الفهرس 
Chapter 1 : IntroductionOnly 14 pages are availabe for public view
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Abstract
reat interests for new antimicrobial agents, in general, come from the increasing rates of resistance to existing antibiotics. This problem extends beyond the clinical application of antimicrobial drugs, such as agricultural microorganisms are also known to have acquired resistance to commonly used antimicrobial chemicals. Endophytic fungi are distinctly distributed throughout the organs and tissues of plants and are associated with various plant structures, such as leaves, branches, stems, roots, shoots. Studies of fungal endophytes in many environments are an active area for research; however, the endophytes in soybean and alfalfa have low interest.
In this study, segments of roots, stems and leaves of Alfalfa and Soybean were screened for isolation of endophytic fungi. A total of 16endophytic fungi were isolated and identified. Endophytic fungi names were Fusarium oxysporum (1), Fusarium oxysporum (2) (from root segments), Acremonium sp. (1), Acremonium sp. (2) (from stem segments), Acremonium strictum and Aspergillus ochraceus (from leaf segments) for Alfalfa, whereas, A. terreus, F. oxysporum (3), F. oxysporum (4), A. fumigates (from root segments), Nigrospora sp. (1), Nigrospora sp. (2), T. harzianum, F. oxysporum (5) (from stem segments), and two isolates of Dark sterile mycelia (from leaf segments) were isolated from Soybean segments. Most of these genera were common endophytic fungi. All of the 16 endophytes isolated from Alfalfa and/or Soybean are belonged to the phylum Ascomycota, except GMEF9 and GMEF10 which isolated from Soybean leaves were types of sterile fungi.
The main purpose of this study was planned to examine the isolated fungal endophytes in their antimicrobial activity in order to provide additional data for the utilization of bioactive metabolites from the endophytic fungi, moreover, determine their potential in production of some important hydrolytic and oxidative enzymes.
So the following points were studied:
A) Isolation and identification of endophytic fungi from segments of alfalfa and soybean.
B) Screening for antimicrobial activity.
C) Evaluation of fungal endophytes for production of hydrolytic and oxidative enzymes.
Production of hydrolytic enzymes on agar plates
Production of oxidative enzymes
Evaluation of antioxidant activity
D) Fermentation assay in liquid medium
Preparation of ethyl acetate extract from fermentation assay in liquid medium (PDB)
Determination of minimum inhibitory concentrations (MIC)
E) Metabolic analysis
The results obtained can be summarized as follows:
16endophytic fungi were isolated and identified (10 from soybean and 6 from alfalfa).
A total of 7 endophytic isolates showed activity against at least one of the tested fungal and/or bacterial pathogens, thus 43.75% (7/16) of the isolates were found to be active (5 from soybean and 2 from alfalfa). 3 isolates (18.75%) of endophytic isolates showed activity against the tested Candida albicans. None of the endophytic isolates showed any ability to inhibit the growth of the two tested bacteria (Bacillus cereus and Serratia marcescens) except Aspergillus ochraceus MSEF6 and A. terreus (GMEF1) isolates (12.5%) (2/16) that showed activity against the two tested bacteria. Generally MSEF6 and GMEF1showed inhibition against most of the tested organisms and could be considered the most potential fungal endophytes in this study.
The ability of production of degrading enzyme by all endophytes was estimated in terms of degradation halo (Colony diameter+clear zone diameter) and fungus colony rations for Enzymatic Index (EI) calculation. Hundred percent of the isolates showed positive activities for CMC-ase, xylanase and amylase activities.
Amylase EI for starch degradation on solid media showed no significantly difference between endophytes except for Nigrospora sp. (2) GMEF6 (EI 3.0) which present very high significantly level in its activity when compared with amylase of the others endophytes.
Results of endophytes’s growth stimulation or inhibition on solid media in presence of different carbon sources compared with glucose as control varied according to isolates and type of C-source. Among the tested endophytes, all isolates were stimulated in colonies growth on CMC- and xylan-agar media except for Trichoderma harzianum (GMEF7) fungal endophyte, the inhibition was investigated in xylan-medium when compared to control amended with glucose only. However, solid medium amended with starch showed inhibition to colony growth for 10 fungal endophytes (~ 62.5% of the total tested endophytes) when compared with control of glucose as a sole C-source.
A total of eight isolates (50%) of the total isolated fungal endophytes showed positive for catalase and peroxidase, whereas, laccase represented by 37.51% (six isolates) in measuring enzyme activity qualitatively.
ABTS oxidation test with and without H2O2 exhibited that peroxidase and laccase activity was diminished in the culture filtrates of the examined isolate MSEF6 compared with enzymes of MGEF1 in quantitatively measuring of enzymes.
In this study, a considerable total antioxidant capacity was observed in EtOAc of both A. ochraceus (MSEF6) (Absorbance, 0.56) and A. terreus (GMEF1) (Absorbance, 0.50), and the reduction power assay showed reduction capacity at considerable values of activity when the standard ascorbic acid was used, whereas, it represented TAC as 28.94±0.05 and 25.78±0.05 μg/mL ascorbic acid equivalent for the EtOAc extracts of A. ochraceus (MSEF6) and A. terreus (GMEF1), respectively
The antimicrobial activity of culture filtrates of the most active isolates A. ochraceus strain MSEF6 and A. terreus strain GMEF1 grown on different growth liquid media on agar plates by well diffusion method. MSEF6 and GMEF1showed antimicrobial activity against the two tested microbes, but only when it was grown in PDB medium.
Result of MIC of A. ochraceus crude EtoAc extracts varied in the range of 15 to 30 mg ml-1 for the tested bacterial/fungal microbes. Among the measured MIC value the extract concentration as antifungal compound was active more. 60% of the concentrated was responsible for MIC of the tested bacterium, whereas, 30% was responsible as MIC concentration in the tested fungal microbe.
Metabolomic profiling of the ethyl acetate extract recovered from PDB culture broth of both A. ochraceus and A. terreus was accessed via LC-HR-MS by employing macros and algorithms that coupled MZmine with databases, specially, the DNP database where, high resolution mass spectra and retention times were used for the identification of dereplicated compounds, which were mainly including phenolics, quinones, benzofurans and isochromanones in good agreement with the isolated and identified compounds from Aspergillus species.
The metabolic diversity of bioactive leads produced by endophytic microbes, particularly fungi, play an important role in protection the plant against pathogens. The inhibition of a wide range of plant and animal pathogens has been previously reported by bioactive leads synthesized by endophytic fungi. Consequently, the isolation and identification of endophytes become extremely important, whereas, the biological activities of medicinal plants can be a result of the capability of their endophytes to produce biologically new active extrolites.