الفهرس 
Chapter 1: IntroductionOnly 14 pages are availabe for public view
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Abstract
Since the beginning of time, natural products have been used to treat human illnesses. Numerous plants, microorganisms, marine or freshwater species, etc., have had their potential as natural medicines investigated. Microbes have made a significant contribution to medicine. Pharmaceutical companies have recently reduced their investment on combinatorial chemistry-based screens in favor of skeletons based on natural products for their discovery efforts. This necessitates the constant exploration of new natural sources and habitats for novel bioactive metabolites.
Plants are regarded as valuable and appealing sources for creating new bioactive chemicals that have a variety of pharmacological actions. Due to its ability to produce numerous beneficial bioactive chemicals, A. cordifolia is regarded as one of the most significant Aptenia species. A thorough research of the fungal endophytes from the genus Aptenia cordifolia is provided in this article, along with information on the chemicals they can produce that have biological functions.
Currently, it is believed that endophytic fungus could be a source of naturally occurring bioactive substances with enormous therapeutic potential. This fungus-plant interaction is made much more challenging by the possibility of the presence of fungi capable of producing both useful biologically active chemicals and mycotoxins in most plant tissue. In general, the need for novel antimicrobial drugs stems from the rising rates of antibiotic resistance. Since agricultural microbes are also known to have developed resistance to frequently used antimicrobial compounds, this issue goes beyond the clinical usage of antimicrobial medications.
The primary objective of this studywas planned to examine the isolated fungal endophytes/pathogens from healthy and diseased Aptenia cordifolia and studying their antimicrobial activity in order to provide additional data for the utilization of these bioactive metabolites from these fungi, moreover, determine the fungal endophytes potential in the growth promotion of Aptenia.
So the following points were studied:
- Isolation and morphological identification of endophytic fungi from Aptenia spp.
- Cultivation of pure endophytic strains in a large scale using different culture media.
- Identification and quantification of bioactive compounds produced by Aptenia.
- Screening of immobilized spore suspensions of the isolated fungal endophytes on growth promotion of Aptenia in pot experiments (in sterile and non- sterile soils).
- Measuring protein and carbohydrates and plant mineral composition in addition to fresh and dry measurements of Aptenia plant, to compare the treated and untreated with endophytes.
-Metabolomic analysis for the extracts of metabolites produced by fungal endophytes.
The results obtained can be summarized as the following:
7 endophytic fungi were isolated and identified morphologically and molecular from healthy Aptenia.
3 pathogenic fungi were isolated and identified morphologically and molecular from diseased Aptenia.
Mostly, the isolated fungal endophytes/pathogens from Aptenia cordifolia 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, The causal agent, of the three isolated pathogens, was re-isolated from all inoculated Aptenia buds host and its microscopic features were similar to the original isolates
Alginate entrapment of fungal spores of endophyte inoculants exhibited improvement of plant growth when compared with either un-inoculated controls (using sterile and non-sterile soil) or Aptenia fungal pathogens. On the other hand, no promotion was noted by the treatments with immobilized pathogens that showing inhibition in the all tested parameters of plant length, shoot and root heights, and the inhibition reached to 30.4, 15.8 and % 61.2%, respectively by the immobilized inoculants of the isolated Aptenia pathogens.
According to number of flowering buds at the 6-week of planting, the most active one R4/ACEFR4/Fusarium oxysporum 2 (in all growth parameters) produced more number of flowers, which reached to 12 flowering buds. On the other hand, no flowering buds were shown either in buds treated with fungal Aptenia pathogens or untreated control under the same condition of experiment.
Most of the tested fungal endophytes were found to have abilities to enhance TAC of plant tissues when buds were inoculated with immobilized spores of these fungi, whereas, TAC value ranged between 24.43-47.9 U. On the other hand, inoculation of Aptenia buds with the isolated pathogens reduced TAC of plant tissues which reached to the same range of control values. The highest TAC promoter was observed in the inoculation of Aptenia buds with Fusarium oxysporum 1 R3/ACEFR3, which presented value of 47.9 U
Values of total soluble protein of Aptenia planttissues that treated with fungal endophytes increased by range of 39%-314.8% compared with untreated control of Aptenia plant tissues soluble protein.
The proline content of the treated buds with fungal pathogens showed highest values ranged between (0.74-0.88 mg/g d.wt), and presented values of 0.74+0.05, 0.80+0.06 and 0.88+0.07 by ACPFD1 Penicillium citrinum, ACPFD2 Aspergillu
s niger 4 and ACPFD3 Fusarium oxysporum 3, respectively, compared to low values of buds treated with fungal endophytes. According to control untreated buds, proline contents were in the same range of values of fungal pathogens treated buds.
No pattern between dry weight and each of total protein, total antioxidant and total proline, in comparing of dry weight of the buds treatments with different fungal endophytes.
The results show of a whole of 10 fungal endophytic/pathogenic isolates of Aptenia cordifolia in a preliminary examination for antimicrobial potentials on solid medium by plugs method (First screening; dual culture method) against the tested five bacteria such as Bacillus cereus, Serratia marcescens, Staphylococcus aureus, Klebsiella pneumoniae and MRSA, one yeast as Candida albicans and two filamentous fungi as Aspergillus flavus and Fusarium oxysporum.
According to fungal genera, Aspergillus represented the highest activity as antibacterial and antifungal especially species of A. niger, that gave size of inhibition clear zone in the first level.
All extracts of the isolated endophytic fungi showed variation in antimicrobial activity against the tested microbes when disc diffusion assay was performed. Additionally, disc diffusion assay represented that Asperigullus niger (3) (R2/ACEFR2) (isolated from Aptenia root) was mostly active as anti-infective against different types of pathogens (human and plant pathogens) as previously mentioned in methods and showing the higher values of Antimicrobial Index (AI) when ethyl acetate extract of filtrates recovered from the fermentation of four different culture media (PDB, PDB-y, Rice and Cz), as well as mycelia extracts. Although, A. niger (3) filtrate extract failed to suppress Candida albicans of the all tested media.
Generally, Aspergillus niger (3) was mostly the potent endophytic isolate as a producer of antifungal and antibacterial metabolites, either in primary or secondary screening experiments. Moreover, potato dextrose media either liquid or solid was mostly preferred for production of these metabolites.
The purpose of thin layer chromatography (TLC) technique is for visualization of metabolites presented in each ethyl acetate extract (filtrate) and methanol extract (mycelium) of the selected four fungal/pathogens isolates (Endophytes, R2, L1, L2; Pathogen, D2) using four different types of media (PDB, PDB-y, Rice, Cz) to verify the most appropriate isolate for enormous production of these metabolites using DCM: MeOH 9:1 solvent system in gradient elution.
Filtrate and mycelium extracts in PDB culture medium of the isolated endophytic and pathogenic fungi were the most convenient isolates to produce numerous metabolites either in short or long wave length UV detection.
No bands have been showed when Extracts of Cz medium was examined. In another TLC plates, methanol extracts of plant tissues treated with the isolated fungal endophytes/pathogens were performed.
Extracts of the treated plants with R2 (A. Niger 3 R2/ACEFR2) and R3 (Fusarium oxysporum 1 R3/ACEFR3) (fungal endophytes) aswell as D2 (Aspergillus niger 4D2/ACPFD2) (pathogen of diseased plant) represented the best appropriate bands visualization of metabolites production in TLC by these fungi.
Metabolomics interpretation of the ethyl acetate extract of A. niger 3 (R2/ACEFR2) employing LC–HR–ESI–MS revealed a wide variety of assorted bioactive compounds which have been exemplified by terpenoids and phenolic compounds (Table 8, Figs. 11 & 12). The detected compounds were identified by coupling MZmine with some databases, namely Marinlit and DNP. In view of that, the mass ion peak at m/z 177.054 for the molecular formula C10H8O3 was identified as 1, 3, 8-Naphthalenetriol (1). This naphthalene diol was previously obtained from Aspergillus fumigatus.
The molecular formula C10H10O3 was identified as 3, 4-Dihydro-8-hydroxy-3-methyl-1H-2-benzopyran-1-one (2) that was previously isolated from Aspergillus sp. Additionally, the mass ion peak at m/z 185.117, corresponding to the proposed molecular formula C10H16O3 was identified as 5-Hydroxy-6-decen-9-olide (3). It was formerly isolated from A. ochraceus. Anishidiol (4) was dereplicated at m/z 221.081 for the suggested molecular formula C12H12O4, previously obtained from A. nishimurae, it exhibited potent antifungal potential. Moreover, the mass ion peak at m/z 273.076 and the predicted molecular formula C15H12O5 was identified as 5,6,8-Trihydroxy-2-methyl-4H-naphtho[2,3-b]pyran-4-one; 6-Me ether (5), which was previously isolated from A. niger.
The metabolic diversity of bioactive leads produced by endophytic microbes, particularly fungi, play an important role in protection the plant against pathogens.
According to Docking Analysis, it showed that the possible anti-microbial mechanism of most of the isolated compounds through inhibition of bacterial topoisomerase II DNA gyrase enzyme. DNA gyrase is an important enzyme in bacteria, and its inhibition results in an interruption in DNA synthesis and thus cell death.
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.