Endophytic fungi from Pecteilis susannae (L.) Rafin (Orchidaceae), a threatened terrestrial orchid in Thailand

Abstract

Eight endophytic fungi were isolated from roots of the threatened terrestrial orchid, Pecteilis susannae (L.) Rafin. Phylogenetic analysis based on an alignment of internal transcribed spacer regions of nuclear rDNA indicated that seven isolates belonged to the genus Epulorhiza and one to Fusarium. All fungal isolates were cultured with orchid seeds collected from three field sites near Doi Suthep-Pui National Park, Chiang Mai, Thailand. Seed germination and protocorm development were evaluated up to 70 days after sowing. Percent symbiotic seed germination was highest (86.2%) when seeds were cultured with Epulorhiza (CMU-Aug 013). The protocorm development was the most advanced up to stage 2, continued embryo enlargement, or rupture of the testa, and the highest percentage was 17.8% when seeds were cultured with Epulorhiza (CMU-Aug 007). Without fungi, seed germination and protocorm development were 62.1% and 11.1%, respectively. The dependency of P. susannae on fungal symbionts for early seedling development is yet to be determined. Optimizing seed germination and seedling fitness will assist the conservation of this threatened orchid in Thailand.

Effects of mycorrhizal fungi on symbiotic seed germination of Pecteilis susannae (L.) Rafin (Orchidaceae), a terrestrial orchid in Thailand

“…Fungal isolates CMU-Aug 007, CMU-Aug 013, CMU-Aug 028, CMU-AU 211 and CMU-AU 212 were isolated as described previously (Chutima et al 2011). The remaining fungai were isolated using a modified procedure of Zhu et al (2008).…”

“…Except for the Epulorhiza isolates CMU-Aug 007, CMUAug 013 and CMU-Aug 028 were selected from our previous study (Chutima et al 2011), the fungal isolates were first identified using morphological characteristics and keys described in Barnett and Hunter (1987), Currah and Zelmer (1992), Currah et al (1997), Robert (1999) and Athipunyakom et al (2004a). DNA required for sequencing the ITS regions was obtained from mycelium as described previously (Chutima et al 2011).…”

“…DNA required for sequencing the ITS regions was obtained from mycelium as described previously (Chutima et al 2011). Briefly, Mycelium was scraped directly off culture plates, transferred into 1.5 ml centrifuge tubes, and freeze dried using a lyophilizer (Dura-Dry TM , USA).…”

“…The internal transcribed spacer (ITS) regions of nuclear rDNA of fungal isolates were amplified by polymerase chain reaction (PCR) with primers ITS4 and ITS5 (White et al 1990) under the thermal conditions described previously (Chutima et al 2011). The PCR products were purified using PCR clean up Gel extraction NucleoSpin® Extract II purification Kit (Macherey-Nagel, Germany) following the manufacturer's protocol.…”

“…Our previous study reported on early seed germination (stage 2 of protocorm development) after sowing in the dark for 70 days with fungal symbionts (Epulorhiza sp.) isolated from roots of P. susannae (Chutima et al 2011). However, Stewart and Zettler (2002) reported symbiotic seed germination of Habenaria repens was up to stage 5 of protocorm development after sowing with Epulorhiza sp.…”