The effect of smoke derivatives and carbon utilisation on symbiotic germination of the endangered Pterostylis despectans

Orchids are highly dependent on exogenous nutritional sources and mycorrhizal associations to survive, particularly when challenged by extreme environmental stress such as bushfires that contribute significantly to its decline in nature. In this study, the effect of smoke derivatives and carbon utilisation was explored to improve germination and seedling establishment of an Australian endangered orchid, Pterostylis despectans (Lowly Greenhood) and its mycorrhizal fungi. Stored seeds were germinated in vitro with pelotonisolated fungal isolates with varying concentrations of smoke water (0–1.0 mL L-1) to simulate fire and sucrose as the carbon source (10 g L-1). Smoke water significantly increased germination, with advanced protocorms and robust seedlings produced. Sucrose inhibited germination such that protocorms and leafing was absent with sucrose inclusion. Fungal isolates were highly variable on its germination efficacy and tolerance to smoke water, highlighting the importance of fungal diversity and supports research-based conservation strategies to circumvent environmental challenges. KEy wORds: in vitro culture, mycorrhizae, smoke water, symbiotic germination doi: https://doi.org/10.15517/lank.v18i3.34534 Received 2 May 2017; accepted for publication 10 September 2018. First published online: 13 September 2018. Licensed under a Creative Commons Attribution-NonCommercial-No Derivs 3.0 Costa Rica License. Introduction. Orchids form minute dust-like seeds that are ideal for wind dispersal. However, they are unable to store nutrients in the embryo and often rely on fungi (predominantly imperfect Rhizoctonia spp.) in order to germinate (Arditti & Ghani 2000, Brundrett et al. 2003, Rasmussen 1995). When fungi colonise orchid seeds, they grow as intracellular tightly coiled hyphal pelotons and a symbiotic relationship is established (Huynh et al. 2004). In this symbiotic relationship, fungi supply the orchid with nutrients including nitrogen (Girlanda et al. 2011) and phosphorus (Cameron et al. 2007) while the orchid supply carbon to the fungus (Cameron, Leake & Read 2006, Látalová & Baláž 2010). Carbon utilisation by mycorrhizal fungi vary with some clades from the same Rhizoctonia species inhibited by sucrose (Wright et al. 2011) resulting in suboptimal seed germination (Huynh et al. 2004, Nikabadi et al. 2014, Wright et al. 2009). Some in vitro studies have successfully germinated orchid seeds asymbiotically (without fungi) using specific stimulants such as growth hormones to promote germination (Huynh et al. 2004, Nikabadi et al. 2014). Despite the germination success of asymbiotic plants, symbiotically germinated orchids established in soil better in the long term than those without fungi (Batty et al. 2001, Rasmussen 1995) which suggests fungal superiority and importance to orchid conservation particularly for plants that reside in depleted nutrient habitats. Fungal specificity of orchids is highly variable between species and different fungi are not equally effective in seed germination or growth (Phillips et al. 2011). Australian orchids generally have higher specificity for symbiotic fungi compared to species from other continents (Batty et al. 2001, Pandey et al. 2013, Phillips et al. 2011, Wright et al. 2009). Moreover, Rhizoctonia diversity in Australia is lower compared to other continents (Brundrett et al. 2003). Patchy fungal distribution in the soil, high fungal-host specificity and the preference for same-site specific fungal selections (Wright et al. 2011) can lead to orchid rarity (Phillips et al. 2011) and is a considerable barrier for the conservation of endangered species, for example Caladenia huegelii (Swarts et al. 2010) and some other Caladenia spp. (Wright et al. 2010) but not others (Bailarote, Lievens & Jacquemyn 2012). Australian orchids reside in fire-prone regions and respond to fire differently ranging from destructive for some species whilst stimulatory for others (Brundrett 2007, Duncan & Coates 2010, Janes, Vaillancourt &