Unlocking a high bacterial diversity in the coralloid root microbiome from the cycad genus Dioon.

Cycads are among the few plants that have developed specialized roots to host nitrogen-fixing bacteria. We describe the bacterial diversity of the coralloid roots from seven Dioon species and their surrounding rhizosphere and soil. Using 16S rRNA gene amplicon sequencing, we found that all coralloid roots are inhabited by a broad diversity of bacterial groups, including cyanobacteria and Rhizobiales among the most abundant groups. The diversity and composition of the endophytes are similar in the six Mexican species of Dioon that we evaluated, suggesting a recent divergence of Dioon populations and/or similar plant-driven restrictions in maintaining the coralloid root microbiome. Botanical garden samples and natural populations have a similar taxonomic composition, although the beta diversity differed between these populations. The rhizosphere surrounding the coralloid root serves as a reservoir and source of mostly diazotroph and plant growth-promoting groups that colonize the coralloid endosphere. In the case of cyanobacteria, the endosphere is enriched with Nostoc spp and Calothrix spp that are closely related to previously reported symbiont genera in cycads and other early divergent plants. The data reported here provide an in-depth taxonomic characterization of the bacterial community associated with coralloid root microbiome. The functional aspects of the endophytes, their biological interactions, and their evolutionary history are the next research step in this recently discovered diversity within the cycad coralloid root microbiome.

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

The nitrogen-fixing bacterium, Nostoc, is a commonly occurring cyanobacterium often found in symbiotic associations. We investigated the potential of cycad cyanobacterial endosymbionts to synthesize microcystin/nodularin. Endosymbiont DNA was screened for the aminotransferase domain of the toxin biosynthesis gene clusters. Five endosymbionts carrying the gene were screened for bioactivity. Extracts of two isolates inhibited protein phosphatase 2A and were further analyzed using electrospray ionization mass spectrometry (ESI-MS)/MS. Nostoc sp. 'Macrozamia riedlei 65.1' and Nostoc sp. 'Macrozamia serpentina 73.1' both contained nodularin. High performance liquid chromatography (HPLC) HESI-MS/MS analysis confirmed the presence of nodularin at 9.55±2.4 ng µg-1 chlorophyll a in Nostoc sp. 'Macrozamia riedlei 65.1' and 12.5±8.4 ng µg-1 Chl a in Nostoc sp. 'Macrozamia serpentina 73.1' extracts. Further scans indicated the presence of the rare isoform [L-Har(2)] nodularin, which contains L-homoarginine instead of L-arginine. Nodularin was also present at 1.34±0.74 ng ml(-1) (approximately 3 pmol per g plant ww) in the methanol root extracts of M. riedlei MZ65, while the presence of [L-Har(2)] nodularin in the roots of M. serpentina MZ73 was suggested by HPLC HESI-MS/MS analysis. The ndaA-B and ndaF genomic regions were sequenced to confirm the presence of the hybrid polyketide/non-ribosomal gene cluster. A seven amino-acid insertion into the NdaA-C1 domain of N. spumigena NSOR10 protein was observed in all endosymbiont-derived sequences, suggesting the transfer of the nda cluster from N. spumigena to terrestrial Nostoc species. This study demonstrates the synthesis of nodularin and [L-Har(2)] nodularin in a non-Nodularia species and the production of cyanobacterial hepatotoxin by a symbiont in planta.

Host selection of symbiotic cyanobacteria in 31 species of the Australian cycad genus: Macrozamia (Zamiaceae).

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.

Differential patterns of evolution and distribution of the symbiotic behaviour in nostocacean cyanobacteria.

Many cyanobacteria commonly identified as belonging to the genus Nostoc are well-known cyanobionts (symbionts) of a wide variety of plants and fungi. They form symbioses with bryophytes, pteridophytes, gymnosperms and angiosperms that are considerably different in the type of reciprocal interaction between the host and the cyanobiont. The phylogenetic and taxonomic relationships among cyanobionts isolated from different hosts and Nostoc strains isolated from free-living conditions are still not well understood. We compared phylogeny and morphology of symbiotic cyanobacteria originating from different host plants (genera Gunnera, Azolla, Cycas, Dioon, Encephalartos, Macrozamia and Anthoceros) with free-living Nostoc isolates originating from different habitats. After preliminary clustering with ARDRA (amplified rDNA restriction analysis), phylogeny was reconstructed on the basis of 16S rRNA gene sequences and compared with morphological characterization, obtaining several supported clusters. Two main Nostoc clusters harboured almost all cyanobionts of Gunnera, Anthoceros and of several cycads, together with free-living strains of the species Nostoc muscorum, Nostoc calcicola, Nostoc edaphicum, Nostoc ellipsosporum and strains related to Nostoc commune. We suggest that the frequent occurrence of symbiotic strains within these clusters is explained by the intensive hormogonia production that was observed in many of the strains studied. However, no evidence for discrimination between symbiotic and free-living strains, either by molecular or morphological approaches, could be found. Sequences of Azolla cyanobiont filaments, taken directly from leaf cavities, clustered tightly with sequences from the planktic cyanobacterium Cylindrospermopsis raciborskii, from the benthic Anabaena cylindrica 133 and from Anabaena oscillarioides HINDAK 1984/43, with high bootstrap values. The phylogenetic analysis showed that two distinct patterns of evolution of symbiotic behaviour might exist for the nostocacean cyanobacteria, one leading to symbioses of Nostoc species with a wide variety of plants, the other leading to the association of a unique cyanobacterial type with the water fern Azolla.

Sequence based data supports a single Nostoc strain in individual coralloid roots of cycads.

The genetic diversity of cyanobacteria associated with cycads was examined using the tRNA(Leu) (UAA) intron as a genetic marker. Coralloid roots of both natural populations of the cycad Macrozamia riedlei (Fischer ex Gaudichaud-Beaupré) C.A. Gardner growing in Perth, Australia and cycads growing in greenhouses, also in Perth, were used and their respective cyanobionts analyzed. Several Nostoc strains were found to be involved in this symbiosis, both in natural populations and greenhouse-originated cycads. However, only one strain was present in individual coralloid roots and in individual plants, even when analyzing different coralloid roots from the same plant. Moreover, when examining plants growing close to each other (female plants and their respective offspring) the same cyanobacterium was consistently present in the different coralloid roots. Whether this reflects a selective mechanisms or merely the availability of Nostoc strains remains to be ascertained. The high cyanobacterial diversity in coralloid roots of cycads revealed by PCR fingerprinting is, therefore, contested. In this study, the potential problems of using different methods (e.g., PCR fingerprinting) to study the genetic diversity of symbiotic cyanobacteria, is also addressed.

Arbuscular mycorrhizas in cycads of southern India.

Root and soil samples of three potted or ground-grown cycads ( Cycas circinalis, C. revoluta, Zamiasp.) were collected between November 1999 and June 2000 and surveyed for arbuscular mycorrhizal (AM) colonization and spore populations. AM fungi were associated with all root systems and rhizosphere samples examined. Root colonization was of a typical Arum type and AM colonization levels differed significantly between species and between potted and ground-grown cycads. Mycorrhizal colonization levels were inversely related to root hair number and length. Spores of nine morphotypes belonging to three genera ( Acaulospora, Glomus, Scutellospora) were extracted from soil. The percentage root length colonized by AM fungi was not related to soil factors, but total AM fungal spore numbers in the rhizosphere soil were inversely related to soil nitrogen and phosphorus levels. AM fungal spore numbers in the soil were linearly related to root length colonized. The co-occurrence of septate non-mycorrhizal fungi was recorded for the first time in cycads. These observations and the relationship between plant mycorrhizal status and soil nutrients are discussed.