No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Nucleic Acid-Based Rapid Detection of Plant-Associated Cyanobacteria
Abstract
Plant-associated cyanobacteria play pivotal roles in diverse ecosystems, impacting plant health, nitrogen fixation, and overall ecosystem dynamics. The rapid and accurate detection of cyanobacteria within plant environments is crucial for understanding their ecological roles and managing associated risks. This chapter provides an overview of nucleic acid-based methods for the rapid detection of cyanobacteria associated with plants. Four distinct techniques, polymerase chain reaction (PCR), quantitative PCR (qPCR), loop-mediated isothermal amplification (LAMP), and metagenomic sequencing are discussed. Each technique’s stepwise procedures, associated materials, and reagents are explained. By employing these techniques, the study aims to expedite the identification and characterization of plant-associated cyanobacteria, contributing to advancements in understanding their role in plant health and ecosystem dynamics.
ResearchGate has not been able to resolve any citations for this publication.
The Next Generation Sequences (NGS) is one of the established metabarcoding methods for analyzing microbial communities. Investigation of the cyanobacterial community in eutrophic freshwater habitats is important, especially for those known as toxic-released cyanobacteria. This study aimed to analyze the structure community of cyanobacteria in Agung and Sunter Barat Lakes in North Jakarta, DKI Jakarta Province using the NGS method. The physico-chemical parameters were also measured. Sampling plots were selected by purposive sampling method. The result of the study successfully analyzed the bacterial community structure and portrayed the dominancy of the cyanobacterial population on the two lakes. Several cyanobacteria genera previously reported in 2008 research were found, including Arthrospira and Planktothrix. The diversity index in Agung Lake (4.7) was higher than in Sunter Barat Lake (3.7). Two dominant populations were found, which were Raphidiopsis (in Agung Lake) and Planktothrix (in Sunter Barat Lake). Both genera have been acknowledged as toxin-releasing cyanobacteria, which could be harmful to the water-biotic community as well as human health. The presence of potentially toxic cyanobacteria in the recreation area of Agung Lake should be taken into consideration for biomonitoring management. Keywords: biomonitoring, cyanobacteria, metabarcoding, microbial community, Sunter Lake
Increasing the accuracy of pathogen identification and reducing the duration of analysis remain relevant for modern molecular diagnostics up to this day. In laboratory and clinical practice, detection of pathogens mostly relies on methods of nucleic acid amplification, among which the polymerase chain reaction (PCR) is considered the "gold standard." Nevertheless, in some cases, isothermal amplification methods act as an alternative to PCR diagnostics. Upon more than thirty years of the development of isothermal DNA synthesis, the appearance of loop-mediated isothermal amplification (LAMP) has enabled new directions of in-field diagnostics of bacterial and viral infections. This review examines the key characteristics of the LAMP method and corresponding features in practice. We discuss the structure of LAMP amplicons with single-stranded loops, which have the sites for primer annealing under isothermal conditions. The latest achievements in the modification of the LAMP method are analyzed, which allow considering it as a unique platform for creating the next-generation diagnostic assays.
The taxonomic identification of organisms based on the amplification of specific genetic markers (metabarcoding) implicitly requires adequate discriminatory information and taxonomic coverage of environmental DNA sequences in taxonomic databases. These requirements were quantitatively examined by comparing the determination of cyanobacteria and microalgae obtained by metabarcoding and light microscopy. We used planktic and biofilm samples collected in 37 lakes and 22 rivers across the Alpine region. We focused on two of the most used and best represented genetic markers in the reference databases, namely the 16S rRNA and 18S rRNA genes. A sequence gap analysis using blastn showed that, in the identity range of 99–100%, approximately 30% (plankton) and 60% (biofilm) of the sequences did not find any close counterpart in the reference databases (NCBI GenBank). Similarly, a taxonomic gap analysis showed that approximately 50% of the cyanobacterial and eukaryotic microalgal species identified by light microscopy were not represented in the reference databases. In both cases, the magnitude of the gaps differed between the major taxonomic groups. Even considering the species determined under the microscope and represented in the reference databases, 22% and 26% were still not included in the results obtained by the blastn at percentage levels of identity ≥95% and ≥97%, respectively. The main causes were the absence of matching sequences due to amplification and/or sequencing failure and potential misidentification in the microscopy step. Our results quantitatively demonstrated that in metabarcoding the main obstacles in the classification of 16S rRNA and 18S rRNA sequences and interpretation of high-throughput sequencing biomonitoring data were due to the existence of important gaps in the taxonomic completeness of the reference databases and the short length of reads. The study focused on the Alpine region, but the extent of the gaps could be much greater in other less investigated geographic areas.
Recurrence and severity of harmful algal blooms (HABs) are increasing due to a number of factors, including human practices and climate change. Sensitive and robust methods that allow for early and expedited HAB detection across large landscape scales are needed. Among the suite of HAB detection tools available, a powerful option exists in genetics-based approaches utilizing environmental sampling, also termed environmental DNA (eDNA). Here we provide a detailed methodological review of three HAB eDNA approaches (quantitative PCR, high throughput sequencing, and isothermal amplification). We then summarize and synthesize recently published eDNA applications covering a variety of HAB surveillance and research objectives, all with a specific emphasis in the detection of two widely problematic freshwater species, Microcystis aeruginosa and Prymnesium parvum. In our summary and conclusion we build on this literature by discussing ways in which eDNA methods could be advanced to improve HAB detection. We also discuss ways in which eDNA data could be used to potentially provide novel insight into the ecology, mitigation, and prediction of HABs.
SYBR Gold is a commonly used and particularly bright fluorescent DNA stain, however, its chemical structure is unknown and its binding mode to DNA remains controversial. Here, we solve the structure of SYBR Gold by NMR and mass spectrometry to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium] and determine its extinction coefficient. We quantitate SYBR Gold binding to DNA using two complementary approaches. First, we use single-molecule magnetic tweezers (MT) to determine the effects of SYBR Gold binding on DNA length and twist. The MT assay reveals systematic lengthening and unwinding of DNA by 19.1° ± 0.7° per molecule upon binding, consistent with intercalation, similar to the related dye SYBR Green I. We complement the MT data with spectroscopic characterization of SYBR Gold. The data are well described by a global binding model for dye concentrations ≤2.5 μM, with parameters that quantitatively agree with the MT results. The fluorescence increases linearly with the number of intercalated SYBR Gold molecules up to dye concentrations of ∼2.5 μM, where quenching and inner filter effects become relevant. In summary, we provide a mechanistic understanding of DNA-SYBR Gold interactions and present practical guidelines for optimal DNA detection and quantitative DNA sensing applications using SYBR Gold.
The detection and monitoring of Vibrio parahaemolyticus pathogen in aquatic foods have become essential for preventing outbreaks. In this study, loop-mediated isothermal amplification (LAMP) assay with the azo dye, hydroxynaphthol blue (HNB) was developed targeting species-specific tlh gene. The assay was carried out on 62 seafood samples that included clam and shrimp and compared with conventional LAMP assay performed with the commonly used fluorescent dye, conventional PCR, and real-time PCR (RT-PCR). Of 62 samples studied for tlh gene, 32 (51.61%) gave positive by HNB-LAMP, which comprised 22 (70.96%) clam samples and 10 (32.25%) shrimp samples. The HNB-LAMP assay was found to be highly sensitive, specific, and superior to conventional PCR (p > 0.05). RT-PCR presented higher sensitivity than HNB-LAMP; however, it has the limitation of being cost-intensive and requiring technical expertise to perform. HNB-LAMP is affordable, rapid, simple, and easy to perform, allowing naked eye visualization.
Soil biochemical properties and microbial communities are usually considered as important indicators of soil health because of their association with plant nutrition. In this study, we investigated the impact of long-term application of bioorganic fertilizer (BOF) on soil biochemical properties and microbial communities in the apple orchard soil of the Loess Plateau. The experiment included three treatments: (1) control without fertilization (CK); (2) chemical fertilizer application (CF); and (3) bioorganic fertilizer application (BOF). The high throughput sequencing was used to examine the bacterial and fungal communities in apple orchard soil. The results showed that the BOF treatment significantly increased the apple yield during the experimental time (2009–2015). The application of BOF significantly increased the activities of catalase and invertase compared to those in CK and CF treatments. The high throughput sequencing data showed that the application of BOF changed the microbial community composition of all soil depths considered (0–20 cm, 20–40 cm, and 40–60 cm), e.g., the relative abundance of bio-control bacteria (Xanthomonadales, Lysobacter, Pseudomonas, and Bacillus), Proteobacteria, Bacteroidetes, Ohtaekwangia, Ilyonectria, and Lecanicillium was increased while that of Acidobacteria, Chloroflexi, Gp4, Gp6 and Sphaerobacter was decreased. The increase in apple yield after the application of BOF might be due to increase in organic matter, total nitrogen and catalase and invertase activities of soil and change in the bacterial community composition by enriching Bacillus, Pseudomonas, Lysobacter, and Ohtaekwangia. These results further enhance the understanding on how BOFs alter soil microbial community composition to stimulate soil productivity.
Cyanobacteria produce an unparalleled variety of toxins that can cause severe health problems or even death in humans, and wild or domestic animals. In the last decade, biosynthetic pathways have been assigned to the majority of the known toxin families. This review summarizes current knowledge about the enzymatic basis for the production of the hepatotoxins microcystin and nodularin, the cytotoxin cylindrospermopsin, the neurotoxins anatoxin and saxitoxin, and the dermatotoxin lyngbyatoxin. Elucidation of the biosynthetic pathways of the toxins has paved the way for the development of molecular techniques for the detection and quantification of the producing cyanobacteria in different environments. Phylogenetic analyses of related clusters from a large number of strains has also allowed for the reconstruction of the evolutionary scenarios that have led to the emergence, diversification, and loss of such gene clusters in different strains and genera of cyanobacteria. Advances in the understanding of toxin biosynthesis and evolution have provided new methods for drinking-water quality control and may inspire the development of techniques for the management of bloom formation in the future.
The last several decades have witnessed dramatic advances in unfolding the diversity and commonality of oceanic diazotrophs and their N(2) -fixing potential. More recently, substantial progress in diazotrophic cell biology has provided a wealth of information on processes and mechanisms involved. The substantial contribution by the diazotrophic cyanobacterial genus Trichodesmium to the nitrogen influx of the global marine ecosystem is by now undisputable and of paramount ecological importance, while the underlying cellular and molecular regulatory physiology has only recently started to unfold. Here, we explore and summarize current knowledge, related to the optimization of its diazotrophic capacity, from genomics to ecophysiological processes, via, for example, cellular differentiation (diazocytes) and temporal regulations, and suggest cellular research avenues that now ought to be explored.
Cyanobacteria are a prolific source of secondary metabolites, including compounds with toxic and enzyme-inhibiting activities. Microcystins and nodularins are the end products of a secondary metabolic pathway comprised of mixed polyketide synthases and nonribosomal peptide synthetases. Both peptides are potent natural toxins produced by distantly related genera of cyanobacteria. Horizontal gene transfer is thought to play a role in the sporadic distribution of microcystin producers among cyanobacteria. Our phylogenetic analyses indicate a coevolution of housekeeping genes and microcystin synthetase genes for the entire evolutionary history of the toxin. Hence they do not corroborate horizontal transfer of genes for microcystin biosynthesis between the genera. The sporadic distribution of microcystin synthetase genes in modern cyanobacteria suggests that the ability to produce the toxin has been lost repeatedly in the more derived lineages of cyanobacteria. The data we present here strongly suggest that the genes encoding nodularin synthetase are recently derived from those encoding microcystin synthetase.
Cell classification and cell counting are essential for the detection, monitoring, forecasting, and management of harmful algae populations. Conventional methods of algae classification and cell counting are known to be time-consuming, labor-intensive, and subjective, depending on the expertise of the observers. The objectives of this study were to classify and quantify five cyanobacteria using the deep learning techniques of a fast regional convolutional neural network (R-CNN) and convolutional neural network (CNN). Water samples taken from the Haman weir of Nakdong River and Baekje weir of the Geum River were observed under the optical microscope. The images captured by the microscope were used to classify cyanobacteria species using the fast R-CNN model. Post-processing of the classified images generated by the model reduced the noises of the cell features, thereby improving the accuracy of the CNN model in quantifying cyanobacteria cells. The distinctive morphological features of the five species were extracted by the fast R-CNN model. This model was able to achieve a reasonable agreement with the manual classification results, yielding average precision (AP) values of 0.929, 0.973, 0.829, 0.890, and 0.890 for Microcystis aeruginosa, Microcystis wesenbergii, Dolichospermum, Oscillatoria, and Aphanizomenon, respectively. The CNN model for the Microcystis species obtained an R² value of 0.775 and RMSE value of 26 cells for training, and an R² of 0.854 and RMSE of 23 cells for validation. A minor underestimation and overestimation for a population with <50 cells and >250 cells were observed, respectively, which are due to the overlapping of cells and the presence of blurry regions in the input images. In conclusion, this study was able to demonstrate the reliable performance of cyanobacteria classification and cell counting using deep learning approaches.
Cyanobacteria (blue-green algae) are Gram-negative oxygenic photosynthetic prokaryotes with a long evolutionary history. They have potential applications in diverse areas, especially in agriculture, as nutrient supplements in agriculture and industry (as biofertilizer, plant growth promoting rhizobacteria and as biocontrol agents). Their role as food supplements/nutraceuticals and in bioremediation and wastewater treatment is an emerging area of interest. In addition, they are known to produce wide array of bioactive compounds (secondary metabolites) with diverse biological activities — including antiviral, antibacterial, antifungal, antimalarial, antitumoral and anti-inflammatory properties, having therapeutic, industrial and agricultural significance. One of the major problems has been regarding their classification being incongruent with the phylogeny, because the phenotype of cyanobacterial strains is known to be altered under different environmental/nutritional conditions. However, because of their simple growth needs, they are the favourite model organisms for deeper understanding of several metabolic processes and for the production of recombinant compounds of medicinal and commercial value. In recent years, cyanobacteria have gained interest for producing third generation biofuels (both biomass and H2 production). With the recent advances in metabolic engineering techniques and availability of genome sequences, novel approaches are being explored for realising the potential of cyanobacteria. Our review provides an overview of the polyphasic approaches used in the analyses of cyanobacterial biodiversity and the potential of these organisms in providing viable solutions to global problems of food, energy and environmental degradation, which need further impetus through adoption of multidisciplinary collaborative programs.
The recent flood of reports using real-time Q-PCR testifies to the transformation of this technology from an experimental tool into the scientific mainstream. Many of the applications of real-time Q-PCR include measuring mRNA expression levels, DNA copy number, transgene copy number and expression analysis, allelic discrimination, and measuring viral titers. The range of applications of real-time Q-PCR is immense and has been fueled in part by the proliferation of lower-cost instrumentation and reagents. Successful application of real-time Q-PCR is not trivial. However, this review will help guide the reader through the variables that can limit the usefulness of this technology. Careful consideration of the assay design, template preparation, and analytical methods are essential for accurate gene quantification.
Molecular cloning. A laboratory manual, 4th edn
- M R Green
- J Sambrook