Plant materials and treatments
Fungi Five species of Trichoderma fungi i.e., (T. harzianum (MW718882), T. lixii (MW719563), T. ghanens (MW719590), T. virens (MW719876), and T. atroviride (MW719255) and their mutant isolates i.e., T. harzianum mutant (NAS108 M1), T. lixii mutant (NAS114-M17), T. ghanens mutant (ON545796), T. virens mutant (NAS115 M17), and T. atroviride mutant (NAS112M2)) were obtained from the Nuclear Agriculture Research Institute. Samples of fungi were given by Dr. S. Shahbazi has collected and identified. Pinto bean (Phaseolus vulgaris), Talash variety selected for research.
Seed treatments by Trichoderma spores
Mixture of spores for seed treatment
spores separated and their population reached in carboxyl methyl cellulose (TMC) solution of 1×108 then added arabic gum (40% w/v).
Suspension of isolates (talc based)
For this treatment, the method was used with a series of changes. Trichoderma spores cultured on wheat. dried at ambient temperature and milled. Then spores were collected. The isolates were added to talc powder till their final population in talc powder was 1×108 spores per gram. The moisture content of talc powder was reduced to 8% and the (Jeyarajan and Nakkeera, 2000). Spores mixture of suspension was added to the pots 7 days after sowing the seeds.
Treatment of spores mixture in soil
Production of this biofertilizer was carried out according to Farvel and others (1985) with a few changes. The base composition of the granule contains TCM solution of spores containing Trichoderma fungi (108 × 1) with 50% kaolin and 2% sodium alginate. Mixture added drop wise into CaCl2 solution (0.1M) pH=4.8. All materials were sterilized before the test and the spherical seeds were dried after distilled water at ambient temperature and stored before use at 4° C (Fravel et al., 1999). Mutant and wild type spores were added to the soil fourteen days before planting.
Seeds biopriming
Seeds were superficially disinfected in ethanol 70% for 1 minute and sodium hypochlorite 2% for 40 second, Finally, washed 3-4 times with sterile distilled water. Each of the treatments had two types (wild type and mutant). treatments are shown in full in the following table with an acronym.
Table 1: Used treatments and their abbreviation
Traetments
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abbreviations
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Mixture of spores for seed treatment by wild type strain
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SW
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Mixture of spores for seed treatment by mutant strain
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SM
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Suspension of isolates (talc based) containing of wild type strain
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TW
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Suspension of isolates (talc based) containing of mutant strain
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TM
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Spores mixture in soil containing of wild type strain
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KW
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Spores mixture in soil containing of mutant strain
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KM
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Control
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-
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All abbreviations throughout the text have been used many times.
The required soil for the pots was placed in bag for 1 hour at a pressure of 50 pounds and a temperature of 121°C autoclave. Then the mixture was mixed with 1: 1: 1, perlite, soil and peat moss. The seeds were planted in soil with two kilograms of pots containing biofertilizer (added to soil on the same day of seeding). The irrigation of the pots was carried out immediately after seeding through a flower bed. The control treatment lacked of any Trichoderma. In the pre-flowering stage, leaf sample was taken to carry out enzymatic and protein analyzes, crushed in liquid nitrogen and stored in a freezer -70 º C.
Soluble protein extraction of leaf
To generalize enzyme activity, total protein content of root extract was used by Bradford method (Bradford, 1976), absorbance measured at 595λ max = nm.
peroxidase enzyme (POD)
Guaiacol substrate, a phenolic compound, was used. Final volume was 1 cc, containing 800 μl of 10 mM Guiacol solution in 50 mM phosphate buffer with pH = 6, 100 μl of enzyme extract at a dilution of 1 to 20 in protein extraction buffer and 100 μM H2O2 at 35 mM. The absorption changes were measured at 25 ° C at 470 nm after each 10 seconds to 2 minutes (Dazy et al., 2008).
polyphenol oxidase (PPO)
Pyrogalol substrate 900 μl in phosphate buffer 10 mM at pH 6.5 and 100 μl of extract at 1 to 2 concentrations in protein buffering buffer and to remove zeroes from pyrogalol in 420 nm absorbance. Then the enzyme extract was added and changes were recorded in 2 minutes and 10 seconds (Raymond et al., 1993).
chlorophyll a, b and carotenoids
0.5 g of fresh weight tissue with 20 ml of acetone 80% homogenized then centrifuged at 6000 rpm for 10 minutes. read the absorbance to read at 663 nm wavelengths for chlorophyll a, 465 nm for chlorophyll b and 470 for carotenoids by spectrophotometer. Finally, using the following formulas, the chlorophyll content and carotenoids are obtained in mg/g (Arnon, 1973)
v = volume of filtered solution (upper solution obtained from centrifugation)
a = absorption of light at wavelengths of 663, 654 and 470 nm
w = sample fresh weight in grams
Proline content
For extraction and measurement of proline (Bates et al., 1973) were used to prepare the ninhydrin reagent, 25.1 g of ninhydrin in 30 ml of glacial acetic acid and dissolved in mild heat, then 20 ml of phosphoric acid 6 M were. Determination of proline content 0.5 g tissue was removed in 10 ml sulfosalicylic acid 3%. 2 ml of the extract diluted with 2 ml of solution ninhydrine and 2 ml of glacial acetic acid were mixed and placed in a water bath (100° C) for 1 hour, then 4 ml of toluene was added to the contents of each tube. The supernatant phase, which included toluene and proline, was separated from the aqueous phase and absorbed by a spectrophotometer apparatus at 520 nm. The standard proline range was 1 to 120 µg/g.
lipid peroxidation, Malondialdehyde (MDA (
0.5 g of fresh leaf weighted and with 5 ml of Trichloroacetic acid (TCA) (0.1%) pulverized, the extract was centrifuged, using a centrifuge machine for 5 minutes, one ml of centrifuge solution with 4.5 ml of TCA solution of 20% Which contains 0.5 g of TBA (in 100 grams) was added and resulting mixture was centrifuged, and the absorbance measured using spectrophotometer at 532 nm and 600 nm. Absorption of the remaining non-specific pigments in 600 nm determined and this amount was deducted (Health and Packer, 1968).
SDS-PAGE
Soluble proteins extracted from leaf samples under SDS-PAGE in 1 mm thick gel plates, described by Laemmli (1970). Electrophoresis performed with discontinuous buffer system in UVP Vertical Electrophoresis Unit . gels was run at 20 mA until bromophenol blue marker reached bottom of gels. Protein molecular masse calculated on basis of Protein Ladder SDS-PAGE Standards. After electrophoresis, gels rinsed out in isopropanol-acetic acid-water then methanol-acetic acid-water solution. Then, gels stained in Coomassie Brilliant Blue R-250, 0.01% (w/v). Afterwards, gels destinedin methanol-acetic acid-water mixture till protein bands became clearly visible.
Protein profile analysis: Gels scanned by GELDOC and molecular weight determined.
Extraction and Assay of Peroxidase (POD)
Peroxidase activity measured according to the method of (Kar and Mishra, 1976) using pyrogallol as substrate. Activity of enzyme (Enzyme Unit i.e. E.U.) calculated. peroxidase isoenzymes analysed from bean leaf.
polyphenoloxidase zymogram
order to evolve the polyphenoloxidase enzyme bands on the polyacrylamide gel, the (Van-Loon, 1971) method was used.
Data analyze
data were analyzed using SPSS software. The comparison of the meanings was done with Duncan's multiple range test with a probability level of 5%. Draw the charts with Excel.