The culture-based approach was used to characterize the fungal endophytes associated with the coralloid roots of the endemic Cycas debaoensis and Cycas fairylakea from various population sites in China. We aim to determine if the assemblages of fungal endophytes inside these endemic plant hosts are distinct and could be explored for bioprospecting. The isolation method yielded a total of 284 culturable fungal strains. Identification based on the analysis of the internal transcribed spacer (ITS) rDNA showed that they belonged to two phyla, five classes, eight orders and 22 families. At least 33 known genera and 62 different species were confirmed based on >97% ITS sequence similarity. The most frequent and observed core taxa in the two host species regardless of their population origin were Talaromyces, Penicillium, Fusarium, Pochonia and Gliocladiopsis. Seventy percent was a rare component of the fungal communities with only one or two recorded isolates. Contrary to common notions, diversity and fungal richness were significantly higher in C. debaoensis and C. fairylakea collected from a botanical garden, while the lowest was observed in C. debaoensis from a natural habitat; this provides evidence that garden management, and to a minor extent, ex-situ conservation practice, could influence fungal endophyte communities. We further selected nineteen fungal isolates and screened for their antagonistic activities via a co-cultivation approach against the phytopathogens, Diaporthe sp. and Colletotrichum sp. Among these, five isolates with high ITS similarity matches with Hypoxylon vinosupulvinatum (GD019, 99.61%), Penicillium sp. (BD022, 100%), Penicillifer diparietisporus (GD008, 99.46%), Clonostachys rogersoniana (BF024, 99.46%) and C. rosea (BF011, 99.1%), which showed exceptional antagonistic activities against the phytopathogenic fungi with a significant inhibition rate of 70–80%. Taken together, our data presented the first and most comprehensive molecular work on culturable fungal endophytes associated with the coralloid roots of cycads. Our study also demonstrated that about 5% of fungal endophytes were not detected by the high-throughput sequencing approach, implying the equal importance of a culture-dependent approach to study fungal communities of cycads. We further highlighted the potential role of endemic and rare plants to discover and isolate unique plant-associated fungal taxa with excellent biocontrol properties.

Endogenously formed coralloid roots in living cycads are known to be infected and maintained by a mutual partnership with photosynthetic cyanobacteria. In this study, two strains of filamentous, nitrogen-fixing endosymbiotic cyanobacteria from the green zone of the coralloid roots of Cycas fairylakea were isolated and grown in culture. The phylogenetic placement based on the 16S rRNA gene region and the secondary structures of the ITS between the 16S and 23S rRNA gene, in addition to traditional morphological characterization, were used to analyse the two strains. Results showed that the 16S rRNA gene sequences of the two strains, CF01 and CF06, displayed ≥97% similarity to the genus Desmonostoc. The phylogeny inferred by maximum likelihood, bayesian inference and neighbour-joining placed the two strains on a separate node away from other species of Desmonostoc. Hormogonial filaments were evident in the two symbiotic species. In addition, a few unique morphological structures in the CF01 and CF06 strains were observed. The D1-D1ʹ, Box-B and V3 helices generated from the ITS of 16S–23S sequences did not fit those of any described species of Desmonostoc, and this led to proposing the two strains as novel species. In compliance with the International Code of Nomenclature for algae, fungi, and plants, the two species are named Desmonostoc lechangense sp. nov. and Desmonostoc aggregatum sp. nov.

Cycads have developed a complex root system categorized either as normal or coralloid roots. Past literatures revealed that a great diversity of key microbes is associated with these roots. This recent study aims to comprehensively determine the diversity and community structure of bacteria and fungi associated with the roots of two Cycas spp. endemic to China, Cycas debaoensis Zhong & Chen and Cycas fairylakea D.Y. Wang using high-throughput amplicon sequencing of the full-length 16S rRNA (V1-V9 hypervariable) and short fragment ITS region. The total DNA from 12 root samples were extracted, amplified, sequenced, and analyzed. Resulting sequences were clustered into 61 bacteria and 2128 fungal OTUs. Analysis of community structure revealed that the coralloid roots were dominated mostly by the nitrogen-fixer Nostocaceae but also contain other non-diazotrophic bacteria. The sequencing of entire 16S rRNA gene identified four different strains of cyanobacteria under the heterocystous genera Nostoc and Desmonostoc. Meanwhile, the top bacterial families in normal roots were Xanthobacteraceae, Burkholderiaceae, and Bacillaceae. Moreover, a diverse fungal community was also found in the roots of cycads and the predominating families were Ophiocordycipitaceae, Nectriaceae, Bionectriaceae, and Trichocomaceae. Our results demonstrated that bacterial diversity in normal roots of C. fairylakea is higher in richness and abundance than C. debaoensis. On the other hand, a slight difference, albeit insignificant, was noted for the diversity of fungi among root types and host species as the number of shared taxa is relatively high (67%). Our results suggested that diverse microbes are present in roots of cycads which potentially interact together to support cycads survival. Our study provided additional knowledge on the microbial diversity and composition in cycads and thus expanding our current knowledge on cycad-microbe association. Our study also considered the possible impact of ex situ conservation on cyanobiont community of cycads.

A novel cyanobacterial strain (CSML-F035) here described as Stenomitos nagquensis sp. nov. was isolated from a plateau meadow wetland habitat in the Tibet Autonomous Region. The novel strain was studied based on a polyphasic approach by combining characteristics of cell morphology and evidence from molecular data and ecology. Phylogenetic analyses based on the 16S rRNA and rbcL genes indicated that the novel strain is a member of the genus Stenomitos (Leptolyngbyaceae) but is positioned in a unique clade, with strongly supported values from BI, ML, and NJ methods. The percentage similarity of the 16S rRNA gene sequence of CSML-F035 compared with known strains of Stenomitos was >97%. Morphologically, S. nagquensis sp. nov. possesses overlapping morphological characteristics with other Stenomitos species, with thin trichomes enclosed in a firm, colorless, hyaline, persistent mucilage. However, the strain appeared to have slightly smaller cells compared to most known species of Stenomitos. Further analyses of the 16S–23S ITS region showed dissimilarity at 11.2–22.8% compared to known species of Stenomitos. The secondary helices—including D1–D1, V2, Box–B, and V3—that were predicted based on the 16S–23S ITS region for Stenomitos nagquensis sp. nov. were also distinct, thereby providing strong additional support for the separation of this species. This study highlights the importance of unexplored habitats in the Tibet region in which cyanobacterial diversity can be deemed high, with many potential new species that remain poorly explored.

Heterocyte-forming cyanobacteria form symbiotic relationships with several lineages of plants. Here, twenty (20) strains of endosymbiotic cyanobacteria (cyanobionts) with Nostoc-like morphologies were isolated from the highly specialized coralloid roots of five host species in Cycadales—Cycas debaoensis, C. fairylakea, C. elongata, Ceratozamia robusta, and Macrozamia moorei. Molecular phylogeny based on the 16S rRNA gene placed these strains into seven different taxa within the Nostocaceae, specifically under the genera Desmonostoc and Dendronalium. The percent dissimilarity and unique patterns in the secondary structures of the D1-D1′, Box-B, V2, and V3 helices, which were based on the 16S–23S rRNA internal transcribed spacer (ITS) regions, supported three distinct species in Desmonostoc. These three morphologically distinct novel species are described in this report: Desmonostoc debaoense sp. nov., Desmonostoc meilinense sp. nov., and Desmonostoc xianhuense sp. nov. Other investigated strains were phylogenetically identified as members of the recently discovered genus Dendronalium and represent the first report of association of that genus with cycads. Our findings suggest that the order Cycadales hosts diverse species of cyanobionts in their coralloid roots and that many potential unreported or novel taxa are present in cycads occurring in their natural habitat and await discovery.