Cyanobacterial 16S rRNA Research Articles

Unexpected cyanobacterial communities in highly heterogeneous toxic blooms from a Mediterranean protected area.

The negative effects associated with cyanobacterial blooms are of particular concern in protected ecosystems, as these areas are ecologically significant and attract a high number of visitors. This study aims to explore the cyanobacterial communities and associated toxicity in three reservoirs located within a Mediterranean National Park with a compromised situation at basin-level. Our results demonstrate the occurrence of dense toxic blooms containing microcystins (reaching values close to 280 μg L-1) and low levels of anatoxin-a and saxitoxins (up to 0.02 μg L-1). Comprehensive metabarcoding analyses based on cyanobacterial 16S rRNA and cyanotoxin-biosynthesis genes (mcyE, anaF and sxtA) unveiled three highly heterogeneous communities, despite the spatial proximity of reservoirs. Additionally, our results suggested the influence of water conductivity on the blooms composition. Among the diverse bloom-forming taxa found, Microcystis sp. and Planktothrix sp. were revealed as the microcystins-producer candidates, and Cuspidothrix issatschenkoi and Dolichospermum/Aphanizomenom sp. as the potential producers of anatoxin-a and saxitoxins, respectively. A polyphasic characterization confirmed the first report of the tropical-related species Planktothrix spiroides in Europe, showing elevated level of dominance. As a whole, we present the scenario of an ecologically important protected area facing significant challenges in the proper management of cyanobacterial blooms.

Biocrust reduces the soil erodibility of coral calcareous sand by regulating microbial community and extracellular polymeric substances on tropical coral island, South China Sea.

Tropical coral islands assume a pivotal role in the conservation of oceanic ecosystem biodiversity. However, their distinctive environmental attributes and limited vegetation render them highly susceptible to soil erosion. The biological soil crust (biocrust), owing to its significant ecological role in soil stabilization and erosion prevention, is deemed an effective means of mitigating soil erosion on coral island. However, existing research on the mechanisms through which biocrusts resist soil erosion has predominantly concentrated on arid and semi-arid regions. Consequently, this study will specifically delve into elucidating the erosion-resistant mechanisms of biocrusts in tropical coral island environments, South China Sea. Specifically, we collected 16 samples of biocrusts and bare soil from Meiji Island. High-throughput amplicon sequencing was executed to analyze the microbial community, including bacteria, fungi, and archaea. Additionally, quantitative PCR was utilized to assess the abundance of the bacterial 16S rRNA, fungal ITS, archaeal 16S rRNA, and cyanobacterial 16S rRNA genes within these samples. Physicochemical measurements and assessments of extracellular polymeric substances (EPSs) were conducted to characterize the soil properties. The study reported a significantly decreased soil erodibility factor after biocrust formation. Compared to bare soil, soil erodibility factor decreased from 0.280 to 0.190 t h MJ-1 mm-1 in the biocrusts. Mechanistically, we measured the microbial EPS contents and revealed a negative correlation between EPS and soil erodibility factor. Consistent with increased EPS, the abundance of bacteria, fungi, archaea, and cyanobacteria were also detected significantly increased with biocrust formation. Correlation analysis detected Cyanobacteria, Chloroflexi, Deinococcota, and Crenarchaeota as potential microbials promoting EPSs and reducing soil erosion. Together, our study presents the evidence that biocrust from tropical coral island in the South China Sea promotes resistance to soil erosion, pinpointing key EPSs-producing microbials against soil erosion. The findings would provide insights for island soil restoration.

Cultivating Resilience in Dryland Soils: An Assisted Migration Approach to Biological Soil Crust Restoration.

Land use practices and climate change have driven substantial soil degradation across global drylands, impacting ecosystem functions and human livelihoods. Biological soil crusts, a common feature of dryland ecosystems, are under extensive exploration for their potential to restore the stability and fertility of degraded soils through the development of inoculants. However, stressful abiotic conditions often result in the failure of inoculation-based restoration in the field and may hinder the long-term success of biocrust restoration efforts. Taking an assisted migration approach, we cultivated biocrust inocula sourced from multiple hot-adapted sites (Mojave and Sonoran Deserts) in an outdoor facility at a cool desert site (Colorado Plateau). In addition to cultivating inoculum from each site, we created an inoculum mixture of biocrust from the Mojave Desert, Sonoran Desert, and Colorado Plateau. We then applied two habitat amelioration treatments to the cultivation site (growth substrate and shading) to enhance soil stability and water availability and reduce UV stress. Using marker gene sequencing, we found that the cultivated mixed inoculum comprised both local- and hot-adapted cyanobacteria at the end of cultivation but had similar cyanobacterial richness as each unmixed inoculum. All cultivated inocula had more cyanobacterial 16S rRNA gene copies and higher cyanobacterial richness when cultivated with a growth substrate and shade. Our work shows that it is possible to field cultivate biocrust inocula sourced from different deserts, but that community composition shifts toward that of the cultivation site unless habitat amelioration is employed. Future assessments of the function of a mixed inoculum in restoration and its resilience in the face of abiotic stressors are needed to determine the relative benefit of assisted migration compared to the challenges and risks of this approach.

Cyanoseq: A database of cyanobacterial 16S rRNA gene sequences with curated taxonomy.

Cyanobacteria are photosynthetic bacteria that occupy various habitats across the globe, playing critical roles in many of Earth's biogeochemical cycles both in both aquatic and terrestrial systems. Despite their well-known significance, their taxonomy remains problematic and is the subject of much research. Taxonomic issues of Cyanobacteria have consequently led to inaccurate curation within known reference databases, ultimately leading to problematic taxonomic assignment during diversity studies. Recent advances in sequencing technologies have increased our ability to characterize and understand microbial communities, leading to the generation of thousands of sequences that require taxonomic assignment. We herein propose CyanoSeq (https://zenodo.org/record/7569105), a database of cyanobacterial 16S rRNA gene sequences with curated taxonomy. The taxonomy of CyanoSeq is based on the current state of cyanobacterial taxonomy, with ranks from the domain to genus level. Files are provided for use with common naive Bayes taxonomic classifiers, such as those included in DADA2 or the QIIME2 platform. Additionally, FASTA files are provided for creation of de novo phylogenetic trees with (near) full-length 16S rRNA gene sequences to determine the phylogenetic relationship of cyanobacterial strains and/or ASV/OTUs. The database currently consists of 5410 cyanobacterial 16S rRNA gene sequences along with 123 Chloroplast, Bacterial, and Vampirovibrionia (formally Melainabacteria) sequences.

Long‐term cyanobacterial dynamics from lake sediment DNA in relation to experimental eutrophication, acidification and climate change

Abstract Cyanobacterial blooms in aquatic environments have impacted ecosystem health, altered food webs and contributed to substantial regional economic losses. The relative impacts of climate change, eutrophication and other environmental stressors on the formation of cyanobacterial blooms remain unclear as a consequence of the lack of long‐term data. The analysis of lake sediment archives can help address such questions. The abundance of target cyanobacterial genes was quantified using droplet digital PCR from two experimental and two reference lakes at the IISD ‐ Experimental Lakes Area, Northwestern Ontario, Canada where various anthropogenic drivers have been manipulated and phytoplankton biomass and taxonomy (via microscopy) tracked over the past 50 years. Based on sediment DNA (sedDNA) records, Lake 227 has seen more than two full orders of magnitude rise in cyanobacterial abundance in response to experimental nutrient (nitrogen and phosphorus) loading since 1969. Lake 223 also experienced a small increase in cyanobacterial abundance during a period of experimental acidification. The sedDNA archives also identified an increase in abundance of mcyE, a gene involved in microcystin synthesis, in Lake 227 after 1990 when experimental nitrogen loading ceased and only phosphorus loading continued. Sequencing of the cyanobacterial 16S rRNA gene from sedDNA revealed a shift towards Nostocales dominance in response to eutrophication in Lake 227; this was also seen in the more recent sediments of the other lakes but to a much lesser extent. When comparing the sedDNA data with historical phytoplankton records of the past ~50 years, moderate‐to‐strong correlations were found between the two. This research validated the use of sedDNA for the analysis of long‐term trends in lakes and the results revealed additional changes that were not recorded from the surface‐water records. Experimental nutrient loading had the greatest influence on the cyanobacterial community in Lake 227. However, climate change, via declines in heating degree days, was also significant in explaining variation in the cyanobacterial communities in the experimental lakes as well as the reference lakes.

Highly-resolved interannual phytoplankton community dynamics of the coastal Northwest Atlantic

Microbial observatories can track phytoplankton at frequencies that resolve monthly, seasonal, and multiyear trends in environmental change from short-lived events. Using 4-years of weekly flow cytometry along with chloroplast and cyanobacterial 16S rRNA gene sequence data from a time-series station in the coastal Northwest Atlantic (Bedford Basin, Nova Scotia, Canada), we analyzed temporal observations for globally-relevant genera (e.g., Bolidomonas, Teleaulax, Minidiscus, Chaetoceros, Synechococcus, and Phaeocystis) in an oceanic region that has been recognized as a likely hotspot for phytoplankton diversity. Contemporaneous Scotian Shelf data also collected during our study established that the major phytoplankton within the Bedford Basin were important in the Scotian Shelf during spring and fall, therefore pointing to their broader significance within the coastal Northwest Atlantic (NWA). Temporal trends revealed a subset of indicator taxa along with their DNA signatures (e.g., Eutreptiella and Synechococcus), whose distribution patterns make them essential for timely detection of environmentally-driven shifts in the NWA. High-resolution sampling was key to identifying important community shifts towards smaller phytoplankton under anomalous environmental conditions, while further providing a detailed molecular view of community compositions underpinning general phytoplankton succession within the coastal NWA. Our study demonstrates the importance of accessible coastal time-series sites where high-frequency DNA sampling allows for the detection of shifting baselines in phytoplankton communities.

Effects of temperature and oxygen on cyanobacterial DNA preservation in sediments: A comparison study of major taxa

AbstractThe analysis of sediment DNA has emerged as a promising alternative to classical paleolimnological proxies, which typically rely on hard fossils to reconstruct environmental change. In recent studies, the timing and causes of apparent increases in toxic cyanobacterial blooms in lakes around the world have been identified by high‐throughput sequencing of cyanobacterial 16S rRNA genes from decadal‐ and centennial‐scale sediment records. However, the reconstruction of ecological time series can be influenced by DNA degradation processes interfering with taxonomic resolution and quantification of target genes. To determine whether some cyanobacterial taxa degrade more rapidly than others, a one‐year incubation experiment was performed to estimate degradation rates in sediments under contrasting environmental conditions of temperature (4°C vs. 25°C) and sediment redox (oxic vs. anoxic). Using sediments collected from an oligotrophic lake, serum bottles were inoculated with either Microcystis aeruginosa, Synechococcus sp., or Trichormus variabilis. These cyanobacteria were chosen because they are widespread yet exhibit contrasting morphologies. The absolute concentrations of the target genes were measured over time through droplet digital PCR. The three taxa exhibited log‐linear declines with different rates of decay. Based on first‐order linear decay models, Microcystis exhibited faster decay under oxic conditions, whereas Synechococcus sp. and Trichormus were both temperature and anoxic dependent. Overall, cyanobacterial decay rates were lowest under 4°C anoxic conditions, corresponding to conditions in deep temperate lakes. Under 4°C, Synechococcus exhibited the lowest degradation rates followed by Trichormus, then Microcystis. These findings will be useful in the interpretation of population and community structure data based on sediment DNA and advances our current understanding of cyanobacterial preservation in sediment.

Cyanobacteria and Algae Meet at the Limits of Their Habitat Ranges in Moderately Acidic Hot Springs

AbstractMicrobial oxygenic photosynthesis in thermal habitats is thought to be performed by Bacteria in circumneutral to alkaline systems (pH > 6) and by Eukarya in acidic systems (pH < 3), yet the predominant oxygenic phototrophs in thermal environments with pH values intermediate to these extremes have received little attention. Sequencing of 16S and 18S rRNA genes was performed on samples from twelve hot springs in Yellowstone National Park (Wyoming, USA) with pH values from 3.0 to 5.5, revealing that Cyanobacteria of the genus Chlorogloeopsis and algae of the genus Cyanidioschyzon (phylum Rhodophyta) coexisted in ten of these springs. Cyanobacterial 16S rRNA genes were more abundant than rhodophyte 18S rRNA genes by 1–7 orders of magnitude, with rhodophyte template abundance approaching that of Cyanobacteria only at the most acidic sites. The ketocarotenoids echinenone and canthaxanthin were identified in samples from all but one spring yielding cyanobacterial sequences and are attributed to pigment synthesis by Cyanobacteria, whereas the absence of detectable chloroplast sequences affiliated with Cyanidioschyzon, pH and temperatures in excess of its limits for growth, and other observations collectively suggested these algae were inactive in many of the springs at the time of sampling. Fluctuations in the supply of meteoric water likely contribute to physicochemical variability in these springs, leading to transitions in microbial community composition. Spatial overlap, but perhaps not temporal overlap, in the habitat ranges of bacterial and eukaryal oxygenic phototrophs indicates that the notion of a sharp transition between these lineages with respect to pH is unwarranted.

Cydrasil 3, a curated 16S rRNA gene reference package and web app for cyanobacterial phylogenetic placement

Cyanobacteria are a widespread and important bacterial phylum, responsible for a significant portion of global carbon and nitrogen fixation. Unfortunately, reliable and accurate automated classification of cyanobacterial 16S rRNA gene sequences is muddled by conflicting systematic frameworks, inconsistent taxonomic definitions (including the phylum itself), and database errors. To address this, we introduce Cydrasil 3 (https://www.cydrasil.org), a curated 16S rRNA gene reference package, database, and web application designed to provide a full phylogenetic perspective for cyanobacterial systematics and routine identification. Cydrasil 3 contains over 1300 manually curated sequences longer than 1100 base pairs and can be used for phylogenetic placement or as a reference sequence set for de novo phylogenetic reconstructions. The web application (utilizing PaPaRA and EPA-ng) can place thousands of sequences into the reference tree and has detailed instructions on how to analyze results. While the Cydrasil web application offers no taxonomic assignments, it instead provides phylogenetic placement, as well as a searchable database with curation notes and metadata, and a mechanism for community feedback.

Comparing Quantitative Methods for Analyzing Sediment DNA Records of Cyanobacteria in Experimental and Reference Lakes.

Sediment DNA (sedDNA) analyses are rapidly emerging as powerful tools for the reconstruction of environmental and evolutionary change. While there are an increasing number of studies using molecular genetic approaches to track changes over time, few studies have compared the coherence between quantitative polymerase chain reaction (PCR) methods and metabarcoding techniques. Primer specificity, bioinformatic analyses, and PCR inhibitors in sediments could affect the quantitative data obtained from these approaches. We compared the performance of droplet digital polymerase chain reaction (ddPCR) and high-throughput sequencing (HTS) for the quantification of target genes of cyanobacteria in lake sediments and tested whether the two techniques similarly reveal expected patterns through time. Absolute concentrations of cyanobacterial 16S rRNA genes were compared between ddPCR and HTS using dated sediment cores collected from two experimental (Lake 227, fertilized since 1969 and Lake 223, acidified from 1976 to 1983) and two reference lakes (Lakes 224 and 442) in the Experimental Lakes Area (ELA), Canada. Relative abundances of Microcystis 16S rRNA (MICR) genes were also compared between the two methods. Moderate to strong positive correlations were found between the molecular approaches among all four cores but results from ddPCR were more consistent with the known history of lake manipulations. A 100-fold increase in ddPCR estimates of cyanobacterial gene abundance beginning in ~1968 occurred in Lake 227, in keeping with experimental addition of nutrients and increase in planktonic cyanobacteria. In contrast, no significant rise in cyanobacterial abundance associated with lake fertilization was observed with HTS. Relative abundances of Microcystis between the two techniques showed moderate to strong levels of coherence in top intervals of the sediment cores. Both ddPCR and HTS approaches are suitable for sedDNA analysis, but studies aiming to quantify absolute abundances from complex environments should consider using ddPCR due to its high tolerance to PCR inhibitors.

Optimized Protocol for Cyanobacterial 16S rRNA Analysis in Danube Delta Lakes

Molecular biology protocols have been more and more accessible to researchers for ecological investigations. However, these protocols always require optimization steps for the analysis of specific types of samples. This study aimed to optimize a molecular protocol to analyze cyanobacterial 16S rRNA in Danube Delta shallows lakes. In this regard, several commercial DNA extraction kits were tested compared to the potassium ethyl xanthogenate extraction method on different matrices. The obtained DNA was further used for 16S rRNA PCR optimization. Finally, an optimized protocol is proposed for the molecular analysis of the cyanobacteria group in freshwater samples. The best DNA extraction method was the potassium xanthogenate extraction from dried cyanobacterial biomass. A dynamic in total genomic eDNA was observed, reflecting the seasonal difference in phytoplankton biomass from the studied lakes. The PCR protocol optimized by us can be successfully applied for the identification of a broad range of cyanobacterial genetic markers.

Microbial inoculation elicited changes in phyllosphere microbial communities and host immunity suppress Magnaporthe oryzae in a susceptible rice cultivar

The hemibiotrophic fungal pathogen Magnaporthe oryzae causes serious crop losses in rice. As phyllospheric microbes share a common habitat with this foliar pathogen, they can be deployed as the change-agents for engineering desired phyllospheric microbial communities to inhibit the disease progression. Comparative evaluation of a set of native microbes, when applied as foliar spray or soil drench was undertaken, showed significant interactive effects on the concentrations of chlorophyll a and b, and carotenoids. A significant enhancement was recorded in the activities of chitosanase, β-1,3-endoglucanase and β-1,4-endoglucanase (CMCase) in the disease challenged plants, as compared to healthy plants. Among the treatments, Bacillus sp. (B1) inoculation recorded lowest values of the activities of all three hydrolytic enzymes, while Bacillus sp. (B4) and Nostoc-Anabaena consortium (C1) led to 30–60% decreases, both as foliar and soil drench modes of application. Distinct changes in the abundances of eubacterial and phylum Cyanobacterial 16S rRNA gene copies, nifH and bacterial amoA illustrated the significance of foliar over the soil drench method. The nifH gene copies were significantly higher due to the foliar method of Calothrix sp. (C2) inoculation, and values were significantly at par values with both the soil drench application of B1 (Bacillus sp.) and C1 (Nostoc-Anabaena consortium) treatments. This study illustrates the significance of the root-shoot linkages in the effective biocontrol of these promising, indigenous microbes, applied as soil drench or foliar agents, which can be useful in abating the incidence of the fungal pathogen in an environment –friendly manner.

Network analysis reveals succession of Microcystis genotypes accompanying distinctive microbial modules with recurrent patterns

Every member of the ecological community is connected via a network of vital and complex relationships, called the web of life. To elucidate the ecological network and interactions among producers, consumers, and decomposers in the Daechung Reservoir, Korea, during cyanobacterial harmful algal blooms (cyanoHAB), especially those involving Microcystis, we investigated the diversity and compositions of the cyanobacterial (16S rRNA gene), including the genotypes of Microcystis (cpcBA-IGS gene), non-cyanobacterial (16S), and eukaryotic (18S) communities through high-throughput sequencing. Microcystis blooms were divided into the Summer Major Bloom and Autumn Minor Bloom with different dominant genotypes of Microcystis. Network analysis demonstrated that the modules involved in the different phases of the Microcystis blooms were categorized into the Pre-Bloom, Bloom, Post-Bloom, and Non-Bloom Groups at all sampling stations. In addition, the non-cyanobacterial components of each Group were classified, while the same Group showed similarity across all stations, suggesting that Microcystis and other microbes were highly interdependent and organized into cyanoHAB-related module units. Importantly, the Microcystis genotype-based sub-network uncovered that Pirellula, Pseudanabaena, and Vampirovibrionales preferred to interact with specific Microcystis genotypes in the Summer Major Bloom than with other genotypes in the Autumn Minor Bloom, while the copepod Skistodiaptomus exhibited the opposite pattern. In conclusion, the transition patterns of cyanoHAB-related modules and their key components could be crucial in the succession of Microcystis genotypes and to enhance the understanding of microbial ecology in an aquatic environment.

Comparison of Microphototrophic Communities Living in Different Soil Environments in the High Arctic

The Arctic region undergoes rapid climate change resulting in soil warming with consequent changes in microbial community structure. Therefore, it is important to gain more knowledge on the pioneer photosynthetic microorganisms and their relations to environmental factors. Here we provide a description of the community composition of microbial phototrophs in three different types of soils in the High Arctic (Svalbard): vegetated soil at a raised marine terrace, biological soil crust (BSC) at high elevation, and poorly-developed BSC in a glacier foreland. The studied sites differed from each other in microclimatic conditions (soil temperature and soil water content), soil chemistry and altitude. Combining morphological (cell biovolume) and molecular methods (NGS amplicon sequencing of cyanobacterial 16S rRNA and eukaryotic 18S rRNA sequences of isolates), we studied the diversity and biovolume of cyanobacteria and eukaryotic microalgae. The results showed that cyanobacteria prevailed in the high altitude BSC as well as in pioneering BSC samples in glacier foreland though with lower biomass. More specifically, filamentous cyanobacteria, mainly Leptolyngbya spp., dominated the BSCs from these two localities. In contrast, coccoid microalgae (green and yellow-green algae) had higher biovolume in low altitude vegetated soils. Thus, the results of this study contribute to a better understanding of microphototrophic communities in different types of Arctic soil environments.

Deciphering the effects of nitrogen, phosphorus, and temperature on cyanobacterial bloom intensification, diversity, and toxicity in western Lake Erie

AbstractAlthough cyanobacterial harmful algal blooms (CHABs) are promoted by nutrient loading and elevated temperatures, the effects of these processes on bloom diversity are unclear. This study used traditional and next‐generation sequencing approaches to assess shifts in phytoplankton, cyanobacterial (16S rRNA), and microcystin‐producing (mcyE) communities during CHABs in western Lake Erie (Maumee and Sandusky Bays) in response to natural and experimental gradients of nitrogen (N), phosphorus (P), and temperature. CHABs were most intense near the Maumee and Sandusky Rivers and were dominated by Microcystis and Planktothrix, respectively. Sequencing of 16S amplicons revealed cryptic cyanobacterial diversity (47 genera) including high abundances of two distinct clades of Synechococcus in both bays and significant differences in community structure between nutrient‐rich nearshore sites and less eutrophic offshore sites. Sequencing of mcyE genes revealed low taxonomic (n = 3) but high genetic diversity (n = 807), with toxigenic strains of Planktothrix being more abundant than Microcystis and more closely paralleling microcystin concentrations. Cyanobacterial abundance significantly increased in response to elevated N, with the greatest increases in combined high N, P, and temperature treatments that concurrently suppressed green and brown algae. N significantly increased microcystin concentrations and the relative abundance of nondiazotrophic genera such as Planktothrix, while diazotrophic genera such as Dolichospermum and Aphanizomenon were, in some cases, enhanced by high P and temperature. While nutrients and elevated temperatures promote CHABs, differing combinations selectively promote individual cyanobacterial genera and strains, indicating management of both N and P will be required to control all cyanobacteria in Lake Erie, particularly as lake temperatures rise.

Contrasting histories of microcystin-producing cyanobacteria in two temperate lakes as inferred from quantitative sediment DNA analyses

Pilon S, Zastepa A, Taranu ZE, Gregory-Eaves I, Racine M, Blais JM, Poulain AJ, Pick FR. 2018. Contrasting histories of microcystin-producing cyanobacteria in two temperate lakes as inferred from quantitative sediment DNA analyses. Lake Reserv Manage. 35:102–117.The incidence and abundance of microcystin-producing cyanobacteria were compared between 2 temperate lakes over the past ∼150–175 yr by combining analyses of sediment DNA (qPCR of 4 target genes and high-throughput sequencing of a cyanobacterial 16S rRNA gene fragment) and sediment microcystin concentrations. In Lake of the Woods (LoW), toxic cyanobacteria were only detected beginning in the early 1980s based on gene copy numbers of the microcystin-synthesizing gene (mcyD), and by the early 2000s based on sediment microcystins; both these proxies for toxic cyanobacteria were significantly correlated. In contrast, in Baptiste Lake (Alberta), both mcyD gene copy numbers and microcystins were detected as far back as ∼1830, before European settlement of the region. Total sediment microcystins in this lake were not correlated with mcyD gene copy numbers, but the latter correlated with 2 other cyanobacterial genes targeted. These results point to a relatively recent emergence and increasing dominance of microcystin-producing cyanobacteria in LoW that coincides with the documented rise in regional temperatures rather than changes in nutrient loading. In contrast, toxic blooms are not a new phenomenon in Baptiste Lake, as microcystin-producing cyanobacteria were clearly part of the ecosystem prior to anthropogenic perturbations; this lake has also experienced a recent increase in microcystins coincident with further nutrient enrichment. High-throughput sequencing of the cyanobacterial 16S rRNA gene indicated that bloom-forming and potentially toxigenic Dolichospermum (including related Nostocales) and Microcystis were present but the former has been the dominant genus over the past 175 yr.

Development and Application of a Quantitative PCR Assay to Assess Genotype Dynamics and Anatoxin Content in Microcoleus autumnalis-Dominated Mats.

Microcoleus is a filamentous cyanobacteria genus with a global distribution. Some species form thick, cohesive mats over large areas of the benthos in rivers and lakes. In New Zealand Microcoleus autumnalis is an anatoxin producer and benthic proliferations are occurring in an increasing number of rivers nationwide. Anatoxin content in M. autumnalis-dominated mats varies spatially and temporally, making understanding and managing proliferations difficult. In this study a M. autumnalis-specific TaqMan probe quantitative PCR (qPCR) assay targeting the anaC gene was developed. The assay was assessed against 26 non-M. autumnalis species. The assay had a detection range over seven orders of magnitude, with a limit of detection of 5.14 × 10−8 ng μL−1. The anaC assay and a cyanobacterial specific 16S rRNA qPCR were then used to determine toxic genotype proportions in 122 environmental samples collected from 19 sites on 10 rivers in New Zealand. Anatoxin contents of the samples were determined using LC-MS/MS and anatoxin quota per toxic cell calculated. The percentage of toxic cells ranged from 0 to 30.3%, with significant (p < 0.05) differences among rivers. The anatoxin content in mats had a significant relationship with the percentage of toxic cells (R2 = 0.38, p < 0.001), indicating that changes in anatoxin content in M. autumnalis-dominated mats are primarily related to the dominance of toxic strains. When applied to more extensive samples sets the assay will enable new insights into how biotic and abiotic parameters influence genotype composition, and if applied to RNA assist in understanding anatoxin production.

Interactive effects of Magnaporthe inoculation and nitrogen doses on the plant enzyme machinery and phyllosphere microbiome of resistant and susceptible rice cultivars.

Severity of plant diseases is often influenced by the availability of nutrients, particularly N; however, its effect on the phyllosphere microbiome in foliar pathogen challenged plants is less investigated in rice. The tripartite interaction among the fungal pathogen (Magnaporthe oryzae), rice cultivars (basmati and non-basmati, blast resistant or susceptible) and nitrogen (N) fertilization (0, 120 and 180N) was investigated. Plant growth, elicitation of defense responses and abundance of microbial members in the rice phyllosphere were monitored using biochemical and molecular methods. In general, photosynthetic pigments were distinct for each cultivar, and optimal N doses led to higher values. The susceptible var. CO-39 and resistant CO-39I exhibited higher contents of photosynthetic pigments and micronutrients such as zinc in leaves in response to N doses. Elicitation of defense and hydrolytic enzymes was significantly influenced by pathogen inoculation and modulated by N doses, but varietal effects were distinct. Scoring indices emphasized the pathogen susceptibility of var. CO-39 and PB-1, which showed almost 40-60% higher values than the resistant cultivars; the interactions of cultivars and N doses was also significant. Characteristic changes were recorded in the abundances of the gene copies, particularly, with an overall increase in the number of cyanobacterial 16S rRNA, and bacterial amoA in pathogen-challenged treatments, while nifH gene copies exhibited a reducing trend with increasing N doses, in the presence or absence of pathogen. The varietal differences in the cause and effect relationships can be valuable in crop protection for more effective foliar application of pesticides or biocontrol agents.

Distribution patterns of marine planktonic cyanobacterial assemblages in transitional marine habitats using 16S rRNA phylogeny

SUMMARYThe community composition of marine planktonic cyanobacteria in transitional marine habitats can influence its overall contribution to aquatic primary production. To understand distribution patterns of marine planktonic cyanobacterial assemblages, phylogenetic and statistical analyses were undertaken on planktonic cyanobacterial 16S rRNA gene sequences from four transitional marine habitats [Baltic Sea (BL), Monterey Bay (MB), South China Sea (SCS) and Sundarbans (SB)]. Out of 3255 sequences analyzed, only 546 sequences were found to be planktonic cyanobacteria and were considered in this study. Among these, 338 sequences representative of Sundarbans, the world's largest mangrove were generated based on Sanger and Illumina sequencing approaches. Based on 16S rRNA phylogeny, four major taxonomic orders of marine planktonic cyanobacteria were recovered in varying proportions with several novel 16S rRNA sequences in each of the four targeted sites. Members of the order Synechococcales were dominant in all the sites (−94% sequences) while the orders Chroococcales and Oscillatoriales were only detected in SB and SCS sites, respectively. In the phylogenetic tree, sequences representing the major marine picocyanobacterial genus Synechococcus showed overwhelming dominance in SB and they were found in three other sites. Prochlorococcus‐like sequences were found in sizeable number in MB and SCS but were absent in SB and coastal BL. Synechococcus‐like sequences were represented by three major marine clusters (5.1, 5.2, and 5.3). Three novel clades as part of Synechococcus cluster were detected only in SB and one novel clade in BL. The majority of OTUs were found to be exclusive to each site, whereas some were shared by two or more sites as revealed by beta‐diversity analysis.

First report of anatoxin-a encoding gene in isolated cyanobacterial strains from Malaysia

Aims: This study focus on the presence of cyanobacterial toxin in Malaysia and anatoxin-a-encoding gene was detected in this study and the status of cyanobacterial toxins in Malaysia can now be clarified. Methodology and results: As part of status determination of cyanobacterial toxins in Malaysia, cyanobacterial strains have been isolated from different environments and identified using cyanobacterial 16S rRNA gene sequence. PCR assay was carried out to detect the presence of cyanobacterial toxin-encoding genes in these isolated strains by amplifying genes encoded for microcystin, anatoxin-a, cylindrospermopsin and saxitoxin. Using molecular identification of 16S rRNA gene sequences, a total of forty-two cyanobacterial strains were identified, which belongs to eighteen different genera of Synechococcus, Cyanobium, Synechocystis, Chroococcidiopsis, Leptolyngbya, Nodosilinea, Limnothrix, Pseudanabaena, Cephalothrix, Aerosakkonema, Oscillatoria, Alkalinema, Pantanalinema, Planktolyngbya, Scytonema, Nostoc, Hapalosiphon and Symphyonemopsis. The toxicity of these strains was tested using PCR amplification of toxin-encoding genes using specific primers. Conclusion, significance and impact of study: Anatoxin-a (ATX) gene, which involved in the biosynthesis of anatoxin-A was detected in two isolated strains namely Limnothrix sp. B15 and Leptolyngbya sp. D1C10. This study focus on the the presence of cyanobacterial toxin in Malaysia can now be determined as potential threat because anatoxin-a-encoding gene was detected in this study and the status of cyanobacterial toxins in Malaysia can now be clarified. © 2018, Universiti Sains Malaysia.