Host Selection of Symbiotic Cyanobacteria in 31 Species of the Australian Cycad Genus: Macrozamia (Zamiaceae)

Abstract

The nitrogen-fixing cyanobacterium Nostoc is a commonly occurring terrestrial and aquatic cyanobacterium often found in symbiosis with a wide range of plant, algal, and fungal species. We investigated the diversity of cyanobacterial species occurring within the coralloid roots of different Macrozamia cycad species at diverse locations throughout Australia. In all, 74 coralloid root samples were processed and 56 endosymbiotic cyanobacteria were cultured. DNA was isolated from unialgal cultures and a segment of the 16S rRNA gene was amplified and sequenced. Microscopic analysis was performed on representative isolates. Twenty-two cyanobacterial species were identified, comprising mostly Nostoc spp. and a Calothrix sp. No correlation was observed between a cycad species and its resident cyanobiont species. The predominant cyanobacterium isolated from 18 root samples occurred over a diverse range of environmental conditions and within 14 different Macrozamia spp. Phylogenetic analysis indicated that endosymbionts were not restricted to previously described terrestrial species. An isolate clustering with Nostoc PCC7120, an aquatic strain, was identified. This is the first comprehensive study to identify the endosymbionts within a cycad genus using samples obtained from their natural habitats. These results indicate that there is negligible host specialization of cyanobacterial endosymbionts within the cycad genus Macrozamia in the wild.

Nodularin, a cyanobacterial toxin, is synthesized in planta by symbiotic Nostoc sp.

“…Detection of mcyE/ndaF and phylogenetic analysis A total of 47 surviving cyanobacterial isolates including 44 Nostoc (19 strains) and 3 Calothrix isolates obtained during a previous cyanobiont diversity study (Table 2 in Gehringer et al, 2010) were screened for the presence of the mcyE/ndaF toxin biosynthesis gene essential for microcystin or nodularin synthesis, respectively. Five of the isolates were found to contain regions indicative of the presence of the mcy or nda gene clusters responsible for the non-ribosomal synthesis of these toxins (Table 1).…”

“…Cyanobacterial isolates (Gehringer et al, 2010) were grown on BG11 medium and incubated at 22 o C on a 16:8 h light:dark cycle of 2000 lux. Isolates used for toxin detection were grown in 500 ml flasks with gentle agitation as above.…”

“…Toxin biosynthesis gene detection DNA was extracted from fresh culture material using the XS buffer extraction method and screened using the primer pair HEPF/R targeting the mcyE/ndaF gene (Jungblut and Neilan, 2006;Figure 2A (Gehringer et al, 2010) and sequenced using the PRISM Big Dye Terminator V3.1 system (Applied Biosystems, Mulgrave, VIC, Australia). Total genomic plant DNA was extracted from the leaves of the cycad plants containing toxin-producing symbiont species, using the Qiagen Plant DNAEasy Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions.…”

“…Methanol extracts of the green symbiotic ring within the remaining coralloid roots were obtained. HPLC HESI/MS/MS analysis identified the presence of nodularin in the coralloid root extract obtained from Macrozamia riedlei MZ65 (Gehringer et al, 2010) (Supplementary Figure S6), the same plant from which Nostoc sp. 'M.…”

“…Nostoc strains are known to associate symbiotically with a wide range of hosts, such as diatoms, bryophytes (lichens and hornworts), pteridophytes (Azolla), gymnosperms (cycads) and angiosperms (Gunnera) (Rai et al, 2000;Bergman et al, 2007). A total of 24 endosymbiont species, mostly Nostoc spp., were recently isolated from the coralloid roots of 74 cycad plants (Gehringer et al, 2010). It has been previously demonstrated that the symbiotic cyanobacteria in Macrozamia riedlei, while photosynthetically inactive, fixed nitrogen and transferred it to the host cycad plant in the form of citrulline and glutamine (Lindblad et al, 1991).…”