الفهرس 
Chapter 1 : IntroductionOnly 14 pages are availabe for public view
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Abstract
This study’s main goal was to test the isolated fungal endophytes/pathogens from healthy and diseased onions and study their antimicrobial activity in order to provide additional information for the use of these fungi’s bioactive metabolites. It also aimed to ascertain the potential of the fungal endophytes in the antagonism of onion rotting fungi So, the following points were studied:
1-Isolation and identification of endophytic/pathogenic fungi from Allium cepa (Onion) using diverse culture media.
2-Pathogenicity test for the isolated fungal pathogens was performed.
3-The antagonistic potential of fungal endophytes against the isolated fungal pathogens of onion.
4-Cultivation of pure endophytic strains in a large scale using different culture media.
5-Extraction of extra- and intra-cellular metabolites from endophyte/pathogen cultures.
6-Investigation of the obtained extracts for their antimicrobial potentials.
7-Metabolomic analyses for the extracts which displayed the promising antimicrobial activities in attempt to characterize the extracted metabolites attributed for these activities.
8-Predicting the mode of action of the most important pure compounds (Docking)
The results obtained can be summarized as the following:
11 endophytic fungi were isolated and identified morphologically and molecular from healthy onion.
3 pathogenic fungi were isolated and identified morphologically and molecular from diseased onion.
Mostly, the isolated fungal endophytes/pathogens from Allium cepa are belonged to the phylum Ascomycetes, and a few belonged to Deutromycetes.
Initial identification was performed based on the shape, structure and color of colonies, conidiophores, conidia and/or sexual fruiting bodies and spores.
Results of pathogenicity test reported that, there was a proportional increase in decay index with increasing either spore suspension concentration or prolongation of incubation period. The greater Decay Index was recorded with high spore suspension concentrations. Moreover, compared to the incubation period of six or Three days, the intensity of bulb rots after 9 days was higher. Aspergillus sp. (ACPF3) was the most virulent pathogen and vigorously infect onion bulbs performing the highest decay index for all examined concentrations.
There are different degrees of antagonism by antagonists (endophytes) against the isolated fungal pathogens of onion. With respect to the studied phytopathogen, the size of the clear zone differed between treatments and fungal endophyte. The highest clear zone (13 mm) was noticed by Penicillium sp. (C3/ACEF8) against Aspergillus sp. (P3/ACPF3) and the lowest (2 mm) was shown against Penicillium sp. (P1/ACEPF1) by the endophyte Fusarium sp. (B2/ACEF5).
In generally, there was an inverse relationship between the development of a clear zone and the existence of overgrowth.
Results of antagonistic action in this study showed inhibition of the three fungal pathogens with varied degrees of Antagonistic Index (AI) according to the type of endophytes and the tested fungal pathogen. There was no correlation between zone of inhibition and values of Antagonistic Index (AI) after 3 days of incubation. Whereas, Penicillium sp. (C2/ACEF7) showed clear zones of 4, 6 and 7 mm against Penicillium sp. (ACPF1), Penicillium sp. (ACPF2) and Aspergillus sp. (ACPF3) respectively, although it gave AI values of 84.6, 12.5 and 12.5, respectively.
The inhibition of onion fungal pathogen by the tested fungal endophytes varied from 11.1 to 75.6% in the test of non-volatile. Diffused metabolites by Aspergillus sp. (A1/ ACEF1), Aspergillus sp. (C1/ ACEF6) and Aspergillus sp. (D1/ ACEF9) The cellophane disc’s incubation for 3 days revealed the most effective growth suppression activity. against the three isolated fungal pathogens compared with diffused metabolites of the other all fungal endophytes.
The results of volatile organic compounds produced by fungal endophytes indicated that the increase in incubation period from 1-3 days improved values of growth inhibition percentage of the tested onion fungal pathogens by the volatized compounds. In addition, the highest values of pathogen growth inhibition by volatile compounds were shown by Aspergillus sp. (C1/ ACEF6) and Aspergillus sp. (D1/ ACEF9), that represented higher values of pathogen growth inhibition by non-volatile compounds as previously mentioned results.
In antimicrobial screening using plug diffusion method a total of thirteen (13/14) endophytic/pathogenic isolates showed activity against at least one of the tested bacterial or fungal pathogens.
According to results of antimicrobial screening assay using plug diffusion method showed the six isolates Fusarium sp. (ACEF3); Aspergillus sp. (ACEF6); Penicillium sp. (ACEF10) and isolated fungal pathogens Penicillium sp. (ACPF1); Penicillium sp. (ACPF2) and Aspergillus sp. (ACPF3) were promising and the most potent as anti-infective against a greater number of plant and human pathogens, therefore, they were chosen to be compared next in disk diffusion assay.
There is no antimicrobial activity against Candida albicans (ATTC 10231) and Klebsiella quasipneumonia (ATTC 700603) and MRSA (33591) except for the endophyte ACEF10 (Penicillium sp.) which was active against MRSA and showed 4 mm.
Disk diffusion assay for filtrate extracts of the six selected fungal endophytes/pathogens represented that Aspergillus sp. (ACEF6), Penicillium sp. (ACEF10) (endophytes isolated from onion leaves) and Penicillium sp. (ACPF2) (pathogen from diseased onion) were mostly active as anti-infective either against a greater number of tested pathogens or high values of inhibition zone and Antimicrobial Index (AI).
Generally, the best activity was observed in PDB and PDB-y media as evidenced by the recorded inhibition zones.
Values of AI against Candida albicans (ATTC 10231) represented promising results for fungal mycelium extracts either by endophytes or pathogens, which were superior on values of AI for standard in many cases and the highest value reached to 1.6 AI. It can be concluded that the three isolates(ACEF6), (ACEF10) (onion endophytes) and (ACPF2) (onion pathogen) were promising and most potent, so that these isolates were chosen for the next investigation of IC50 measuring.
IC50 is measured by the dose that causes death of 50% (the LD50), a more reproducible measure of toxicity than the dose that will kill all. IC50 was measured using agar-well diffusion method.
IC50 gave ranged values between 1.1 to 73.78µg/ml-1 when tested against the bacterial/fungal human pathogens. The results revealed that IC50 values of Penicillium sp. (LCEF10) presented the best antibacterial value against MRSA (ATTC 33591) and Klebsiella quasipneumoniae (ATTC 700603) 1.11 μg/ mL, 3.89 μg /mL respectively. Among the measured IC50 values, concentrations of the extracts as antibacterial compounds were more active
In TLC, mycelium extracts in PDA culture medium of the all-isolated fungi (14 isolates endophytes/pathogens), the most potent endophytic/pathogenic fungi (Fusarium sp. ACEF3; Aspergillus sp. ACEF6; Penicillium sp. ACEF10 and isolated fungal pathogens Penicillium sp. ACPF1; Penicillium sp. ACPF2 and Aspergillus sp. ACPF3) were the most convenient isolates to produce numerous metabolites either in short or long wave length UV detection.
Metabolomic profiling of the ethyl acetate extracts recovered from most active isolates were 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 in good agreement with previously isolated and identified compounds from Aspergillus, Penicillium and Fusarium sp. species.
The metabolic diversity of bioactive leads produced by endophytic microbes, particularly fungi, play an important role in protection the plant against pathogens.
Based on the IC50 results, metabolomics profiling of the ethyl acetate extract of Penicillium sp. ACEF10 was carried out using LC-HR-ESI-MS for dereplication purposes in order to explore its metabolic pool which has resulted in the identification of twenty-one compounds. Different classes of metabolites have been characterized in the ethyl acetate extract of Penicillium sp. ACEF10 like terpenoids, phenolics, macrolides, and polyketides. Additionally, in silico molecular docking study was carried out in order to predict which compounds most likely responsible for their antimicrobial potential. Interestingly, all dereplicated metabolites were able to interact with the key amino acids residues of the catalytic domain. Whereas, compounds striatisporin A (Verma R et al.2021), hynapene-A (11), (-)-A-26771B (12) and 4-(4-formyl-2-methoxy phenoxy) butanoic acid (21) showed the lowest energy values revealing their responsibilities for the antimicrobial potential.
On the other hand, Metabolomics analysis of the crude extract of Penicillium sp. ACEF10 using LC–HR–ESI–MS for dereplication purposes has resulted in the identification of a range of varied secondary metabolites that were dominated by terpenoides and polyketides.
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 that would be recommended in medicine and in pharmaceutical industries.