Identification of Endophytic Fungi Strain R2 from Ginkgo biloba and Its Antimicrobial Activity

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1 http / /xuebao. jxau. edu. cn Acta Agriculturae Universitatis Jiangxiensis E - mail ndxb7775@ sina. com R * Staphylococcus aureus Escherichia coli Bacillus subtilis 3 Ginkgo biloba L. 36 ITS - rdna L1 L4 L5 L7 T2 T3 R1 R2 8 R2 20 mm 20 mm 21 mm ITS - rdna R2 Cladosporium oxysporum S A Identification of Endophytic Fungi Strain R2 from Ginkgo biloba and Its Antimicrobial Activity WANG Ya 1 LI Xi-xi 2 ZHANG Zhi-bin 3 YAN Ri-ming 1 3 ZENG Qing-gui 3 ZHU Du 1 3* 1. Key Laboratory for Research on Active Ingredients in Natural Medicine of Jiangxi Province Yichun University Yichun China 2. College of Science and Technology Jiangxi University of Traditional Chinese Medicine Nanchang China 3. Key Laboratory of Protection and Utilization of Subtropic Plant Resources of Jiangxi Province Jiangxi Normal University Nanchang China Abstract The antimicrobial activity of 35 endophytic fungi isolated from traditional Chinese medicinal plant Ginkgo biloba were detected by the method of cylinder plate test with Staphylococcus aureus Escherichia coli and Bacillus subtilis as indicator microorganisms. Observation on the morphological characters and internal transcribed spacers ribosomal DNA ITS-rDNA sequence analysis were conducted to identify the strain. The results showed that the extracts of 8 isolated entophytic fungi strain L1 L4 L5 L7 T2 T3 R1 and R2 had antibiotic activity against some indicator microorganisms. Especially the extract of strain R2 obviously exhibited strongest antibiotic activity against those indicator microorganisms the inhibition zones diameters were 20 mm 20 mm and 21 mm respectively. Based on the morphological characters and ITS-rDNA sequence analysis the endophytic fungi strain R2 was identified as Cladosporium oxysporum YRD E - mail jxwangya@ 126. com 1987 * E - mail zhudu12@ 163. com

2 4 R2 719 Key words Ginkgo biloba endophytic fungi antimicrobial activity identification Taxomyces andreanae 4 Shiraia sp. Slf14 5 Aspergillus flavus LF40 6 bioprospecting 7 8 Ginkgo biloba L Staphylococcus aureus Escherichia coli Bacillus subtilis PDA 12 PDB φ = 75% 1 min 3 φ = 0. 1% 2 min cm 0. 5 cm PDA d PDA 3 PDA PDB 500 ml 120 ml r /min 10 d 95% 24 h CFU /ml LB 4 6 mm 150 μl 3 5 μ /ml ~ 24 h R2 1 R2 15 PDA d 16 FEI - Quanta200F 2 R2 ITS - rdna ITS - rdna PCR ITS1 5'-TCCGTAGGTGAACCTGCGG - 3' ITS4 5' - TCCTCCGCTTATTGATATGC - 3' 3

3 DNA CTAB 17 PCR DNA 2 μl 10 PCR buffer 5 μl Taq DNA 2. 5 Unit dntp 2. 5 mmol /L 4 μl 3 μl 50 μl 94 5 min s s s min PCR 10 g /L 3 ITS - rdna ITS-rDNA NCBI BLAST GenBank ITS MEGA 4. 0 ClustalX Neighbour - joining Bootstrap PDA % L1 R1 R2 Tab. 1 1 Antibacteria activity of endophytic fungi Isolation endophytic fungi component Staphylococcus aureus Escherichia coli Bacillus subtilis L1 L1 Fermentation extract L4 L4 Fermentation extract L5 L5 Fermentation extract L7 L7 Fermentation extract T2 T2 Fermentation extract T3 T3 Fermentation extract R1 R1 Fermentation extract R2 R2 Fermentation extract > 20 mm mm < < 20 mm + < 10 mm Diameter of inhibition zone > 20 mm mm < Diameter of inhibition zone < 20 mm + Diameter of inhibition zone < 10 mm - Without inhibition zone. A B C 1 R1 2 R2 3 L1 4 5 U /ml A Staphylococcus aureus B Escherichia coli C Bacillus subtilis 1 The strain R1 2 The strain R2 3 The strain L1 4 Gentamycin sulfate for positive control 5 U /ml. 1 Fig. 1 Antimicrobial activity of endophytic fungi isolated from Ginkgo biloba

4 4 R R2 20 mm 20 mm 21 mm 2. 3 R R2 PDA 28 5 d 30 ~ 40 mm 2 - A B 3 ~ 6 μm 2 - C 2 - D 2 - E 15 4 ~ 8 μm 2 - F 18 R2 Cladosporium sp. A PDA B C Bar = 20μm D E F Bar = 5 μm A Colony characteristics of R2 on PDA B C Conidiophores Bar = 20 μm D E F Conidiospores Bar = 5 μm. Fig. 2 2 R2 Macro and micro characteristics of strain R ITS - rdna ITS1 ITS4 R2 DNA 529 bp GenBank HQ Blast GenBank R2 rdna - ITS Cladosporium oxysporum 99% 3 R2 Cladosporium sp. Fig. 3 3 R2 ITS - rdna Phylogenetic analysis of strain R2 based on ITS-rDNA sequences Cladosporium oxysporum R2 ITS - rdna R2 Cladosporium sp. Cladosporium oxysporum Cladosporium oxysporum R2

5 Taxomyces andreanae 4 Shiraia sp. Slf14 Aspergillus flavus 5 - LF Xylaria sp. YX R2 ITS - rdna C. oxysporum C. oxysporum Stone J K Bacon C W White J F. An overview of endophytic microbes Endophytism Defined M / /Bacon C W White J. Microbial Endophytes New York Marcel Dekker J Wang Y Zeng Q G Zhang Z B et al. Isolation and characterization of endophytic Huperzine A-producing fungi from Huperzia serrata J. J Ind Microbiol Biotechnol Stierle A Strobel G Stierle D. Taxol and taxane production by Taxomyces andreanae an endophytic fungus of Pacific Yew J. Science Zhu D Wang J Zeng Q et al. A novel endophytic Huperzine A producing fungus Shiraia sp. Slf14 isolated from Huperzia serrata J. J Appl Microbiol J Strobel G Daisy B Castillo U et a1. Natural products from endophytic microorganisms J. J Nat Prod J J J J J S-221 J J M M

6 4 R Guo L D Hyde K D Liew E C Y. Identification of endophytic fungi from Livistona chinensis based on morphology and rd- NA sequences J. New Phytol Tasic S Miladinovic-Tasic N. Cladosporium spp. cause of opportunistic mycoses J. Acta Fac Med Naiss J J Liu X L Dong M S Chen X H et al. Antimicrobial activity of an endophytic Xylaria sp. YX-28 and identification of its antimicrobial compound 7-amino-4-methylcoumarin J. Appl Microbiol Biotechnol Zhang Z B Zeng Q G Yan R M et al. Endophytic fungus Cladosporium cladosporioides LF70 from Huperzia serrata produces Huperzine A J. World J Microbiol Biotechnol Zhang P Zhou P P Yu L J. An endophytic taxol-producing fungus from Taxus media Cladosporium cladosporioides MD2 J. Curr Microbiol 櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗櫗 Bagchi G D Jain D C Kumar et al. The phytotoxic effects of the artemisinin related compounds of Artemisia annua J. Journal of Medicinal and Aromatic Plant Sciences Dayan F E Hernandez A Allen S N et al. Comparative phytotoxicity of artemisinin and several sesquiterpene analogs J. Phytochemistry J Paramanik R C Chikkaswamy B K Roy D G et al. Effect of biochemicals of Artemisia annua in plants J. Journal of Phytological Research Jassbi A R Zamanizadehnajari S Baldwin I T. Phytotoxic volatiles in the roots and shoots of Artemisia tridentata as detected by headspace solid - phase microextraction and gas chromatographic - mass spectrometry analysis J. Journal of Chemical Ecology Shalinder K Daizy R B. Assessment of allelopathic potential of Artemisia scoparia against some plants J. The Bioscan J J Barratt B I P Lorimer S D Perry N B et al. A bioassay to evaluate native plant extracts as slug feeding deterrents C. Proceedings of the Forty Sixth New Zealand Plant Protection Conference Christchurch New Zealand Clark S J Dodds C J Henderson I F et al. A bioassay for screening materials influencing feeding in the field slug Deroceras reticulatum Muller Mollusca Pulmonata J. Annals of Applied Biology Sherbiny A H EL Omar A M Sisi A G et al. Natural botanical extracts as repellants for the house sparrow Passer domesticus. II. Efficacy under rice field conditions J. Annals of Agricultural Science Moshtohor Bakry F A Mohamed R T Hommossany K. Biological and biochemical responses of Biomphalaria alexandrina to some extracts of the plants Solanum siniacum and Artemisia judaica L. J. Pesticide Biochemistry and Physiology