bean (Phaseolus vulgaris cv Bronko) under salinity may help in programs that aimed to improve the grain yield under salinity stress. Therefore, the aim of this study was to assess the effect of biofertilizers and ascorbic acid as presoaking integrated with foliar spray on the changes in growth and green yield, physio-biochemical components, and the antioxidant defense systems of common bean plants grown under salt stress.

MATERIALS AND METHODS
Seeds of common bean (Phaseolus vulgaris cv. Bronko) were obtained from the Seed Center affiliated with the Directorate of Agriculture in Minia. Seeds were selected for uniformity by choosing those of equal size and with the same color. The selected seeds were washed with distilled water, sterilized with 1% sodium hypochlorite solution for about 2 min and thoroughly washed again with distilled water. The seeds were left to dry at room temperature at (28-30 0 C) on filter paper for about two days and then divided into three groups, one of them was soaked in water for three hours, and the second group was soaked in ascorbic acid (200 ppm) also for three hours, the third group was coated with Tricoderma harzianum (T24) during sowing. Five uniforms air-dried common bean seeds (Phaseolus vulgaris cv. Bronko) were sown along a centre row in each pot at 30-mm depth in plastic pots, each filled with about 4 kg mixed (clay sandy soil) within a proportion of 2:1 (v:v), respectively in order to reduce compaction and improve drainage. Plants were left to grow for 3 weeks and then treated with different concentrations of NaCl (0.0, 50, 100, 150 and 200 mM of NaCl) and irrigated with tap water, according to its measured field capacity with top irrigation, then left to grow further for 60 days from sowing.
Plant samples were collected after 60 days from sowing for measurement of some growth parameters i.e. (leaf area, plant height, root length, fresh and dry weights of shoots and roots) photosynthetic pigments, carotenoids, soluble sugars, soluble proteins, total free amino acids, MDA, proline, total phenolics, total flavonoids, DPPH% activity, Ascorbic acid, Esterase profile. Chlorophyll a, chlorophyll b and carotenoids were determined using the spectrophotometric method described by Lichtenthaler, (1987). Total free amino acids were determined according to the method of Moore and Stein (1948), soluble sugars by anthrone sulphuric acid method which was carried out by Fales (1951) and Schlegel (1956) and adopted by Badour (1959. The soluble proteins were determined according to the method adopted by Lowery et al. (1951). Leaf areas were carried out according to McKee, (1974). The level of lipid peroxidation was measured in terms of malondialdehyde (MDA) content. MDA is a product of lipid peroxidation following the method of Heath and Packer (1968), expressed as n mol (MDA) g -1 (fresh weight). Total phenolic contents according to Reis et al, 2012, and Total flavonoids according to Jia et al., 1999, 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH˙) were assayed according to Yi et al. 2008 and ascorbic acid was assayed according (Ismail et al., 2014). Preparation of polyacrylamide gel electrophoresis (PAGE) and staining of esterases, the plant tissue was homogenized in buffer (0.1 M Tris-HCl with 2 mM EDTA pH7.8) and centrifuged at 13000 g for 15 min. Esterase (EST, EC 3.1.1.x) isoforms were detected by staining with α-naphthyl acetate/Fast blue RR according to Tanksley and Orton (1983).

Statistical analysis:
The data of all experiments were subjected to a one-way analysis of variance and means were compared using the least significant difference test (L.S.D.) using a statistical program (Sta. Base. Exe.) on the computer (Steel and Torrie 1960).

RESULTS
The data in Figure (1) showed the differential effects of NaCl on (fresh matter, dry matter and plant length) in both shoot and root of Phaseolus vulgaris cv Bronko presoaked in either water or ascorbic acid and others inoculated with Trichoderma harzianum (T24). In water presoaked plants salinity had a remarkable inhibitory effect only at 200 mM NaCl which reduced fresh, dry and shoot length to (56.57%, 47.96% and 88.64%) and (66.72%, 61.54%, 76.19%) in both shoot and root respectively compared to absolute control. However lower and moderate salinity levels had a slight promoting effect on these parameters. Treatment of plants with either ascorbic acid or Trichoderma enhanced these parameters which reached (119.40% and 114.44%) with Trichoderma at 50 mM NaCl in shoot fresh and dry matter respectively compared to absolute control. In roots treated with ascorbic acid or inoculated with Trichoderma the promotion of these parameters was pronounced at almost salinity levels used. The data presented in Figure (2) concluded that all pigment contents (Chl.a, Chl.b, Chl.a+b and carotenoids) were enhanced at lower and higher salinity levels in plants presoaked in water, however moderate salinity levels keep the values near to absolute control. At lower salinity levels 50 mM NaCl, the significant enhancement reached to 149.13%, 140%, 147.77% and 139.37% in both (Chl.a, Chl.b, Chl.a+b and carotenoids) respectively compared with absolute control. At higher salinity levels 200 mM NaCl all pigment fractions gained higher values reached to (114.24%, 108.86%, 113.41% and 110.78%) respectively comparable with absolute control. The treatment of plants with either ascorbic acid or Trichoderma did not affect the pigment contents whatever the treatment used or pigment fraction analyzed except for carotenoids which exhibited a slight promotion at moderate and higher levels of salinity with either ascorbic or Trichoderma. This reflects the protecting role of this fraction under stress conditions. It is worth mentioning that the promotion effect of Trichoderma is better than ascorbic acid, especially at moderate salinity levels used. The data in Table (1) revealed the effect of presoaking with water or ascorbic acid and others inoculated with Trichoderma on water content and leaf area. In water presoaked plants, salinity had various effects on both shoot and root water content, increasing it slightly in shoots and pronouncedly enhancing it in roots reached to 112.54% from absolute control at 200 mM NaCl. The leaf area remained unchanged at lower and moderate levels of salinity but reduced sharply at higher levels of salinity in plants presoaked in water. Treatment of plants with ascorbic acid enhanced leaf area only at a higher salinity level (200 mM NaCl) compared with reference control values. The data in Figure (3) illustrated soluble proteins, soluble carbohydrates and amino acids. All fractions were increased in shoots and decreased in roots with increasing salinity except for soluble proteins which remained more or less unchanged along the salinity levels used. Treatment of plants with either ascorbic acid or Trichoderma resulted in a reduction of these components in shoots except for soluble proteins which increased slightly with Trichoderma treatments. In roots, both soluble proteins and amino acids were increased, however, soluble carbohydrates were decreased under the same treatments. It is worth mentioning that the amount of soluble carbohydrates in shoots is much higher than that of roots especially plants presoaked in water compared to others treated with Trichoderma or ascorbic acid. The data in Table (2) showed the effect of salt stress and combination with ascorbic acid or Trichoderma on MDA and proline contents in both plant organs of Phaseolus vulgaris cv Bronko. The data clearly demonstrated that both proline and MDA were increased with increasing salinity in both shoots and roots of water-presoaked plants. Treatment of plants with either ascorbic acid or Trichoderma decreased MDA and significantly increased proline in both shoots and roots. It is worthy to mention that, proline accumulated with higher content in shoots with either ascorbic or Trichoderma reaching 286.9% and 320.1% at 200 mM NaCl with ascorbic or Trichoderma respectively comparable with absolute control values.  The data in Figure (4), is concerned with phenols, flavonoids and ascorbic acid in both shoots and roots of common bean plants. Phenols and flavonoids increased in both shoots and roots with increasing salinity, especially at lower and moderate levels of salt, however, ascorbic acid remained more or less unchanged with all treatments in reference plants but exhibited a remarkable increase in both organs with Trichoderma treatments reached to 116.67% and 108.3% in both shoots and roots respectively at 200 mM NaCl. Treatment of plants with either ascorbic acid or Trichoderma exhibited a reduction in phenols and increment in flavonoids, especially with Trichoderma at all salinity levels used. The data in Table (3) showed the effect of salt treatment on plants presoaked in water or ascorbic acid and others inoculated with Trichoderma on the percentage activity of DPPH in both organs. Salinity increased the DPPH activity in both organs shoots and roots at all salinity levels used, however treatment with either ascorbic acid or Trichoderma significantly enhanced the scavenging activity of DPPH% in both organs along the salt levels used.
The assessment of esterase isozyme profiles on 7.5% native polyacrylamide gel revealed the presence of 6isoforms in Phaseolus vulgaris cv Bronko in response to water presoaking and treatments with either ascorbic acid or Trichoderma under different concentrations of NaCl Figure (5). The increase of band intensity, the appearance of new bands and the disappearance of esterase bands may be an indication of an increase in activity response or tolerant strategy to treatments with ascorbic acid or Trichoderma under different concentrations of NaCl. The difference in density and number of bands are more pronounced under different concentrations of salinity which is more obvious under treatments of ascorbic acid and Trichoderma, however, Trichoderma was more effective than all salinity levels. The presence of 6 isozymes under control and treated plants reflects the role of these enzyme isoforms in salt stress resistance.

Various morphological and physiological factors in common bean
Phaseolus vulgaris cv Bronko were negatively affected by salinity stress, while treatment with either ascorbic acid or Trichoderma harzianum (T24) mitigated these effects. Both fresh, dry, as well as shoot and root lengths, were decreased with increasing salinity. The reduction in this parameter reached 56.57%, 47.69%, 88.64%, 66.72%, 61.54 and 76.19 at 200 mM NaCl in both shoot and root respectively compared with absolute control. Treatment with either ascorbic acid or Trichoderma enhanced all these parameters. The enhancement with Trichoderma reached to 74.63%, 71.12%, 95.45%, 76.03%, 83.08% and 85.71% at 200 mM NaCl respectively in both shoot and root compared with absolute control. Salinity stress induces osmotic and ionic stress that leads to retarded growth in terms of both shoot and root length, fresh and dry weight, reduced pigment content and hampers uptake of mineral elements (Ahmad et al., 2014;Ma et al., 2020). In the same manner, Younis et al. (2010) reported that the growth reduction caused by salinity stress is due to inhibition of apical growth in plants as well as an imbalance of endogenous hormones. The enhanced production of ROS during salinity stress leads to progressive oxidative damage and ultimately cell death and growth suppression (Ruiz-Lozano et al., 2012).
AsA treatment increases the content of IAA, which stimulates cell division and/or cell enlargement and this, in turn, improves plant growth (Khan et al. 2011). Working with bean plants treated with Trichoderma velutinum, Mayo et al., (2016) found that plants inoculated with this fungus showed a significant increase in dry weight in both shoots and roots by inducing the expression of defense-related genes. The enhancement of plant biomass by promoting the growth of lateral root has been observed in many plant species treated with Trichoderma spp. and this effect has also been related to the production of indole-3-acetic acid (IAA) or auxin analogues Vinale et al., (2012). According to Ahmad et al. (2015), the application of T. harzianum restored mustard plant height. Similar results were obtained by Hashem et al. (2011), who mentioned that stigmasterol caused stimulation of plant height and leaf area.
All pigment fractions (Chl.a, Chl.b, Chl.a+b and carotenoids) were enhanced at lower and higher salinity levels in plants presoaked in water, however moderate salinity levels keep the values near-absolute control. The treatment of plants with either ascorbic acid or Trichoderma enhanced carotenoids at moderate and higher levels of salinity which synergistically function with ascorbic acid to provide an effective barrier against oxidation under salinity stress. The reduction of chlorophylls under salinity stress was attributed to salt-induced acceleration of enzymes responsible for chlorophyll degradation and the instability of the pigment-protein complex (Hernandez andAlmansa 2002, Duarte et al., 2013). Directly or indirectly the reduction of photosynthesis affects the pigments and chlorophyll contents of plants (Parida and Das 2005). Among the positive effects of ascorbic acid in the counteraction of the adverse effects of salt, and stress is the stabilization and protection of the photosynthetic pigments and the photosynthetic apparatus from oxidization damage (Khan et al., 2011). Application of Trichoderma harzianum has restored the chlorophyll and carotenoids content to appreciable levels in the present study and the results corroborate with the findings of Zhang et al. (2013). Trichoderma harzianum increases the uptake of essential elements especially Mg2+ that are negatively affected by NaCl stress; hence the chlorophyll synthesis increases in Trichoderma inoculated plants especially we found an increment in Mg +2 contents in our results data not shown.
Water content increased in both shoots and roots with increasing salinity however, treatments with either ascorbic acid or Trichoderma enhanced the water content . Zhang et al., 2016, postulated that wheat seeds treated with Trichoderma longibrachiatum (T6) increased relative water content in the leaves and roots. In the same context, Khafagy et al. (2009), andAzzedine et al. (2011) reported that ascorbate mitigated the inhibitory effect of salt stress on plant growth due to increased leaf area, improved chlorophyll and carotenoid contents, and enhanced antioxidant accumulation. The primary plant response to salinity is a reduced leaf surface expansion, followed by a diverse closure of stomata as the stress intensifies and finally leads to photosynthesis inhibition (Parida and Das, 2004).
Soluble proteins, soluble carbohydrates and amino acids were increased in shoots and decreased in roots with increasing salinity. Treatment of plants with either ascorbic acid or Trichoderma resulted in a reduction of these components in shoots except for soluble proteins. In roots, both soluble proteins and amino acids were increased, however, soluble carbohydrates were decreased under the same treatments. Accumulation of soluble carbohydrates, proteins and amino acids might have a physiologically important role in energy supply, osmotic adjustment to maintain leaf water potential, increase stress tolerance and decrease osmotic shock (Babaeian et al., 2011;Ma et al., 2020). Ejaz et al. (2012) stated that sugarcane soluble protein contents were increased by salt stress. Ebrahim, 2005 found that salinity decreased the soluble protein content of Vicia faba and sugar beet exposed to salt stress. According to the plant species or cultivar, the stress conditions make changes in soluble protein contents due to the cell structural modifications Wimmer et al., 2003. Proline increased significantly with increasing salinity in both shoots and roots of water-presoaked plants however MDA increased in both shoots and roots at all salinity levels used compared with absolute control. Treatment of plants with either ascorbic acid or Trichoderma decreased MDA in both shoots and roots and increased proline in the same organs. Malondialdehyde content can serve as an indicator of the rate of oxidative processes in cells. In contrast, salinity stress increased malondialdehyde whether in leaf tissue or root. Malondialdehyde results indicated that lipid peroxidation started to increase at 100 mM NaCl in roots. Accumulation of malondialdehyde was reported in a number of salt-sensitive plants (Luna et al., 2002, Gehlot et al., 2003. In this study, proline content increased in NaCl-treated plants and reached 267.94% in shoots at 200 mM NaCl compared to absolute control confirming its contribution to osmotic adjustment (Delauney andVerma, 1993, Mervat et al., 2013). Proline protects membranes and proteins against the adverse effects of high concentrations of inorganic ions and temperature extremes (Rudolph et al., 1986;Santoro et al., 1992;Ma et al., 2020). Proline may also function as a hydroxyl radical scavenger (Smirnoff and Cumbes, 1989)). Proline is also an important osmolyte that maintains the cell osmoregulation in NaCl stress (Rasool et al., 2013 andYan et al., 2017).
Phenols and flavonoids increased in both shoots and roots, with increasing salinity, especially at lower and moderate levels of salt, however ascorbic acid remained more or less unchanged with all treatments in reference plants but exhibited a remarkable increase in both organs with Trichoderma treatment. We found differences in the accumulation of flavonoids, phenols and ascorbic acid in both plant parts depending on the interaction conditions used either ascorbic or Trichoderma. Plant with a higher amount of ascorbic acid content showed better protection against oxidative stress, influencing many enzyme activities, and minimizing the oxidative damage through synergetic function with other antioxidants, Foyer and Noctar, (2005). Flavonoids protect plants against various biotic and abiotic stresses, playing an important role in the interaction between the plant and the environment [Pourcel, et al., 2007]. The differences in the pattern of flavonoid accumulation observed in the metabolome of the bean could reflect the different metabolic pathways induced by the beneficial fungus Trichoderma or treatment with ascorbic acid. We found that the application of ascorbic acid or Trichoderma at lower salinity levels of 50 mM NaCl increased total phenolics and total flavonoids. These results confirmed those of Jaleel et al., 2009 andSalama et al., 2014 in Phaseolus vulgaris under salt stress. Application of ascorbic acid at a dose of 200 or 400 mg via foliar spray significantly increased total phenolics, total flavonoids, and total tannins in Phaseolus vulgaris under water stress conditions Alaa et al. (2020). In general, the production of phenol compounds in plants under abiotic stress depended on the type of stress, stress intensity, stress duration, plant development stages and plant part type, i.e., whole seedlings or plant parts, namely roots or leaves Weidner, et al., (2011).
Antioxidant activity (free radical scavenging activity by DPPH%) increased in both organs shoots and roots at all salinity levels used, however treatment with either ascorbic acid or Trichoderma increased the scavenging activity of DPPH% in both organs along the salt levels used. The decrease in absorbance of the DPPH% radical caused by antioxidants can be ascribed to the action between an antioxidant molecule and the radicle. In addition, the further increase in DPPH activity and endogenous ascorbic acid was a result of strengthening the antioxidative defense system followed by an increased tolerance of bean plants to salt stress. Ascorbic acid may be participated in the up-regulation of soluble sugars and proline synthesis to enhance tolerance mechanisms under salt stress. The increase in the antioxidant activity was attributed to the increases of secondary metabolites (phenolics and flavonoids) which are considered a tolerance advantage of biotic stresses as reported by El-Amier et al. (2019).
The presence of 6 esterase isoforms in Phaseolus vulgaris cv Bronko presoaked in water or treated with either ascorbic acid or Trichoderma under different concentrations of NaCl, the increase of band intensity, the appearance of new bands and disappearance of other esterase bands may be an indication of an increase in activity response to presoaking in water or treatment with either ascorbic acid or inoculation with Trichoderma under various NaCl concentrations. Gigova. et al. (2012) reported the isoenzyme pattern and changes in the esterase activity of Synechocystis under different growth conditions four clearly visible bands and eight weaker bands of EST activity were detected by gels analysis. Phaseolus vulgaris cv Bronko contains several forms of esterase with a broad range of functions, hydrolyzing ester bonds in different types of metabolites. Multiple forms of esterase and their differential expression during stress conditions suggested their important role in several physiological processes Coppens and Dewitte (1990). Hydrolytic enzymes such as esterase participate in altering cell walls Cosgrove, (2001). The role of esterase in many cases is associated with cell wall metabolism but little is known about their intracellular function. One of their most interesting functions in the cytoplasm is the metabolism of many pesticides and pollutants entering the cell Cummins et al, (2001). Mukherjee et al. (2004) reported that esterase variation of Lemna minor is a potential biomarker of heavy metal pollution. Precise localization, isolation and characterization of Trichoderma or ascorbic acid-induced esterase isozymes are required for the understanding of their role during salt stress. These results are in agreement with Hassanein (1999), who found that salinity increases esterase isozymes and the highest numbers of esterase isozymes were detected under the highest NaCl concentration. Also, El-Sayed et al. (2007) found that salinity and gamma rays caused the appearance and disappearance of bands in two wheat cultivars. These results are similar to Mohamed (2005) who found that under salt stress, 150 mM NaCl caused enhancement of the esterase isozyme bands in shoots and roots of maize plants.

Conclusion:
Most of the studies emphasize the role of plant growth-promoting rhizofungi against biotic and abiotic stresses. Nevertheless, the current study revealed the synergistic effects between rhizofungi Trichoderma harzinum and non-enzymatic antioxidant (ascorbic acid) in mitigation stress in Phaseolus vulgaris cv Bronko which proved to be beneficial in most cases in imparting resistance through improved uptake of essential elements, increased carotenoids which played a protective role against stress, modulation of osmolytes (sugars, proteins and proline), antioxidants, enhanced scavenging activity and also esterase isozymes which played a vital role under salt stress conditions. Also, water presoaking is an efficient mechanism to promote some physiological activities parallel to Trichoderma and ascorbic acid in our results.