نوع مقاله : مقاله پژوهشی

نویسندگان

• امید اتقیا ¹
• خلیل‌بِردی فتوحی فر ¹
• محسن فرزانه ²
• محمد جوان نیکخواه ¹

¹ گروه گیاهپزشکی، دانشکده کشاورزی، دانشکدگان کشاورزی و منابع طبیعی، دانشگاه تهران، کرج. ایران

² پژوهشکده گیاهان و مواد اولیه دارویی، دانشگاه شهید بهشتی، تهران

چکیده

کلیدواژه‌ها

• متابولیت ثانویه
• آرایه
• ریختشناسی
• اسپکتروفتومتر
• واکاوی تبارزایی

عنوان مقاله [English]

Evaluation of total phenolic and flavonoid content in some endophytic fungi of Damask Rose (Rosa damascena Mill.) in Iran

نویسندگان [English]

• Omid Atghia ¹
• Khalil-Berdi Fotouhifar ¹
• Mohsen Farzaneh ²
• Mohammad Javan Nikkhah ¹

کلیدواژه‌ها [English]

• secondary metabolite
• taxon
• morphology
• spectrophotometry
• molecular phylogeny

Extended Abstract

Introduction

      The genus Rosa belongs to the Rosaceae family, including over 200 species. Among them, Rosa damascena Mill. is a unique species used in health, pharmaceutical, and food industries. Endophytes are a wide range of plant pathogenic and saprophytic fungi that have a long latent period before the onset of external symptoms, which are necessary for the establishment of a symbiotic relationship. Many endophytes have the potential to synthesize various bioactive metabolites such as alkaloids, terpenoids, phenolics, quinines, flavonoids, and steroids. So far, no study has been conducted on the identification of endophytic fungi of the Damask rose plant, nor on the investigation of total phenolic and flavonoid content produced by its endophytic fungi in Iran. Therefore, the current research can provide new information in this field.

Materials and Methods

       To isolate and identification of endophytic fungi of Damask rose in Iran, during the autumn of 2016 and 2017, some specimens from healthy twigs and leaves of the bushes were collected from East Azerbaijan, West Azerbaijan, Isfahan, Alborz, Khorasan Razavi, Zanjan, Kerman, Kermanshah, Mazandaran, and Yazd Provinces. After washing with running tap water for 15 min and four and five times with sterile distilled water, plant materials surface sterilized with 70% ethanol for 1 min, 2% sodium hypochlorite solution for 1 min for leaves and 3 min for twigs, then rinsing with sterile distilled water for 1 min and 70% ethanol for 1 min, followed by final rinsing with sterile distilled water for 1 min. Plant materials were cut into small pieces after drying and were placed onto the 2% WA amended with chloramphenicol, gentamicin, and were kept at 25ºC in continuous dark conditions for 7-40 days. Fungal isolates were purified using the hyphal tip method. isolates were grouped according to the major morphological and cultural characteristics. The molecular analysis was performed based on nucleotide sequences of ITS-rDNA and tef1-α genomic regions. The pathogenicity test of the selected isolates was performed on the seedlings obtained from the tissue culture of the Damask rose by creating a wound on the branch (branch inoculation). PDA plugs from 14-day-old cultures were placed on the wounds and then were wrapped with parafilm to prevent desiccation. Fresh Agar plugs without fungal mycelium were used as control treatments. The selected endophytic fungi were grown on PDA culture medium, and after seven days, one to three mycelial plugs were transferred into the potato dextrose broth (PDB) culture medium. The inoculated culture media were kept at 25 ˚C to 27 ˚C for 14 days inside the shaker incubator at the speed of 120 rpm. Screening of fungal isolates was done based on the production of phenolic acids (total phenolics) and flavonoids in the culture medium using the spectrophotometry method. Statistical analysis was done using the SAS software version 9.4.

Results and Discussion

     A total of 58 fungal isolates were recovered from the Damask rose. Based on the morphological characteristics and molecular analysis of ITS-rDNA and tef1-α genomic sequences, Fusarium globosum, Sarocladium kiliense, Colletotrichum karsti, Lophotrichus ampullus, Ascotricha chartarum, Hyphodontia setulosa, and Kalmusia variispora were isolated and identified. The evaluation of the symptoms on the branches after 25 days from inoculation proved that the isolates are non-infectious on the Damask rose plants. The screening of endophytic fungal isolates was done based on the production of phenolic acids (total phenolics) and flavonoids in the culture medium using the spectrophotometry method. After analyzing the obtained results, it was found that the two isolates, Rd45 (L. ampullus) and Rd393 (K. variispora), have the highest and lowest amount of total flavonoids, respectively. In addition, two isolates, Rd36 (H. setulosa) and Rd45, also produced the highest and lowest amount of total phenolics, respectively.

Conclusion

     Since endophytic fungi are potential sources of various phenolic and flavonoid compounds, the present study aims to identify endophytic fungi of Damask rose plants. The findings of this investigation showed that all identified taxa are new endophytic fungi of the Damask rose in the world. This study is the first report of L. ampullus for fungi of Iran. In the present study, a large number of endophytic fungal isolates were recovered, which could produce phenolic and flavonoid compounds.