Endophytic Fungi Isolated from Crocus sativus L. (Saffron) as a Source of Bioactive Secondary Metabolites

Abstract

Introduction: Endophytic fungi are becoming an important source of new natural bioactive products. Many interesting endophytic fungi have been isolated from traditional medicinal plants. Crocus sativus L. (saffron) is one of the most expensive and rarest spices in the world, used as a dye, aroma and for medicinal purposes. This study reports on the molecular characterization of endophytic fungi isolated from roots of saffron plants growing in Taliouine-Morocco, and the examination of the antibacterial and antioxidant activities of secondary metabolites extracted from these endophytes. Methods and Material: The fungi were isolated from sterilized saffron root fragments and identified using internal transcribed spacer (ITS) rRNA gene sequence analysis. Three endophytes were fermented and extracted with ethyl acetate (EtOAc) and chloroform (CHCl 3). The antibacterial activity of fungi extracts was evaluated using the Agar Diffusion Method against six strains, Bacillus sp., Stenotrophomonas sp., Pseudomonas putida, Pantoea sp., Luteibacter sp. and Escherichia coli. The antioxidant activity was tested by using the α,α-diphenyl-β-picrylhydrazyl (DPPH) analysis and β-Carotene bleaching test (BCBT) methods. Results: Sixty fungal isolates were recovered and purified from saffron roots. ITS rDNA sequences were 99-100% identical to three different species: Rhizopus oryzae, Aspergillus fumigatiaffinis and Aspergillus niger. Both Rhizopus oryzae extracts had high antibacterial activity against most of the bacteria tested, while Aspergillus niger and Rhizopus oryzae extracts showed an antioxidant capacity using DPPH and BCBT methods, respectively. Conclusion: The fungal endophytes inhabiting saffron roots could be a potential source of natural plant bioactive secondary metabolites especially for antibacterial and antioxidant purposes.

Microbiome Fingerprint as Biomarker for Geographical Origin and Heredity in Crocus sativus: A Feasibility Study

“…Amycolatopsis, Kribbella, Streptosporangium, Geodermatophilus, and Aeromicrobium) have been reported earlier from saffron rhizosphere in our previous studies from Kashmir and Kishtwar in India and Taliouine in Morocco (Ambardar et al, 2021 unpublished data;Chamkhi et al, 2018). However, the bacteria reported from saffron for the first time have been reported from other plants.…”

“…This explains the dominance of Proteobacteria, Actinobacteria, and Bacteroidetes in the cormosphere, which are among the most dominant phyla in the soil and maybe getting transferred from the soil, just like in rhizosphere (Miyashita, 2015;Fierer, 2017;Compant et al, 2019). To our knowledge, cormosphere microbiome has not been studied in any other plant, except for saffron reported previously by our group (Ambardar and Vakhlu, 2013;Ambardar, 2014;Ambardar et al, 2014Ambardar et al, , 2016Kour et al, 2018) from Kashmir, India (Chamkhi et al, 2018), and from Taliouine, Morocco. Previous reports by our group have also confirmed the dominance of Proteobacteria in Kashmir saffron rhizosphere and cormosphere, even though the method used was cultivation dependent and 16S targeted metagenome sequencing was different than the methods used in the present study (Ambardar and Vakhlu, 2013;Ambardar et al, 2014) (Supplementary Material 1).…”

“…Streptomyces have been previously reported from the saffron rhizosphere also using cultivation independent approaches (Ambardar et al, 2014). In addition, to Pseudomonas and Streptomyces, 53 have also been reported previously in saffron in Kashmir and Morocco (Ambardar and Vakhlu, 2013;Ambardar et al, 2014Ambardar et al, , 2016Chamkhi et al, 2018;Kour et al, 2018) (Supplementary Material 2). Interestingly, we found that 24 genera were common to all, representing 32.8% of collective bacteriome in the four samples (three field and one laboratory sample).…”

“…In addition, 22 bacterial genera (out of 24) representing the core microbiome of the C. sativus in the present study were also reported previously in Saffron grown in Kashmir, India, by our group, out of which 19 are PGPB in saffron and other plants (Ambardar and Vakhlu, 2013;Kour et al, 2018;Umadevi et al, 2018;Vergani et al, 2019) (Supplementary Material 3). Agrobacterium, Bacillus, Delftia, Luteibacter, Pantoea, Pseudomonas, Rahnella, Rhizobium, and Variovorax from rhizosphere and Agrobacterium, Pseudomonas, Rhizobium, and Variovorax from cormosphere of saffron grown in Morocco using cultivation dependent methods have been reported earlier as well (Chamkhi et al, 2018). Core microbiome enriched with plant growth promoting bacteria indicates that the plant selects the core microbiome based on their function (Supplementary Material 3).…”

“…Pseudomonas isolates have been reported to solubilize phosphate, produce siderophores, ammonia, and indole-3-acetic acid, and colonize the roots of tomato plants (Qessaoui et al, 2019) and also have been reported to induce salt tolerance in Arabidopsis thaliana (Chu et al, 2019). Pseudomonas has also been reported to be dominant in saffron rhizosphere grown in Kashmir, India, analyzed using both cultivation dependent and cultivation independent method (Ambardar and Vakhlu, 2013;Ambardar et al, 2014), and Morocco, analyzed using cultivation dependent approaches (Chamkhi et al, 2018). Streptomyces is the largest genus of Actinobacteria phylum comprising 500 species and has been found to promote plant growth (Lehr et al, 2007(Lehr et al, , 2008Schrey and Tarkka, 2008;Viaene et al, 2016).…”