Structure and Biosynthesis of Desmamides A-C, Lipoglycopeptides from the Endophytic Cyanobacterium Desmonostoc muscorum LEGE 12446.

Certain cyanobacteria of the secondary metabolite-rich order Nostocales can establish permanent symbioses with a large number of cycads, by accumulating in their coralloid roots and shifting their metabolism to dinitrogen fixation. Here, we report the discovery of two new lipoglycopeptides, desmamides A (1) and B (2), together with their aglycone desmamide C (3), from the nostocalean cyanobacterium Desmonostoc muscorum LEGE 12446 isolated from a cycad (Cycas revoluta) coralloid root. The chemical structures of the compounds were elucidated using a combination of 1D and 2D NMR spectroscopy and mass spectrometry. The desmamides are decapeptides featuring O-glycosylation of tyrosine (in 1 and 2) and an unusual 3,5-dihydroxy-2-methyldecanoic acid residue. The biosynthesis of the desmamides was studied by substrate incubation experiments and bioinformatics. We describe herein the dsm biosynthetic gene cluster and propose it to be associated with desmamide production. The discovery of this class of very abundant (>1.5% d.w.) bacterial lipoglycopeptides paves the way for exploration of their potential role in root endosymbiosis.

Niche differentiation rather than biogeography shapes the diversity and composition of microbiome of Cycas panzhihuaensis.

BACKGROUND: Given their adaptation to nutrient-poor and drought environments, cycads are vital models for plant-microbiome interaction research because they are likely to host an important reservoir of beneficial microbes that may support cycad survival. However, a comprehensive understanding of the diversity and community composition of microbiome associated with different plant compartments as well as bulk soils of cycad species remains elusive. METHOD: An extensive investigation of species diversity and community composition of bacterial and fungal microbiome in roots, seeds, unfertilized seeds, ovules, pollens, and soils of Cycas panzhihuaensis L. Zhou & S. Y. Yang has been conducted by high-through sequencing technology. Moreover, principal component analysis (PCA), hierarchical cluster analysis (HCA), and heatmap analysis were applied to test the niche-specific effect and biogeography factor among different sample types of this cycad species. RESULTS: Highly diverse microbiota and significant variation of community structure were found among different compartments of C. panzhihuaensis. Soils exhibited a remarkable differentiation of bacterial community composition compared to the other five plant organs as revealed by PCA, HCA, and heatmap analyses. Different compartments possessed unique core microbial taxa with Pseudomonadaceae and Nectriaceae shared among them. According to the indicator species analysis, there was almost no differentiation of dominant microbiomes with regard to the geography of the host cycad. Two main transmission models existed in the C. panzhihuaensis. CONCLUSIONS: Each sample type represented a unique niche and hosted a niche-specific core microbial taxa. Contrary to previous surveys, biogeography hardly exerted impact on microbial community variation in this study. The majority of the cycad-associated microbes were horizontally derived from soils and/or air environments with the rest vertically inherited from maternal plants via seeds. This study offers a robust knowledge of plant-microbiome interaction across various plant compartments and soils and lends guidelines to the investigation of adaptation mechanism of cycads in arid and nutrient-poor environments as well as their evolutionary conservation.

Highly diverse endophytes in roots of Cycas bifida (Cycadaceae), an ancient but endangered gymnosperm.

As an ancient seed plant, cycads are one of the few gymnosperms that develop a root symbiosis with cyanobacteria, which has allowed cycads to cope with harsh geologic and climatic conditions during the evolutionary process. However, the endophytic microbes in cycad roots remain poorly identified. In this study, using next-generation sequencing techniques, we investigated the microbial diversity and composition of both the coralloid and regular roots of Cycas bifida (Dyer) K.D. Hill. Highly diverse endophytic communities were observed in both the coralloid and regular roots. Of the associated bacteria, the top five families were the Nostocaceae, Sinobacteraceae, Bradyrhizobiaceae, Bacillaceae, and Hyphomicrobiaceae. The Nectriaceae, Trichocomaceae, and Incertae sedis were the predominant fungal families in all root samples. A significant difference in the endophytic bacterial community was detected between coralloid roots and regular roots, but no difference was observed between the fungal communities in the two root types. Cyanobacteria were more dominant in coralloid roots than in regular roots. The divergence of cycad root structures and the modified physiological processes may have contributed to the abundance of cyanobionts in coralloid roots. Consequently, the colonization of cyanobacteria inhibits the assemblage of other endophytes. Our results contribute to an understanding of the species diversity and composition of the cycad-endophyte microbiome and provide an abbreviated list of potential ecological roles of the core microbes present.

Genetic diversity of symbiotic cyanobacteria in Cycas revoluta (Cycadaceae).

The diversity of cyanobacterial species within the coralloid roots of an individual and populations of Cycas revoluta was investigated based on 16S rRNA gene sequences. Sixty-six coralloid roots were collected from nine natural populations of cycads on Kyushu and the Ryukyu Islands, covering the entire distribution range of the species. Approximately 400 bp of the 5'-end of 16S rRNA genes was amplified, and each was identified by denaturing gradient gel electrophoresis. Most coralloid roots harbored only one cyanobiont, Nostoc, whereas some contained two or three, representing cyanobiont diversity within a single coralloid root isolated from a natural habitat. Genotypes of Nostoc within a natural population were occasionally highly diverged and lacked DNA sequence similarity, implying genetic divergence of Nostoc. On the other hand, Nostoc genotypes showed no phylogeographic structure across the distribution range, while host cycads exhibited distinct north-south differentiation. Cycads may exist in symbiosis with either single or multiple Nostoc strains in natural soil habitats.

Comparative analysis of cyanobacteria in the rhizosphere and as endosymbionts of cycads in drought-affected soils.

Does the diversity of cyanobacteria in the cycad rhizosphere relate to the cyanobiont species found in the coralloid roots of these ancient plants? The aim of this study was to identify the diversity of soil cyanobacteria occurring in the immediate vicinity of 22 colonized coralloid roots belonging to members of the cycad genera: Macrozamia, Lepidozamia, Bowenia and Cycas. The majority of coralloid roots were sampled at depths > 10 cm below the soil surface. A total of 32 cyanobacterial isolates were cultured and their 16S rRNA gene partially sequenced. Phylogenetic analysis revealed nine operational taxonomic units of soil cyanobacteria comprising 30 Nostoc spp., a Tolypothrix sp. and a Leptolyngbya sp. Microscopy indicated that all isolates were unialgal and confirmed their genus identity. Rhizospheric diversity was compared to existing data on cyanobionts isolated at the same time from the cycad coralloid root. The same isolate was present in both the cycad coralloid root and rhizosphere at only six sites. Phylogenetic evidence indicates that most rhizosphere isolates were distinct from root cyanobionts. This weak relationship between the soil cyanobacteria and cycad cyanobionts might indicate that changes in the soil community composition are due to environmental factors.

Influence of hydraulic retention time in a two-phase upflow anaerobic sludge blanket reactor treating textile dyeing effluent using sago effluent as the co-substrate.

PURPOSE: Textile dyeing and sago industries are the most polluting industries in South India, especially in industrial cities like Salem, Tamil Nadu, where textile dyeing and sago industries are clumped together geographically. Conventional physicochemical treatment followed by biological processes for the effluent generated from these industries are ineffective, costlier and produce huge quantities of hazardous sludge and harmful by-products which requires further treatment and safe disposal. Hence, the development of an alternative treatment method will become important. The main objective of this investigation is to establish a sustainable biotreatment technology for the treatment of textile dyeing effluent using sago effluent as co-substrate in a two-phase upflow anaerobic sludge blanket (UASB) reactor. METHODS: In this study, influence of hydraulic retention time (HRT) in a two-phase UASB reactor treating textile dyeing effluent using sago effluent as co-substrate was investigated with different HRTs (36, 30, 24 and 18 h) with an optimum mixing ratio of 70:30 (sago to textile dye wastewaters). RESULTS: The results revealed that the HRT had a high influence on the chemical oxygen demand (COD) and colour removal. The maximum COD removal efficiency of 39.4% and 88.5% and colour removal efficiency of 43.7% and 84.4% in the acidogenic and methanogenic reactors, respectively was achieved at 24 h of HRT. The biogas production was 312 L/day. CONCLUSION: The biphasic UASB reactor could be a very feasible alternative, cost-effective, eco-friendly and sustainable treatment system for textile dyeing effluent with sago effluent as a co-substrate.

Cycas micronesica (Cycadales) plants devoid of endophytic cyanobacteria increase in beta-methylamino-L-alanine.

Cycads are among the most ancient of extant Spermatophytes, and are known for their pharmacologically active compounds. beta-methylamino-l-alanine (BMAA) is one metabolite that been implicated as causal of human neurodegenerative diseases in Guam. We grew Cycas micronesica seedlings without endophytic cyanobacteria symbiosis, and quantified initial and ending BMAA in various plant tissues. BMAA increased 79% during nine months of seedling growth, and root tissue contained 75% of the ultimate BMAA pool. Endophytic cyanobacteria symbionts were not the source of BMAA increase in these seedlings, which contradicts previously reported claims that biosynthesis of this toxin by cyanobacteria initiates its accumulation in the Guam environment. The preferential loading of root tissue with BMAA does not support earlier reports that this toxin serves a defensive role against herbivory of leaf or seed tissues. The long history of conflicting results in Guam's cycad toxin research continues, and recent developments underscore the sense of urgency in continued research as this endangered cycad population approaches extirpation from the island.

Cyanobacterial chemotaxis to extracts of host and nonhost plants.

Chemotaxis may be important when forming cyanobacterial symbioses. However, knowledge of cyanobacterial attraction towards plants and factors affecting chemotaxis is limited. Chemo-attraction was observed in Nostoc strains 8964:3 and PCC 73102 towards exudate or crushed extract of the natural hosts Gunnera manicata, Cycas revoluta and Blasia pusilla, and the nonhost plants Trifolium repens, Arabidopsis thaliana and Oryza sativa. As all tested plant extracts generated chemotaxis, the possibility to attract cyanobacteria may be widespread in plants. Chemotaxis was reduced by increased temperature and darkness and was stimulated by phosphorous and iron starvation and elevated salt concentration. Sugars (arabinose, galactose, and glucose) had a positive effect on chemotaxis, whereas flavonoids (chrysin and naringenin) and amino acids (methionine, glycine, serine, phenylalanine, glutamine, and lysine) had no effect.

Return of the cycad hypothesis - does the amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC) of Guam have new implications for global health?

Recently published work provides evidence in support of the cycad hypothesis for Lytico--Bodig, the Guamanian amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC), based on a new understanding of Chamorro food practices, a cyanobacterial origin of beta-methylaminoalanine (BMAA) in cycad tissue, and a possible mechanism of biomagnification of this neurotoxic amino acid in the food chain. BMAA is one of two cycad chemicals with known neurotoxic properties (the other is cycasin, a proven developmental neurotoxin) among the many substances that exist in these highly poisonous plants, the seeds of which are used by Chamorros for food and medicine. The traditional diet includes the fruit bat, a species that feeds on cycad seed components and reportedly bioaccumulates BMAA. Plant and animal proteins provide a previously unrecognized reservoir for the slow release of this toxin. BMAA is reported in the brain tissue of Guam patients and early data suggest that some Northern American patients dying of Alzheimer's disease (AD) have detectable brain levels of BMAA. The possible role of cyanobacterial toxicity in sporadic neurodegenerative disease is therefore worthy of consideration. Recent neuropathology studies of ALS/PDC confirm understanding of this disorder as a 'tangle' disease, based on variable anatomical burden, and showing biochemical characteristics of 'AD-like' combined 3R and 4R tau species. This model mirrors the emerging view that other neurodegenerative disease spectra comprise clusters of related syndromes, owing to common molecular pathology, with variable anatomical distribution in the nervous system giving rise to different clinical phenotypes. Evidence for 'ubiquitin-only' inclusions in ALS/PDC is weak. Similarly, although there is evidence for alpha-synucleinopathy in ALS/PDC, the parkinsonian component of the disease is not caused by Lewy body disease. The spectrum of sporadic AD includes involvement of the substantia nigra and a high prevalence of 'incidental'alpha-synucleinopathy in sporadic AD is reported. Therefore the pathogenesis of Lytico-Bodig appears still to have most pertinence to the ongoing investigation of the pathogenesis of AD and other tauopathies.

A mechanism for slow release of biomagnified cyanobacterial neurotoxins and neurodegenerative disease in Guam.

As root symbionts of cycad trees, cyanobacteria of the genus Nostoc produce beta-methylamino-l-alanine (BMAA), a neurotoxic nonprotein amino acid. The biomagnification of BMAA through the Guam ecosystem fits a classic triangle of increasing concentrations of toxic compounds up the food chain. However, because BMAA is polar and nonlipophilic, a mechanism for its biomagnification through increasing trophic levels has been unclear. We report that BMAA occurs not only as a free amino acid in the Guam ecosystem but also can be released from a bound form by acid hydrolysis. After first removing free amino acids from tissue samples of various trophic levels (cyanobacteria, root symbioses, cycad seeds, cycad flour, flying foxes eaten by the Chamorro people, and brain tissues of Chamorros who died from amyotrophic lateral sclerosis/Parkinsonism dementia complex), we then hydrolyzed the remaining fraction and found BMAA concentrations increased 10- to 240-fold. This bound form of BMAA may function as an endogenous neurotoxic reservoir, accumulating and being transported between trophic levels and subsequently being released during digestion and protein metabolism. Within brain tissues, the endogenous neurotoxic reservoir can slowly release free BMAA, thereby causing incipient and recurrent neurological damage over years or even decades, which may explain the observed long latency period for neurological disease onset among the Chamorro people. The presence of BMAA in brain tissues from Canadian patients who died of Alzheimer's disease suggests that exposure to cyanobacterial neurotoxins occurs outside of Guam.

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.