that Produces Hypericin

Transcription

1 1 2 An Endophytic Fungus from Hypericum perforatum that Produces Hypericin 3 Souvik Kusari, Marc Lamshöft, Sebastian Zühlke, and Michael Spiteller* 4 5 Institute of Environmental Research (INFU), University of Dortmund, Otto-Hahn-Strasse 6, D Dortmund, Germany. 6 7 * To whom correspondence should be addressed. Tel: +49(0) Fax: +49(0) m.spiteller@infu.uni-dortmund.de. 9 Supporting Information 10 Table of Contents 11 Figure S1. Hypericum perforatum L. and its endophytic fungus INFU/Hp/KF/34B. 12 S2. LSU (28S) rdna Sequence obtained from the endophytic fungus INFU/Hp/KF/34B. 13 S3. Morphological description on Potato Dextrose Agar (PDA). 14 S4. Isolation of total genomic DNA, PCR amplification of LSU (28S) rdna and Sequencing. 15 S5. Culture conditions and morphological description on Potato Dextrose Broth (PDB). 1

2 17 Figure S1. Hypericum perforatum L. and its endophytic fungus INFU/Hp/KF/34B (a) (b) (c) (a) Hypericum perforatum L. (b) Endophytic mycelia growing out from cut areas of surface sterilized Hypericum stem segments on streptomycin supplemented WA plate. (c) Endophytic fungi, INFU/Hp/KF/34B, growing on rich medium (PDA) plate. 28 S2. LSU (28S) rdna Sequence obtained from the endophytic fungus INFU/Hp/KF/34B TTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCCCTAGTAACGGCGAG TGAAGCGGCAACAGCTCAAATTTGAAATCTGGTCTTCGGGCCCGAGTTGTAATTTGTAGAG GAAGCTTTTGGCGCGGTACCTTCTGAGTCCCCTGGAACGGGGCGCCATAGAGGGTGAGAGC CCCGTATAGTTGGATACCAAGCCTGTGTAAAGCTCCTTCGACGAGTCGAGTAGTTTGGGAA TGCTGCTCAAAATGGGAGGTAAATTTCTTCTAAAGCTAAATACCGGCCAGAGACCGATAGC GCACAAGTAGAGTGATCGAAAGATGAAAAGCACTTTGAAAAGAGGGTTAAATAGCACGTG AAATTGTTGAAAGGGAAGCGCTTGTGACCAGACTTGCGCCGGGCTGATCATCCGGTGTTCT CACCGGTGCACTCTGCCCGGCTCAGGCCAGCATCGGTTCTCGCGGGGGGACAAAGGTCCTG GGAACGTAGCTCCTCCGGGAGTGTTATAGCCCAGGGCGTAATGCCCCCGCGGGGACCGAG GTTCGCGCTCTGCAAGGATGCTGGCGTAATGGTCACCAGCGACCCGTCTTGAAACACGGAC CAAGGAGTCAAGGTTTTGCGCGAGTGTTTGGGTGTAAAACCCGCACGCGTAATGAAAGTGA ACGTAGGTGAGAGCTTCGGCGCATCATCGACCGATCCTGATGTATTCGGATGGATTTGAGT AGGAGCGTTAAGCCTTGGACCCGAAAGATGGTGAACTATGCTTGGATAGGGTGAAGCCAG AGGAAACTCTGGTGGAGGCTCGCAGCGGTTCTGACGTGCAAATCGATCGTCAAATCTGAGC ATGGGGGCGAAAGACTAATCGAACCATCTAGTAGCTGGTTACCGCCGAAGTTTCCCTCAGG ATAGCAGTGTTGTCTTCAGTTTTATGAGGTAAAGCGAATGATTAGGGACTCGGGGGCGCTT TTTAGCCTTCATCCATTCTCAAACTTTAAATATGTAAGAAGCCCTTGTTACTTAGCTGAACG TGGGCATTCGAATGTACCAACACTAGTGGGCCATTTTTGGTAAGCAGAACTGGCGATGCGG GATGAACCGAACGCGGGGTTAAGGTGCCGGAGTGGACGCTCATCAGACACCACAAAAGGC GTTAGTACATCTTGACAGCAGGACGGTGGCCATGGAAGTCGGAATCCGCTAAGGACTGTGT AACAACTCACCTGCCGAATGTACTAGCCCTGAAAATGGATGGCGCTCAAGCGTCCCACCCA TACCCCGCCCTCAGGGTAGAAACGATGCCCTGAGGAGTAGGCGGCCGTGGAGGTCAGTGA CGAAGCCTAGGGCG 2

3 The LSU (28S) rdna sequence obtained from the endophyte fungus strain that produces hypericin (1) and emodin (2) in culture. The sequence was obtained using the methods described in the Experimental Section. The sequence has been submitted to the EMBL-Bank under accession number AM S3. Morphological description on Potato Dextrose Agar (PDA) The fungus produces copious amounts of aerial, surficial and submerged hyphae on rich medium (PDA) that reached 6 cm diameter in about 5 days at 28 ± 2 C. The mycelia, initially white and cottony, gradually turn dark grey to black with white to light grey, cottony centers. The aerial hyphae are almost all medium to dark grey, slender, with pointed tips. The surface hyphae are light to dark grey or black, slender with pointed growing tips. Light microscopic studies have revealed that the hyphae are intertwined into rope-like strands, sometimes coiled, and branched. The hyphae preferably grows on the surface of the media, while sill developing hyphal coils, hyphal stranding and right-angle branching. Each strand contains uniform, elongated, rectangular hyphal cells; the terminal cell almost always oval to pointed (towards the periphery) giving the overall appearance of pointed tips. Branching is by structural changes in single terminal hyphal cells that start dividing bi-directionally first followed by uniform growth in both directions. Branching is right-angled most of the times, though branching at lesser angular distance is visible in most peripheral hyphae. The overall colony appearance is fan-like, spreading from the center towards the edges. No sporulation was observed in rich medium (PDA). Microscopic studies confirmed the absence of sporulation in the above media. The fungus did not even sporulate in other rich mycological media as well, like Sabouraud Agar (SA) and Malt Extract Agar (MEA), though the characteristic color development of the mycelia are always prominent at later stages of growth. Whether sporulation is possible (if at all) under special conditions (nutrient requirement changes, ph changes, spiking media with host plant extract, etc.) is currently being studied along with electron microscopic and optimization studies (as mentioned in the Experimental Section). From the reverse side of the Petri dish, the color is tan or brown to black. 76 S4. Isolation of total genomic DNA, PCR amplification of LSU (28S) rdna and Sequencing 3

4 Total DNA was isolated from the mycelial mass using the Macherey Nagel (MN) Food DNA extraction kit. About 100 mg of fungal tissue were scraped from the agar surface and transferred to a tube for homogenizing using a homogenizer. The homogenized samples were transferred into MN Tube Strips (lysis) and added 1 ml buffer (CF, supplied with MN kit) preheated to 65 C, 10 µl of proteinase K solution, 10 µl of RNase A and mixed vigorously. The setup was incubated at 65 C for 30 min. The samples were centrifuged for 20 min at 5600 x g. 300 µl clear supernatant was transferred to a roundwell block, added 300 µl buffer (C4, supplied with MN kit) and 200 µl ethanol. The contents were mixed by vigorous vortexing for s followed by a brief spin for 30 s at 1500 x g to collect any sample from cap strips. The samples were then transferred into the wells of the NucleoSpin Food Binding Strips. 100 µl pre-warmed TE buffer (10 mmol Tris-HCl, ph 8.0, 1 mmol EDTA, 70 C) was dispensed to each well of the NucleoSpin Food Binding Strips directly onto the membranes followed by incubation at room temperature for 2-3 min. Finally, centrifugation was performed at 5600 g for 2 min to obtain the genomic DNA. The obtained genomic DNA was subjected to PCR analysis (ABI GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA) using primers directed to the D2 region (variable) of the large subunit (LSU) (28S) rdna. The PCR performed in 25 µl reaction volume contained 2.5 µl of 10x Taq Polymerase reaction buffer (Fermentas GmbH, St. Leon-Rot, Germany), 2.5 µl of 25 mm MgCl 2, 0.2 µl of Taq Polymerase enzyme (Fermentas GmbH, St. Leon-Rot, Germany), 1 µl of 100x BSA (Fermentas GmbH, St. Leon-Rot, Germany), 2 µl of 10 µm dntps (Fermentas GmbH, St. Leon-Rot, Germany), 1.25 µl of 100 pm forward primer LR0R (ACCCGCTGAACTTAAGC), 1.25 µl of 100 pm reverse primer LR7 (TACTACCACCAAGATCT), 1.30 µl of 100 pm control primer/template LR5 (TCCTGAGGGAAACTTCG), 0.5 µl extracted genomic DNA (suspension), and 12.5 µl of water. The control was added as per manufacturer s guidelines. The results from the control helped in determining whether failed reactions (if any) were the result of poor primer quality or reaction failure. The reaction conditions were as follows: 95 C for 15 min, 34 cycles (95 C 45 s, 45 C 45 s, 72 C 90 s) and 72 C for 7 min followed by cooling to 4 C. PCR product was sequenced on an ABI 3730xl DNA Analyzer (Applied Biosystems, Foster City, 4

5 CA) using BigDye Terminator v 3.1 Cycle sequencing kit (Applied Biosystems, Foster City, CA). Briefly, the amplified DNA was resuspended in BigDye Terminator sequencing buffer and Hi-Di formamide (Applied Biosystems, Foster City, CA), and loaded into the instrument capillary array (50 cm) by electrokinetic injection with the help of an autosampler. The array was pre-filled with a special polymer called POP-7 (Applied Biosystems, Foster City, CA), a medium that prevents the DNA fragments to stick together. The DNA sequence fragments were separated by size as they travelled through the polymer-filled capillary array (electrophoresis). As they reached the detection window, a laser beam was used to excite the molecules and emissions from samples were collected simultaneously and spectrally separated by a spectrograph. The emissions were focused as columns of light onto the attached CCD camera which was read and interpreted by the 3730xl Data Collection software (Applied Biosystems, Foster City, CA) and displayed as an electropherogram. 114 S5. Culture conditions and morphological description on Potato Dextrose Broth (PDB) The fungus was inoculated into 500 ml conical flasks with four indentations, containing 100 ml Potato Dextrose Broth (PDB) medium (DIFCO, cat. no ) adjusted to ph 5.6 before autoclaving. These flasks were incubated at 28 ± 2 C with shaking (200 rpm) on a rotary shaker (Heidolph UNIMAX 2010, Germany) for 5 days. Under shake flask conditions in PDB (200 rpm rotation, 28 ± 2 C), the fungus grew as white, medium to big, non-sticky, round balls. Pellicle formation at the edge of the flask was light and slightly sticky. Interestingly, the fungal mycelium did not show any coloration unlike on solid medium (PDA). 5