Presymbiotic growth and sporal morphology are affected in the arbuscular mycorrhizal fungus Gigaspora margarita cured of its endobacteria

Abstract

SummarySome arbuscular mycorrhizal fungi contain endocellular bacteria. In Gigaspora margarita BEG 34, a homogenous population of b-Proteobacteria is hosted inside the fungal spore. The bacteria, named Candidatus Glomeribacter gigasporarum, are vertically transmitted through fungal spore generations. Here we report how a protocol based on repeated passages through single-spore inocula caused dilution of the initial bacterial population eventually leading to cured spores. Spores of this line had a distinct phenotype regarding cytoplasm organization, vacuole morphology, cell wall organization, lipid bodies and pigment granules. The absence of bacteria severely affected presymbiotic fungal growth such as hyphal elongation and branching after root exudate treatment, suggesting that Ca. Glomeribacter gigasporarum is important for optimal development of its fungal host. Under laboratory conditions, the cured fungus could be propagated, i.e. could form mycorrhizae and sporulate, and can therefore be considered as a stable variant of the wild type. The results demonstrated that -at least for the G. margarita BEG 34 isolate -the absence of endobacteria affects the spore phenotype of the fungal host, and causes delays in the growth of germinating mycelium, possibly affecting its ecological fitness. This cured line is the first manipulated and stable isolate of an arbuscular mycorrhizal fungus.

Detection of a novel intracellular microbiome hosted in arbuscular mycorrhizal fungi

“…Phenotypic consequences of CaGg removal from the host include important morphological changes as well as reduced proliferation of host presymbiotic hyphae. Yet, the host is not obligately dependent on the bacteria (Lumini et al, 2007;Mondo et al, 2012). These features suggest that Glomeribacter endobacteria are mutualistic associates of AMF (Lumini et al, 2007).…”

“…Yet, the host is not obligately dependent on the bacteria (Lumini et al, 2007;Mondo et al, 2012). These features suggest that Glomeribacter endobacteria are mutualistic associates of AMF (Lumini et al, 2007). Comparisons of host and symbiont phylogenies indicate that, while CaGg is a heritable endosymbiont (Bianciotto et al, 2004), it also engages in recombination and host switching, which play an important role in stabilizing this 400-million-year-old association (Mondo et al, 2012).…”

“…The spores were recovered from pot cultures by wet sieving (Gerdemann and Nicolson, 1963) and surface sterilized (Lumini et al, 2007). The spore samples were morphologically identified as Gigaspora margarita following Bentivenga and Morton (1995).…”

“…DNA extraction, amplification and clone library analysis DNA extractions were performed by crushing either individual spores or groups of five or ten spores according to Lumini et al (2007). Three fragments of the fungal ribosomal gene cluster, namely 18S, ITS and 28S, were amplified.…”

“…To ensure proper preservation of endosymbiont cells and fungal organelles, which could be jeopardized by the very thick fungal cell wall (12-16 mm, Lumini et al, 2007), we used high pressure and freeze substitution specimen preparation. On the basis of the previous molecular analyses, two isolates of G. margarita (CM23 and CM47) were selected for this experiment.…”