Biodiversity and biocatalyst activity of culturable hydrocarbonoclastic fungi isolated from Marac-Moruga mud volcano in South Trinidad.

Mud volcanoes (MVs) are visible signs of oil and gas reserves present deep beneath land and sea. The Marac MV in Trinidad is the only MV associated with natural hydrocarbon seeps. Petrogenic polyaromatic hydrocarbons (PAHs) in its sediments must undergo biogeochemical cycles of detoxification as they can enter the water table and aquifers threatening ecosystems and biota. Recurrent hydrocarbon seep activity of MVs consolidates the growth of hydrocarbonoclastic fungal communities. Fungi possess advantageous metabolic and ecophysiological features for remediation but are underexplored compared to bacteria. Additionally, indigenous fungi are more efficient at PAH detoxification than commercial/foreign counterparts and remediation strategies remain site-specific. Few studies have focused on hydrocarbonoclastic fungal incidence and potential in MVs, an aspect that has not been explored in Trinidad. This study determined the unique biodiversity of culturable fungi from the Marac MV capable of metabolizing PAHs in vitro and investigated their extracellular peroxidase activity to utilize different substrates ergo their extracellular oxidoreductase activity (> 50% of the strains decolourized of methylene blue dye). Dothideomycetes and Eurotiomycetes (89% combined incidence) were predominantly isolated. ITS rDNA sequence cluster analysis confirmed strain identities. 18 indigenous hydrocarbonoclastic strains not previously reported in the literature and some of which were biosurfactant-producing, were identified. Intra-strain variability was apparent for PAH utilization, oil-tolerance and hydroxylase substrate specificity. Comparatively high levels of extracellular protein were detected for strains that demonstrated low substrate specificity. Halotolerant strains were also recovered which indicated marine-mixed substrata of the MV as a result of deep sea conduits. This work highlighted novel MV fungal strains as potential bioremediators and biocatalysts with a broad industrial applications.

Genetic identification of medicinally used Salacia species by nrDNA ITS sequences and a PCR-RFLP assay for authentication of Salacia-related health foods.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots and stems of several Salacia species have been used as traditional medicines, especially in Ayurvedic medical system for the treatment of diabetes, rheumatism, gonorrhea, amenorrhea, skin diseases, etc. Due to reported evidence supporting Salacia's beneficial effects in early-stage diabetes and other lifestyle-related diseases, Salacia-based dietary supplements and health foods have been gaining popularity in Japan and other countries in recent years. However, due to the morphological similarities between Salacia plants, particularly in the medicinally used parts (roots and stems), the authentication of the botanical identities of Salacia-derived products is challenging. AIM OF THIS STUDY: This study aims to develop a genetic approach to authenticate the medicinally used Salacia species and to determine the botanical sources of the commercially available Salacia-derived products. MATERIALS AND METHODS: The sequences of nuclear DNA internal transcribed spacer (ITS) and chloroplast trnK-rps16 region were determined and compared between 10 plant specimens from three medicinally used Salacia species as well as 48 samples of commercial crude drugs. Moreover, a PCR-restriction fragment length polymorphism (RFLP) assay was developed for rapid identification based on the ITS sequences. RESULTS: The plant specimens from the three medicinally used Salacia species showed three main types of sequences in both ITS (types I, II, III) and trnK-rps16 (i, ii, iii) regions. Combined the sequences of ITS and trnK-rps16 regions, S. reticulata and S. oblonga had type I-i and type III-iii or similar sequences, respectively. S. chinensis had type II-ii or II(536M)-i sequences. Forty-eight samples of commercial crude drugs were identified based on ITS and trnK-rps16 DNA barcode. A convenient PCR-RFLP assay using Cac8I restriction enzyme was established and applied to identify the botanical sources of health food products purchased from online retailers. All the twelve samples were identified as S. chinensis. CONCLUSION: The nrDNA ITS sequences provided useful information to authenticate Salacia species and to elucidate the phylogenetic relationship within the Salacia genus. Genetic identification results revealed that S. chinensis and S. reticulata are the major sources of commercially available Salacia-products. Based on the ITS sequences, a convenient PCR-RFLP assay was established for the identification of the medicinally used Salacia species as well as their derived health food products.

The ITS region provides a reliable DNA barcode for identifying reishi/lingzhi (Ganoderma) from herbal supplements.

The dietary supplement industry is rapidly growing yet, a recent study revealed that up to 60% of supplements may have substituted ingredients, some of which can be harmful contaminants or additives. When ingredients cannot be verified morphologically or biochemically, DNA barcoding complemented with a molecular phylogenetic analysis can be a powerful method for species authentication. We employed a molecular phylogenetic analysis for species authentication of the commonly used fungal supplement, reishi (Ganoderma lingzhi), by amplifying and sequencing the nuclear ribosomal internal transcribed spacer regions (ITS) with genus-specific primers. PCR of six powdered samples and one dried sample all sold as G. lucidum representing independent suppliers produced single, strong amplification products in the expected size-range for Ganoderma. Both best-hit BLAST and molecular phylogenetic analyses clearly identified the presence of G. lingzhi DNA in all seven herbal supplements. We detected variation in the ITS sequences among our samples, but all herbal supplement samples fall within a large clade of G. lingzhi ITS sequences. ITS-based phylogenetic analysis is a successful and cost-effective method for DNA-based species authentication that could be used in the herbal supplement industry for this and other fungal and plant species that are otherwise difficult to identify.

Land use shapes the resistance of the soil microbial community and the C cycling response to drought in a semi-arid area.

The aim of this study was to understand the responses of the microbial community of soil under different land uses to drought in a semi-arid Mediterranean area. In a laboratory incubation, soil samples from different land uses (natural forest, drip-irrigated orchard, rain-fed almond tree cultivation and abandoned area) were maintained at 20% and 60% of the WHC. The microbial biomass and potential enzyme activities were determined after four and fifty days of soil incubation. The diversity and composition of the microbial community were studied after 50 days of incubation. The total mineralisation of soil organic C (SOC), as well as, the mineralisation of fresh organic matter (FOM) and the "priming effect" were analysed after addition of 13C-enriched plant tissue. Both land use and drought had significant effects in the soil microbial community, but the effect of land use was stronger than that of drought. The PLFA content (microbial biomass) of the forests soil was greater under drought. After 50 days of soil incubation, the microbial biomass and most of potential enzyme activities of the almond tree and abandoned soil samples were not significantly affected by drought contrary to those in orchard soil. The total and FOM mineralisation were on average lower in soil under drought than under optimal moisture for all land uses. However, the responses of the priming effect to drought were dependent on the land use. Overall, we conclude that the resistance to drought of the soil microbial community from an agroecosystem having a semi-arid climate is strongly influenced by the previous land use.

Rapid Authentication of the Herbal Medicine Plant Species Aralia continentalis Kitag. and Angelica biserrata C.Q. Yuan and R.H. Shan Using ITS2 Sequences and Multiplex-SCAR Markers.

Accurate identification of the plant species that are present in herbal medicines is important for quality control. Although the dried roots of Aralia continentalis (Araliae Continentalis Radix) and Angelica biserrata (Angelicae Pubescentis Radix) are used in the same traditional medicine, namely Dok-Hwal in Korean and Du-Huo in Chinese, the medicines are described differently in the national pharmacopeia. Further confusion arises from the distribution of dried Levisticum officinale and Heracleum moellendorffii roots as the same medicine. Medicinal ingredients from all four plants are morphologically similar, and discrimination is difficult using conventional methods. Molecular identification methods offer rapidity and accuracy. The internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal RNA gene (rDNA) was sequenced in all four plant species, and the sequences were used to design species-specific primers. Primers for each species were then combined to allow sample analysis in a single PCR reaction. Commercial herbal medicine samples were obtained from Korea and China and analyzed using the multiplex assay. The assay successfully identified authentic medicines and also identified inauthentic or adulterated samples. The multiplex assay will be a useful tool for identification of authentic Araliae Continentalis Radix and/or Angelicae Pubescentis Radix preparations in Korea and China.

[Study on identification of Sarcandra glabra and Chloranthus spicatus's leaves by PCR amplification of specific alleles].

The paper is aimed to identify SNP in Sarcandra glabra and Chloranthus spicatus, and authenticate S. glabra from Ch. spicatus and the mixture by using PCR amplification of specific alleles. SNPs in the ITS sequences of S. glabra and Ch. spicatus were found by ClustulX 2. 1 program and Bioedit software. Primers for authentic S. glabra and Ch. spicatus was designed according to the SNP site, and ITS sequence universal primers plus to the authentic primer to construct a multi-PCR reaction system, and then optimized the PCR reaction system. Five hundred and eighty band special for S. glabra and 470 bp band special for Ch. spicatus were found by using multi-PCR reaction. The multi-PCR reaction system could be applied to identify S. glabra and Ch. spicatus's leaves.

Genetic polymorphism of medicinally-used Codonopsis species in an internal transcribed spacer sequence of nuclear ribosomal DNA and its application to authenticate Codonopsis Radix.

Codonopsis Radix has been prescribed as the roots of Codonopsis pilosula, C. pilosula var. modesta and C. tangshen in Chinese Pharmacopoeia. In order to find out genetic markers for identifying the 3 taxa and to authenticate Codonopsis Radix, the molecular analysis of the internal transcribed spacer sequence of nuclear ribosomal DNA was conducted on Codonopsis plants collected widely from Gansu Prov. and Chongqing city of China, the main producing areas of Codonopsis Radix. Significant genetic polymorphism was observed, represented by 11 types of ITS sequences in C. pilosula, 5 types in C. pilosula var. modesta and 5 types in C. tangshen. Among the determined sequences, 1, 1 and 2 types were thought to be of pure lines of each taxon, respectively, designated as types P0, PM0, T1 and T3, and the rest might be derived from hybridization. Hybrid lines were inferred to be resulting from the combination of these pure lines. The informative sites for discriminating the 3 taxa were detected at the nucleotide positions 122nd, 226th, 441st and 489th from upstream of the ITS sequence. For discrimination of the types of C. tangshen, including one type T0 registered in GenBank, the nucleotides at positions 135th, 489th and 500th were informative. Botanical sources of the crude drugs produced in a wide range of the southeast Gansu Prov. were C. pilosula, just those from Wenxian of Gansu Prov. were C. pilosula var. modesta. The crude drugs produced in Chongqing were derived from C. tangshen.

Botanical origin of dietary supplements labeled as "Kwao Keur", a folk medicine from Thailand.

In the course of our study on the quality of dietary supplements in Japan, both the internal transcribed spacer (ITS) sequence of nrDNA and the rps16 intron sequence of cpDNA of products labeled as "Kwao Keur" were investigated. As a result, the DNA sequence of Pueraria candollei var. mirifica, which is the source plant of Kwao Keur, was observed in only about half of the products. Inferred from the determined sequences, source plants in the other products included Medicago sativa, Glycyrrhiza uralensis, Pachyrhizus erosus, and Ipomoea batatas, etc. These inferior products are estimated to lack the efficacy implied by their labeling. In order to guarantee the quality of dietary supplements, it is important to identify the source materials exactly; in addition, an infrastructure that can exclude these inferior products from the market is needed for the protection of consumers from potential damage to their health and finances. The DNA analysis performed in this study is useful for this purpose.

Effect of dietary prebiotic (mannan oligosaccharide) supplementation on the caecal bacterial community structure of turkeys.

The identification of specific bacterial species influenced by mannan oligosaccharide (MOS) supplementation may assist in the formulation of new and improved diets that promote intestinal health and improve bird performance, offering suitable alternatives to antimicrobials in feed for sustainable poultry production. This study has been conducted to evaluate the use of a MOS compound derived from the yeast cell wall of Saccharomyces cerevisiae on turkey performance, bacterial community structure and their phylogenetic associations. A 42-day turkey trial was carried out on birds fed control and MOS-supplemented diets. Bird performance data (weight gains, feed consumption and feed efficiency ratios) were collected, and caecal contents were extracted from randomly caught poults on days 28, 35 and 42 posthatch. Bird performance data showed no improvements as a result of dietary supplementation. Automated ribosomal intergenic spacer analysis (ARISA) revealed the bacterial community structure to be significantly altered on days 28 and 35 posthatch but not day 42 as a result of dietary supplementation. This technique was coupled with 16S rRNA gene sequence analysis to elucidate phylogenetic identities of bacteria. The dominant bacteria of the caecum on all days in both treatment groups were members of phylum Firmicutes, followed by the Bacteroidetes and Proteobacteria phyla, respectively. Statistical analysis of the 16S rRNA gene libraries showed that the composition of the MOS clone library differed significantly to the control on day 35 posthatch. It can be concluded that MOS alters the bacterial community structure in the turkey caecum.

Authentication of Angelica anomala Avé-Lall cultivars through DNA barcodes.

Angelica anomala Avé-Lall (Chuanbaizhi in Chinese) is an important medicinal plant which can be used in traditional Chinese medicines; however, there are no authentic and universal methods to differentiate this Sichuan famous-region drug of A. anomala from a large number of non-famous-region and false drugs. It has been demonstrated that DNA barcoding is a molecular diagnostic method for species identification, which uses a single standardized DNA fragment. In this study, we tested five DNA barcoding candidates (matK, ITS, ITS2, rbcL, and psbA-trnH), and we found that ITS was the best candidate to authenticate the famous-region drug of A. anomala. Moreover, through comparative analysis of these five DNA barcodes between A. anomala and Angelica dahurica, we found that ITS had the most and ITS2 had more variable regions, but the psbA-trnH, rbcL, and matK regions were identical. Hence, we suggest ITS as the DNA barcoding to identify A. anomala and A. dahurica. Moreover, we are determined to adopt the A. anomala as the accurate Latin name of Chuanbaizhi.

Induction of apoptosis against cancer cell lines by four ascomycetes (endophytes) from Malaysian rainforest.

Endophytic fungi have been shown to be a promising source of biologically active natural products. In the present study, extracts of four endophytic fungi isolated from plants of the National Park, Pahang were evaluated for their cytotoxic activity and the nature of their active compounds determined. Those extracts exhibiting activity with IC(50) values less than 17 µg/ml against HCT116, MCF-7 and K562 cell lines were shown to induce apoptosis in these cell lines. Molecular analysis, based on sequences of the rDNA internal transcribed spacers ITS1 and ITS4, revealed all four endophytic fungi to be ascomycetes: three sordariomycetes and a dothideomycete. Six known compounds, cytochalasin J, dechlorogriseofulvin, demethylharzianic-acid, griseofulvin, harzianic acid and 2-hexylidene-3-methyl-succinic acid were identified from a rapid dereplication technique for fungal metabolites using an in-house UV library. The results from the present study suggest the potential of endophytic fungi as cytotoxic agents, and there is an indication that the isolates contain bioactive compounds that mainly kill cancer cells by apoptosis.

[PCR-SSCP molecular identification of Panax ginseng and P. quinquefolius based on ITS2 bar coding SNPs].

OBJECTIVE: To establish a method for identifying Panax ginseng and P. quinquefolius with PCR-SSCP, on the basis of specific SNP identification sites of their ITS2 bar codes. METHOD: ITS2 sequences of P. ginseng and P. quinquefolius recorded in GenBank were searched to conduct a comparative analysis and screen out specific SNP identification sites of their ITS2 bar codes. Based on that, the Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) method was adopted for analyzing 11 P. ginseng samples and 10 P. quinquefolius samples and verifying sequencing of their PCR products. RESULT: The P. ginseng and P. quinquefolius samples had the same agarose mages of PCR-SSCP with the standard medicines. There were significant differences between the PCR-SSCP agarose mages of P. ginseng and P. quinquefolius, with identifical identification results between PCR-SSCP and sequencing. CONCLUSION: Compared with the sequencing method, PCR-SSCP is so rapid and accurate that it can be used for identifying P. ginseng and P. quinquefolius medicines.

[Study on ITS sequences of Aconitum vilmorinianum and its medicinal adulterant].

OBJECTIVE: To analyze and compare the ITS sequences of Aconitum vilmorinianum and its medicinal adulterant Aconitum austroyunnanense. METHODS: Total genomic DNA were extracted from sample materials by improved CTAB method, ITS sequences of samples were amplified using PCR systems, directly sequenced and analyzed using software DNAStar, ClustalX1.81 and MEGA 4.0. RESULTS: 299 consistent sites, 19 variable sites and 13 informative sites were found in ITS1 sequences, 162 consistent sites, 2 variable sites and 1 informative sites were found in 5.8S sequences, 217 consistent sites, 3 variable sites and 1 informative site were found in ITS2 sequences. Base transition and transversion was not found only in 5.8S sequences, 2 sites transition and 1 site transversion were found in ITS1 sequences, only 1 site transversion was found in ITS2 sequences comparting the ITS sequences data matrix. By analyzing the ITS sequences data matrix from 2 population of Aconitum vilmorinianum and 3 population of Aconitum austroyunnanense, we found a stable informative site at the 596th base in ITS2 sequences, in all the samples of Aconitum vilmorinianum the base was C, and in all the samples of Aconitum austroyunnanense the base was A. CONCLUSION: Aconitum vilmorinianum and Aconitum austroyunnanense can be identified by their characters of ITS sequences, and the variable sites in ITS1 sequences are more than in ITS2 sequences.

Aislamiento y caracterización de dos cepas de Fusarium oxysporum causantes de la pudrición seca en Solanum tuberosum en Colombia / Isolation and characterization of two strains of Fusarium oxysporum causing potato dry rot in Solanum tuberosum in Colombia

resumen(AU)

Background. Fusarium oxysporum has worldwide distribution and causes severe vascular wilt or root rot in many plants. Strains are classified into formae speciales based on their high degree of host specificity, of which multilocus sequence typing provides a fairly good estimate. Aims. The main aim of this study was to identify the causal agent of an infected potato tuber in Colombia. Methods. Two F. oxysporum isolates were recovered from a potato tuber showing symptoms of dry rot. Both macroscopic and microscopic morphology differences were observed between the two isolates. Koch's postulates were verified and in quantitative tuber pathogenecity trials, both isolates induced moderate dry rot. Ribosomal internal transcribed spacer (ITS) and partial intergenic spacer region (IGS) sequences were PCR-amplified, sequenced and shown to be identical for the two isolates. A maximum parsimony phylogeny was created using F. oxysporum IGS sequences available in the Genebank database, which does not include sequences from the formae speciales tuberosi. Results. Our two isolates were most closely related to a red clover (Trifolium pratense) pathogenic isolate and two non-pathogenic F. oxysporum isolates from birdsfoot trefoil (Lotus corniculatus) and Lycopersicon sp. rhyzosphere (99% identity). Conclusions. These experiments showed that our isolates are not restricted to potato and that a molecular marker is needed to differentiate the formae speciales since the IGS and EF-1alpha do not have the power to do it(AU)

Marichromatium litoris sp. nov. and Marichromatium chrysaorae sp. nov. isolated from beach sand and from a jelly fish (Chrysaora colorata).

Three strains (JA349(T), JA553(T), JA439) of phototrophic sulphur bacteria were isolated from marine habitats of India. 16S rRNA gene sequence of the three strains clustered phylogenetically with members of the genus Marichromatium of the family Chromatiaceae belonging to the class Gammaproteobacteria. All the strains shared highest sequence similarity with the type strains of Marichromatium spp. (96-99% sequence similarity) and the new strains were characterized based on polyphasic taxonomy. Strains JA349(T) and JA553(T) can be distinguished from closest relative species of the genus Marichromatium with respect to distinct differences in cellular polar lipids, fatty acids and carbon/nitrogen sources utilization. Both strains were distinctly related (<50% based on DNA-DNA hybridization) with the type strains of the genus Marichromatium. Multilocus Sequence Analysis (MLSA) of the concatenated five protein coding genes (fusA, pufM, dnaK, recA, soxB) along with internal transcribed spacer (ITS; 16S-23S rRNA) had sequence similarity of less than 92% with the type strains of Marichromatium spp. Distinct phenotypic, chemotaxonomic and molecular differences allow the separation of strains JA349(T) and JA553(T) into new species of the genus Marichromatium for which, we propose the names Marichromatium litoris sp. nov. and Marichromatium chrysaorae sp. nov., respectively.

Desulfovibrio africanus subsp. uniflagellum subsp. nov., a sulfate-reducing bacterium from a uranium-contaminated subsurface aquifer.

The bacterial strain SR-1(T) was isolated from subsurface sediments of a uranium-contaminated site in Shiprock, New Mexico, USA. Cells are vibrioid and motile by means of a single polar flagellum. Strain SR-1(T) grows on sulfate, oxidizing formate, lactate and H2, but not malate, and ferments pyruvate. The DNA sequences of the 16S rRNA gene and the 16S-23S internal transcribed spacer of strain SR-1(T) showed 99.9 and 99.4 % similarity, respectively, to those of the type strain Desulfovibrio africanus DSM 2603(T). The DNA sequence of the ITS region is 300 bases in length and contains two tRNA genes (tRNA(Ile), tRNA(Ala)). The partial DNA sequence of the dsrAB gene showed 94.6 % amino acid sequence similarity to that of D. africanus. The DNA G+C content of strain SR-1(T) was 62.4 mol% and it showed 72 % DNA-DNA similarity to D. africanus. DNA typing methods that target gene clusters and whole genomes revealed characteristic genomic fingerprints for strain SR-1(T). A small plasmid was detected by gel electrophoresis. On the basis of distinct phenotypic and genotypic characteristics, strain SR-1(T) represents a novel subspecies of D. africanus, for which the name Desulfovibrio africanus subsp. uniflagellum subsp. nov. is proposed. The type strain is SR-1(T) (=JCM 15510(T) =LS KCTC 5649(T)).

Pythium stipitatum sp. nov. isolated from soil and plant debris taken in France, Tunisia, Turkey, and India.

Pythium stipitatum is a slow-growing oomycete and has been isolated from soil samples and plant materials from France, Tunisia, Turkey and India. Its morphological characteristics are reminiscent of those of Pythium ramificatum, discovered in Algeria by the corresponding author. Unfortunately, the Algerian isolate was not deposited in any culture collection and ultimately got lost. Those were the days when molecular description of fungi was not a fashion; hence, no molecular characteristics of the Algerian isolates were deposited to the GenBank. Moreover, its coralloid antheridial branches made it an easy prey to be considered as synonymous to Pythium minus. Because there are no living strains of P. ramificatum, and no sequence at the GenBank, it is being treated as 'nomen invalidum' here. However, we have now isolated the same type of oomycete from four different countries and we have sufficient evidence, both molecular and morphological, to describe it as a new species, quite different from P. minus. In this article, we are giving the morphological and molecular evidence to separate it as a distinct species, P. stipitatum, belonging to the 'Clade E' of the genus Pythium. Taxonomic description of this oomycete, its comparison with related species, and the sequence of the internal transcribed spacer region of its rRNA gene, are discussed here.

PCR detection of potato cyst nematode.

Potato cyst nematode (PCN) is responsible for losses in potato production totalling millions of euros every year in the EC. It is important for growers to know which species is present in their land as this determines its subsequent use. The two species Globodera pallida and Globodera rostochiensis can be differentiated using an allele-specific PCR.

Mycorrhizal status and diversity of fungal endophytes in roots of common buckwheat (Fagopyrum esculentum) and tartary buckwheat (F. tataricum).

To determine the mycorrhizal status and to identify the fungi colonising the roots of the plants, common buckwheat (Fagopyrum esculentum) and tartary buckwheat (F. tataricum) were inoculated with an indigenous fungal mixture from a buckwheat field. Root colonisation was characterised by the hyphae and distinct microsclerotia of dark septate endophytes, with occasional arbuscules and vesicles of arbuscular mycorrhizal fungi. Sequences of arbuscular mycorrhizal fungi colonising tartary buckwheat clustered close to the Glomus species group A. Sequences with similarity to the Ceratobasidium/Rhizoctonia complex, a putative dark septate endophyte fungus, were amplified from the roots of both common and tartary buckwheat. To the best of our knowledge, this is the first report of arbuscular mycorrhizal colonisation in tartary buckwheat and the first molecular characterisation of these fungi that can colonise both of these economically important plant species.

Genetic and morphological characterization of Rivularia and Calothrix (Nostocales, Cyanobacteria) from running water.

In this study, a polyphasic approach was adopted to investigate natural freshwater (river and stream) samples of Rivularia colonies and isolated strains of cyanobacteria with a high degree of trichome tapering (genera Rivularia and Calothrix). Analysis of the phycocyanin (PC) operon and the intervening intergenic spacer (cpcBA-IGS) and 16S rRNA gene sequences were used for genetic characterization. In addition, a molecular fingerprinting method, temperature-gradient gel electrophoresis, which allows sequence-dependent separation of PCR products, was used to assess genotypic diversity in environmental samples and isolated strains. The results showed a high variability of the PC-IGS among the genotypes that was not associated with the morphologies observed. This study underlines the importance of choosing a low-nutrient-content culture medium, especially one with a low phosphorus concentration, for studying typical morphological features of Rivularia for taxonomic purposes. Molecular fingerprinting methods and morphological analyses confirmed the diversity in Rivularia colonial structure and trichome features corresponding to genetic diversity within a single colony. Phylogenetic analysis of cpcBA-IGS was largely consistent with that obtained from 16S rRNA gene sequence analysis and confirmed the high level of divergence between genotypes. The sequences of Rivularia and Calothrix from this study and database sequences showed great heterogeneity and were clearly not monophyletic. The results of this genetic and morphological study of field samples and fresh isolates indicated that the current classification of these genera needs to be revised.