identification and Comparison silymarin inendophytic fungi isolated from seed of Silybum marianum (L) Gaert.)

Document Type : scientific research article

Authors

1 Department of Horticulture, Faculty of Crop Sciences, Sari Agricultural Sciences and Natural Resources University, Sari, Iran,

2 Department of Horticulture, Faculty of Crop Sciences, Sari Agricultural Sciences and Natural Resources University, Sari, Iran

3 Department of Plant Protection, Faculty of Crop Sciences, Agricultural Sciences and Natural Resources, Sari, Iran

4 Genetic and Agricultural biotechnology Institute of Tabarestan, Sari Agricultural Sciences and Natural Resources University, Sari, Iran

Abstract

Introduction: Silybum marianum (L) Gaert.) Is a member of Asteraceae family, and silymarin is known as a valua effective substance in the plant, which is used to treat a number of diseases, such as liver disease. Chalcone synthase is one of the key enzymes in the production of silymarin. The fungal endophytes live in the tissues, without infection, and have a beneficial effect on the production of secondary metabolites of plants. Recently, several endophytic fungi from milk thistle (Silybum marianum (L) Gaert) have been isolated and studies were the chemical and molecular on its fungus has been considered. Therefore, this study aimed to isolate endophytic fungi from the seeds of milk thistle plant, and comparing the production of fungi and plant silymarin, as well as investigating the Chalcone synthase amino acid sequences associated with the L Silybum marianum and several other fungi from the NCBI database (http: //www.ncbi.nlm.nih.gov).
Materials and Methods: Therefore, this research was carried out to isolate endophytic fungi from the seeds of milk thistle plant, and to compare the production of fungal and plant silymarin. For this purpose, milk thistle seeds were collected from the area of Sari Agricultural and Natural Resources University and after disinfection, They sterilized in conditions, cultivated on MS medium. After appearance of fungi in the culture medium, the fungi were transferred to the PDA medium under sterile conditions the next for purification to WA medium. Fungal growth isolates were transferred to PDB medium and after 10 days of silymarin extraction Molecular identification of endophyte fungi isolates was performed by amplification of ribosomal areas, then the Alignment of the obtained sequence with the sequences on the NCBI site was compared with BLAST online software and identified with phylogeny tree of endophytic fungus. Investigate the bioinformatics of Chalcone synthase in L. Silybum marianum and several other fungi species,is use the NCBI database, was obtained by comparing domains and similar positions of Chalcone synthase, and was Investigating with the clustalo uniprot database. Then it was analyzed with CLC Genomics Workbench5.5.2 software and their specifications table was drawn up. The phylogeny tree of these sequences was drawn using the Mega7 software, using the Neighbor- joining
Results: The results of high performance liquid chromatography confirmed the silymarin production of fungi. The amount of silymarin for leaf samples of intact plant was 33.079 ppm and for the in vitro grown plant was 17.183 ppm and for the endophyte fungus was 7. 29 ppm. Using ITS, molecular identification of isolates was carried out and its species was identified as Alternaria alternata. Also, analysis phylogenic relationship of chalcone synthase, as a key enzyme for production of silymarin, between milk thistle and a few fungi showed its closest proximity to to Alternaria sp.
Conclusion: The study of the production of silymarin in fungi isolated from the seeds of milk thistle , in vitro and in vivo plants, along with the bioinformatics study of the pathway of production of Chalcone synthase enzyme, determined the role of endophytic fungi in the production of silymarin. The fungal isolate was also able to produce silymarin in laboratory conditions

Keywords

References

1.Al-Hawamdeh, F.M., Shibli, R.A. andAl-Qudah, T.S. 2013. In vitro production of silymarin from Silybum marianum L. Med Aromat Plants. 1: 001. 2167-0412.
2.Ataei Salami, Tajik Ghanbari, Babayizad, M. and Mehdian, P. 2017. Isolation and identification of endophytic fungi in Sarakhs forest from Shahid Zareh forest. Third National Conference on Applied Microbiology. Babolsar. Mazandaran University. (In Persian)
3.Babakhanzadeh Sajirani, A. and Hasan Abadi, M. 2010. Interaction between Plant Growth Promoting Rhizobacteria (PGPR) and Endophytes with Medicinal Plants to Increase Production of Chemicals and Secondary Metabolites, National Conference on Environment and Plant Production. Semnan. Islamic Azad University. pp. 21-17. (In Persian)
4.Das, M., Prakash, H.S. and Nalini, M.S. 2017. Antioxidative and antibacterial potentials of fungal endophytes from Justicia wynaadensis Heyne: an ethnomedicinal rain forest species of western Ghats. Asian J. Pharm. Clin. Res. 10: 6. 203-209.
5.Devari, S., Jaglan, S., Kumar, M., Deshidi, R., Guru, S., Bhushan, S., Kushwaha, M., Gupta, A.P., Gandhi, S.G., Sharma, J.P. and Taneja, S.C. 2014. Capsaicin production by Alternaria alternata, an endophytic fungus from Capsicum annum; LC–ESI–MS/MS analysis. Phytochem. 98: 183-189.
6.El-Elimat, T., Raja, H.A., Graf, T.N., Faeth, S.H., Cech, N.B. and Oberlies, N.H. 2014. Flavonolignans from Aspergillus iizukae, a fungal endophyte of milk thistle (Silybum marianum). J. Nat. Prod. 77: 2. 193-199.
7.Fallah Huseini, H., Hemati, A.R. and Alavian, S.M. 2004. A review of herbal medicine: Silybum marianum. J. Med Plant. 3: 11. 14-24. (In Persian)
8.Hasanloo, T., Kossari, M., Mohajeri Naraghi, S. and Bagheri, M. 2008. Effect of Different Strains of Trichoderma harzianum on the Silymarin Content of Milk thistle (Silybum marianum) 6th Iranian Horticultural Science Congress, Rasht, Guilan University. (In Persian)
9.Hasanloo, T., Ahmadi, M., Khayyan Nequei, S.M. and Salehi Jouzani, G.R. 2013. Elicitation effects of fungal extracts on Silymarin accumulation in Silybum marianum (L.) gaertn hairy root culture. J. Med Plant. 4: 48. 25-39. (In Persian)
10.Kaul, S., Wani, M., Dhar, K.L. and Dhar, M.K. 2008. Production and GC-MS trace analysis of methyl eugenol from endophytic isolate of Alternaria from rose. Ann. Micro. 58: 3. 443-445.
11.Khalighi Sigaroodi, F., Jarvandi, S. and Taghizadeh M. 2013. Application of Medicinal Plants. Arjmand. Press, 248p. (In Persian)
12.Kordi, H., Aghdasi, M. and Khalafi, M. 2013. An investigation on flavonolignans in different organs of Silybium marianum L. in Gorgan region. Iran. J. Med Arom. Plant. 29: 3. 651-664.(In Persian)
13.Lou, J., Fu, L., Peng, Y. and Zhou, L. 2013. Metabolites from Alternaria    fungi and their bioactivities. Molecules.       18: 5891-5935.
14.Nasiri Madiseh, Z., Mofid, M., Ebrahimi, M., Khyam Nekouee, S.M. and Khosravi Ahahli, M. 2010. Isolation of Taxol-producing endophytes fungi from Iranian yew (Taxus baccata L.).J. Shahrekord Univ. Med. Sci.11: 4. 101-106. (In Persian)
15.Omidbaigi, R. 2009. Production and Processing of Medicinal Plant. Behnashr. Press, 440p. (In Persian)
16.Rahimi, N., Nazeri, S. and Mirzaei, S. 2013. Taxol production by endophytic fungi isolated from Corylus avellana. Arak. Med. Univ. J. 16: 37-44. (In Persian)
17.Raja, H.A., Kaur, A., El-Elimat, T., Figueroa, M., Kumar, R., Deep, G., Agarwal, R., Faeth, S.H., Cech, N.B. and Oberlies, N.H. 2015. Phylogenetic and chemical diversity of fungal endophytes isolated from Silybum marianum (L) Gaertn.(milk thistle). Mycology. 6: 1. 8-27.
18.Schmid, J., Doerner, P.W., Clouse, S.D., Dixon, R.A. and Lamb, C.J. 1990. Developmental and environmental regulation of a bean chalcone synthase promoter in transgenic tobacco. The Plant Cell. 2: 7. 619-631.
19.Seshime, Y., Juvvadi, P.R., Fujii, I. and Kitamoto, K. 2005. Discovery of a novel superfamily of type III polyketide synthases in Aspergillus oryzae. Biochem. Biophys. Res. Commun.331: 253-260.
20.Strobel, G. and Daisy, B. 2003. Bioprospecting for microbial endophytes and their natural products. Bioprospecting for microbial endophytes and their natural products. Microbiol. Mol. Biol. Rev. 67: 4. 491-502.
21.Torres, M. and Corchete, P. 2016. Gene expression and flavonolignan production in fruits and cell cultures of Silybum marianum. J. Plant Physiol. 192: 111-117.
Journal of Plant Production Research
Volume 27, Issue 3
December 2020
Pages 23-36

Files

Share

How to cite

Statistics