الفهرس 
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Abstract
The current study aims to use the bioinoculum Trichoderma harzianum and non-enzymatic antioxidant ascorbic acid to alleviate salinity stress in two broad bean cultivars Phaseolus vulgaris cv Nebraka and Phaseolus vulgaris cv Bronko grown for 60 days from sowing under different concentrations of NaCl (0.0, 50, 100, 150 and 200) mM NaCl.
Physiological results
1-Seed Germination:
- The final germination percentage at 4th day does not affect by salt stress in reference control plants of Phaseolus vulgaris cv Nebraska: while it was decreased by increasing salinity especially at moderate and higher concentration in Phaseolus vulgaris cv Bronko.
- Treatment of plants with either ascorbic or Trichoderma of two Phaseolus vulgaris (cv Nebraka and cv Bronko) resulted in a slight increase in germination percentage at higher concentration of NaCl.
2-Growth criteria:
- Salinity reduced both fresh and dry weight and plant length (shoots and roots) of two common bean cultivers Phaseolus vulgaris cv Bronko and Phaseolus vulgaris cv Nebraska, especially at 200 mM NaCl in water presoaked seeds (control), this reduction were more pronounced in Nebraska than bronco. Seed presoaked with ascorbic acid or coated with T. harzianum resulted in a pronounced increase in all these parameters in both cultivers, especially with ascorbic acid in cv Nebraska.
- The water content was slightly increased in shoots and roots with increasing salinity in control plants of both culivars. Seed presoaking with ascorbic acid or coated with T. harzianum resulted in a pronounced increase in water content in cv Bronko while a negligible effect in water content in cv Nebraska.
- Leaf area exhibited a marked decrease with increasing salinity in reference plants of cv Nebraska, it was remained unchanged at lower and moderate levels of salinity but reduced sharply at higher levels in cv bronko. Treatment of plants with either ascorbic or Trichoderma enhanced leaf area only at moderate and higher salinity levels (150 and 200 mM NaCl) in cv Nebraska.
3-Pigment analysis
In Phaseolus vulgaris cv Nebraska:
- All pigment fractions (Chl a, b, a+b and carotenoids) decreased with increasing salinity, especially at 200 mM NaCl. Seed treatment with ascorbic acid or inoculated with T. harzianuma resulted in a remarkable increase in all pigments, especially at 100 mM NaCl, Of interest, pigment fractions with ascorbic acid treatment have a higher values than other treatments.
In Phaseolus vulgaris cv Bronko:
- 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 to absolute control. The treatment of plants with either ascorbic acid or Trichoderma did not affect the pigment contents what ever 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.- It is worth to mention that the promotion effect of Trichoderma is better than ascorbic acid especially at moderate salinity levels used.
4-Soluble carbohydrates, soluble proteins and free amino acids
- Soluble carbohydrates decreased in shoots and increased in roots with increasing salinity in control plants of cv Nebraska, while they increased in shoots and decreased in roots with increasing salinity in control plants of cv Bronko. Treatments with ascorbic acid or Trichoderma had an increasing effect at almost treatments, especially in roots of cv Nebraska while it had a depressive effect on soluble carbohydrates especially in roots comparable with reference control of cv Bronko.
- Soluble proteins, both shoots and roots exhibited an increasing trend with increasing salinity in plants presoaked with water (Reference control) especially shoots of cv Nebraska, in cv Bronko, in reference plants, in shoots it remains more or less unchanged with increasing salinity, however in roots it tends to increase especially at lower and moderate salinity levels used. Treatments of plants of cv Nebraska with either ascorbic or Trichoderma decreased the soluble protein fraction in both organs which more pronounced in shoots than roots. Treatments of plants of cv Bronko with either ascorbic acid or Trichoderma had an stimulatory effect on soluble protein fraction especially with Trichoderma.
- The contents of free amino acids were markedly increased in both organs of cv Nebrsaka which more obvious in roots than shoots, while in cv Bronko free amino acids increased in shoots and decreased in roots with increasing salinity in reference control plant (plants presoaked in water). In cv Nebraska neither ascorbic acid nor Trichoderma had a positive effect in amino acid accumulation whatever the organ analyzed or treatment used, however, treatment of plants of cv Bronko with either ascorbic acid or Trichoderma resulted in a significant increase in free amino acids especially with Trichoderma.
5-MDA and proline:
- MDA increased in shoots and roots in both cultivers in control plants. Treatment of plants with either ascorbic acid or Trichoderma had a remarkable decreasing effect on MDA in both shoots and roots of both cultivers.
- Proline content significantly increased with increasing salinity in shoots and roots of both cultivars, till 150 mM NaCl in control plants. Treatment of plants with ascorbic acid or Trichoderma decreased proline content of cv Nebraska in roots and slightly increased it at certain concentrations in shoots with increasing salinity compared to untreated plants, While, in cv Bronko proline significantly increased in both shoots and roots.
6-Phenols, Flavonoids and Ascorbic acids
- Both phenols and flavonoids in shoots of cv Nebraska, slightly decreased with increasing salinity; however, in roots, phenols decreased, and flavonoids significantly increased. In cv Bronko they increased in both shoots and roots with increasing salinity especially at lower and moderate levels of salt. Treatment with Trichoderma in cv Nebraska slightly decreased both components, especially at higher concentrations. Ascorbic acid treatment enhanced phenols only at higher concentrations in shoots in the same cultivar. In cv Bronko, both treatments exhibited a reduction in phenols and increment in flavonoids especially with Trichoderma at all salinity levels used.
- Ascorbic acid content decreased in roots and remained unchanged in shoots of cv Nebraska under reference conditions. However, in cv bronko, it remained more or less unchanged with all treatments. Treatment of cv Nebraska with either ascorbic acid or Trichoderma exhibited a marked increase in ascorbic acid with increasing salinity especially in shoots that reached to 131.4% with ascorbic acid compared to control at 150 mM NaCl. In cv Bronko, these treatments exhibited a remarkable increase in ascorbic acid in both organs with Trichoderma treatments reached to 116.67% and 108.3% in both shoots and roots respectively at 200 mM NaCl.
7-DPPH % (2,2 diphenyle 1-picryl hydrazyl hydrate)
Both shoots and roots exhibited a marked increase in DPPH% by increasing salinity in both cultivars, where they reached to (143.3% and 123.8%) at 200 mM NaCl in both shoot and root of cv Nebraska respectively in control plant.Treatment of plants of cv Nebraska with either ascorbic acid or Trichoderma decreased the percentage of DPPH activity in shoots and slightly increased it in roots which more pronounced with Trichoderma treatment. In cv Bronko, these treatments significantly enhanced the scavenging activity of DPPH% in both organs along the salt levels measured.
8- Mineral composition:
A-Na+ and K+ contents
- Both shoots and roots of cv Nebraska exhibited a marked increase in Na+ content reached to 145% from reference values at 200 mM NaCl in shoots and 122.58% at the same treatment in roots; however, K+ content increased in shoots and decreased in roots with increasing salt concentration. In cv Bronko, both Na and K contents were increased in both shoots and roots at almost cases. Treatment with either ascorbic acid or Trichoderma in cv Nebraska decreased Na+ in both organs (shoots and roots) and increased K+ in all organs analyzed. In cv Bronko, these treatments significantly increased K in both shoots and roots, while Na increased in shoots and decreased in roots.
B- Ca+2 and Mg+2 contents
- In cv Nebraska both Ca and Mg were increased with increasing salinity in control plants reached to 159.3% and 142.9 at 200 mM NaCl respectively in shoot. In roots both two cations decreased with increasing salt concentration. In cv Bronko Ca and Mg contents decreased in shoots and increased in roots with increasing salt. Treatment of plants of cv Nebraska with either ascorbic acid or Trichoderma resulted in a significant increase in both elements almostly at lower and moderate levels of salinity except for treatment with ascorbic acid which have no changes at lower and moderate salinity levels, while in cv Bronko, these treatments resulted in most cases in a promotion of both elements in shoots and roots.
9-Mineral Ratios:
- In shoots of cv Nebraska, K/Na ratio remained more or less unchanged and tended to decrease at higher salinity levels 200 mM NaCl of reference control plants. However the Ca/Na ratio was increased with increasing salinity reached to 123.7 at 150 mM NaCl. In roots, both ratios decreased with increasing salinity regardless the ratio determined. In cv Bronko, K/Na and Ca/Na ratios decreased slightly with increasing salinity in shoots of reference control plants.
- Treatment of plants of cv Nebraska with either ascorbic acid or Trichoderma resulted in pronouncing increase in both ratios in shoots and roots except the Ca/Na ratio in roots which exhibited a slight decrease. In cv Bronko these treatments negatively affected these ratios in shoots. In roots both ratios were increased especially at lower and moderate salinity levels used, treatments with either ascorbic acid or Trichoderma promoted these ratios especially with Trichoderma which reached to 250.4% at 200 mM NaCl in K/Na ratio.
10-Enzyme profile:
A)- Peroxidase Enzyme:
The electrophoresis profile of POX showed two isoforms (POX1 and POX2) under salinity and treatments with either ascorbic acid or Trichoderma in both cultivars. In cv Nebraska, POX1 was appeared under all different salt concentrations and with other treatments. On the other hand, pox2 appeared only at 200 mM NaCl and with all selected Trichoderma treatments. In cv Bronko, treatments with either ascorbic acid or Trichoderma decreased the activity of enzyme in most cases except at (100 mM NaCl+ Trichoderma and 200 mM NaCl+ Trichoderma).
B-Esterase Enzyme:
In Phaseolus vulgaries cv Nebraska. Five bands were appeared with different densities and intensity under reference control, ascorbic acid and Trichoderma treatments. This difference are more pronounced under control and 100 mM NaCl treatments. While, Esterase isozyme profiles revealed the presence of 6 isoforms in response to water presoaking and treatments with either ascorbic acid or Trichoderma under different concentrations of NaCl in Phaseolus vulgaris cv Bronko.
11- Electrophoretic Protein profile:
(1) In Phaseolus vulgaries cv Nebraska:
One band of protein profile was detected at 43 kDa at all treatments used. The band intensity increased with increasing salinity till 200 mM NaCl. The band intensity was more obvious with both treatments (Ascorbic acid or Trichoderma).
(2) Phaseolus vulgaries cv Bronka:
One band of protein profile was detected at 35 kDa at all treatments used. The band intensity increased with increasing salinity till 200 mM NaCl. The band intensity was more obvious with both treatments (Ascorbic or Trichoderma). It is clearly obvious that P. Nebraska had a salt resistant protein with high relative density compared with P. Bronko (4.11 comapred with 3.47).
12-RNA and RT-PCR
The RNA was isolated and its concentration was estimated using a NanoDROP Spectrophotometer. The SOS1 gene was selected as one of the genes responsible for the removal of excess sodium ions from the cytoplasm of the cell to the outside, by measuring the expression of this target gene under salt stress in the plants under study using a technique RT-PCR. The results showed a decrease in the gene expression of SOS1 in the common bean plants (phaseolus vulgaris cv. Nebraska) with the following different treatments (Control + Ascorbic Acid = 0.9) and (Control + Trichoderma = 1.1) plants compared to the reference control (1.2).also showed an increase in gene expression of SOS1 in these treatments (150 mM NaCl + ascorbic acid = 1.7) and (150 mM NaCl + Trichoderma = 1.9) compared to the reference control (1.2) and its equivalent of salt (1.5).while the results in (phaseolus vulgaris cv. Bronko) showed an increase in gene expression of SOS1 in control plants (2.3). Significant increase with plants treated with Trichoderma (2.6), and in most of the treatments with ascorbic acid (2.4) and Trichoderma under the influence of salt stress, an increase in gene expression was observed.