Current Insight into Culture-Dependent and Culture-Independent Methods in Discovering Ascomycetous Taxa.
Ontology highlight
ABSTRACT:
SUBMITTER: Wijayawardene NN
PROVIDER: S-EPMC8467358 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Airborne microorganisms in the waste associated environments are more active and complex compared to other places. However, the diversity and structure of airborne bacteria in waste-associated environments are still not clearly understood. The purpose of this study was to assess airborne bacterial community in electronic waste dismantling site and a waste transfer station based on culture-dependent and culture-independent methods. A total of 229 isolates were obtained from four airborne sites collected from residential area, electronic industrial park, and office area in or near an electronic waste dismantling site and a waste transfer station in Southern China in the morning, afternoon, and evening. Most of the isolates were isolated from air for the first time and 14 potentially novel species were identified by Sanger sequencing. Bacterial communities in waste-associated bioaerosols were predominated by Proteobacteria and Bacteroidetes. Abundant genera (>1%) included Paracaedibacteraceae (uncultured EF667926), Ralstonia, Chroococcidiopsis, Chitinophagaceae (uncultured FN428761), Sphingobium, and Heliimonas. One-third of the species in these genera were uncultured approximately. Differences community structure existed in airborne bacterial diversity among different sampling sites. These results showed that waste-associated environments have unique bacterial diversity. Further studies on such environments could provide new insights into bacterial community.
| S-EPMC8055949 | biostudies-literature
Project description:Vibrios represent a natural contaminant of seafood products. V. alginolyticus, V. cholerae, V. parahaemolyticus and V. vulnificus are the most hazardous species to human health. Given the worldwide consumption of mollusc products, reliable detection of Vibrio species is recommended to prevent human vibriosis. In this study, culture-dependent and -independent methods were compared and integrated to implement knowledge of the Manila clam Vibrio community composition. Here, 16S and recA-pyrH metabarcoding were applied to compare the microbial communities of homogenate clam samples (culture-independent method) and their culture-derived samples plated on three different media (culture-dependent method). In addition, a subset of plated clam samples was investigated using shotgun metagenomics. Homogenate metabarcoding characterized the most abundant taxa (16S) and Vibrio species (recA-pyrH). Culture-dependent metabarcoding detected the cultivable taxa, including rare species. Moreover, marine agar medium was found to be a useful substrate for the recovery of several Vibrio species, including the main human pathogenic ones. The culture-dependent shotgun metagenomics detected all the main human pathogenic Vibrio species and a higher number of vibrios with respect to the recA-pyrH metabarcoding. The study revealed that integration of culture-dependent and culture-independent methods might be a valid approach for the characterization of Vibrio biodiversity.
| S-EPMC8228196 | biostudies-literature
Project description:PurposeTo date, molecular methods that circumvent the limitations of traditional culture methods have not been used to describe the vaginal microflora in India. Here, we compared culture and culture-independent molecular methods in characterizing the vaginal microbiota in Indian women.MethodologyCulture methods involved traditional cultivation on Rogosa and sheep blood agar, whereas culture-independent methods bypassed a culturing step by performing broadrange 16S rDNA PCR on DNA isolated directly from vaginal swabs.ResultsA total of 13 women were included in the study, of which five were characterized as healthy, two were bacterial vaginosis intermediate and six were bacterial vaginosis positive according to Nugent scoring. Lactobacillus jensenii was detected most frequently when using culture methods. On the other hand, Lactobacillus iners, which was not detected by culture methods, was the most common Lactobacillus sp. detected using cultivation-independent methods.ConclusionWe found little overlap between the species found using cultivation-dependent and cultivation-independent methods. Rather, culture-dependent and culture-independent methods were found to be complementary in describing the vaginal microflora among South Indian women. Culture-independent methods were found to be superior in detecting clinically relevant vaginal flora.
| S-EPMC7147076 | biostudies-literature
Project description:Many orchid species are endangered due to anthropogenic pressures such as habitat destruction and overharvesting, meanwhile, all orchids rely on orchid mycorrhizal fungi (OMF) for seed germination and seedling growth. Therefore, a better understanding of this intimate association is crucial for orchid conservation. Isolation and identification of OMF remain challenging as many fungi are unculturable. In our study, we tested the efficiency of both culture-dependent and culture-independent methods to describe OMF diversity in multiple temperate orchids and assessed any phylogenetic patterns in cultivability. The culture-dependent method involved the cultivation and identification of single pelotons (intracellular hyphal coils), while the culture-independent method used next-generation sequencing (NGS) to identify root-associated fungal communities. We found that most orchid species were associated with multiple fungi, and the orchid host had a greater impact than locality on the variability in fungal communities. The culture-independent method revealed greater fungal diversity than the culture-dependent one, but despite the lower detection, the isolated fungal strains were the most abundant OMF in adult roots. Additionally, the abundance of NGS reads of cultured OTUs was correlated with the extent of mycorrhizal root colonization in orchid plants. Finally, this limited-scale study tentatively suggests that the cultivability character of OMF may be randomly distributed along the phylogenetic trees of the rhizoctonian families.
| S-EPMC10890429 | biostudies-literature
Project description:Microbial endophytes are well known to protect host plants against pathogens, thus representing a promising strategy for the control of xylem-colonizing pathogens. To date, the vast majority of microbial communities inhabiting the olive xylem are unknown; therefore, this work pursues the characterization of the xylem-limited microbiome and determines whether the culture isolation medium, olive genotype, and the plant material used to analyze it can have an effect on the bacterial populations retrieved. Macerated xylem tissue and xylem sap extracted with the Scholander chamber from olive branches obtained from two cultivated and a wild olive genotypes were analyzed using culture-dependent and -independent approaches. In the culture-dependent approach using four solid culture media, a total of 261 bacterial isolates were identified after performing Sanger sequencing of 16S rRNA. Culturable bacteria clustered into 34 genera, with some effect of culture media for bacterial isolation. The cultivated bacteria belonged to four phyla and the most abundant genera included Frigoribacterium (18.8%), Methylobacterium (16.4%), and Sphingomonas (14.6%). On the other hand, in the culture-independent approach conducted using Illumina MiSeq 16S rRNA amplicon sequencing [next-generation sequencing (NGS)] of the xylem extracts, we identified a total of 48 operational taxonomic units (OTUs) belonging to five phyla, being Sphingomonas (30.1%), Hymenobacter (24.1%) and Methylobacterium (22.4%) the most representative genera (>76% of reads). In addition, the results indicated significant differences in the bacterial communities detected in the xylem sap depending on the genotype of the olive tree studied and, to a minor extent, on the type of sap extraction method used. Among the total genera identified using NGS, 14 (41.2%) were recovered in the culture collection, whereas 20 (58.8%) in the culture collection were not captured by the NGS approach. Some of the xylem-inhabiting bacteria isolated are known biocontrol agents of plant pathogens, whereas for others little information is known and are first reported for olive. Consequently, the potential role of these bacteria in conferring olive tree protection against xylem pathogens should be explored in future research.
| S-EPMC6988092 | biostudies-literature
Project description:Although a number of different methods have been used to quantify soil bacteria, identifying the optimal method(s) for soil bacterial abundance is still in question. No single method exists for undertaking an absolute microbial count using culture-dependent methods (CDMs) or even culture-independent methods (CIMs). This study investigated soil storage and pretreatment methods for optimal bacterial counts. Appropriate storage temperature (4°C) and optimal pretreatment methods (sonication time for 3 min and centrifugation at 1400 g) were necessary to preserve bacterial cell viability and eliminate interference from soil particles. To better estimate soil bacterial numbers under various cellular state and respiration, this study also evaluated three CDMs (i.e., colony forming unit, spotting, and most probable number (MPN) and three CIMs (i.e., flow cytometry (FCM), epifluorescence microscopy (EM) count, and DNA quantitation). Each counting method was tested using 72 soil samples collected from a local arable farm site at three different depths (i.e., 10-20, 90-100, and 180-190 cm). Among all CDMs, MPN was found to be rapid, simple, and reliable. However, the number of bacteria quantified by MPN was 1-2 orders lower than that quantified by CIMs, likely due to the inability of MPN to count anaerobic bacteria. The DNA quantitation method appeared to overestimate soil bacterial numbers, which may be attributed to DNA from dead bacteria and free DNA in the soil matrix. FCM was found to be ineffective in counting soil bacteria as it was difficult to separate the bacterial cells from the soil particles. Dyes used in FCM stained the bacterial DNA and clay particles. The EM count was deemed a highly effective method as it provided information on soil mineral particles, live bacteria, and dead bacteria; however, it was a time-consuming and labor-intensive process. Combining both types of methods was considered the best approach to acquire better information on the characteristics of indigenous soil microorganisms (aerobic versus anaerobic, live versus dead).
| S-EPMC7875414 | biostudies-literature
Project description:In this study, the microbial ecology of three naturally fermented sausages produced in northeast Italy was studied by culture-dependent and -independent methods. By plating analysis, the predominance of lactic acid bacteria populations was pointed out, as well as the importance of coagulase-negative cocci. Also in the case of one fermentation, the fecal enterocci reached significant counts, highlighting their contribution to the particular transformation process. Yeast counts were higher than the detection limit (> 100 CFU/g) in only one fermented sausage. Analysis of the denaturing gradient gel electrophoresis (DGGE) patterns and sequencing of the bands allowed profiling of the microbial populations present in the sausages during fermentation. The bacterial ecology was mainly characterized by the stable presence of Lactobacillus curvatus and Lactobacillus sakei, but Lactobacillus paracasei was also repeatedly detected. An important piece of evidence was the presence of Lactococcus garvieae, which clearly contributed in two fermentations. Several species of Staphylococcus were also detected. Regarding other bacterial groups, Bacillus sp., Ruminococcus sp., and Macrococcus caseolyticus were also identified at the beginning of the transformations. In addition, yeast species belonging to Debaryomyces hansenii, several Candida species, and Willopsis saturnus were observed in the DGGE gels. Finally, cluster analysis of the bacterial and yeast DGGE profiles highlighted the uniqueness of the fermentation processes studied.
| S-EPMC1082564 | biostudies-literature
Project description:Bioremediation is a cost-effective and sustainable approach for treating polluted soils, but our ability to improve on current bioremediation strategies depends on our ability to isolate microorganisms from these soils. Although culturing is widely used in bioremediation research and applications, it is unknown whether the composition of cultured isolates closely mirrors the indigenous microbial community from contaminated soils. To assess this, we paired culture-independent (454-pyrosequencing of total soil DNA) with culture-dependent (isolation using seven different growth media) techniques to analyse the bacterial and fungal communities from hydrocarbon-contaminated soils. Although bacterial and fungal rarefaction curves were saturated for both methods, only 2.4% and 8.2% of the bacterial and fungal OTUs, respectively, were shared between datasets. Isolated taxa increased the total recovered species richness by only 2% for bacteria and 5% for fungi. Interestingly, none of the bacteria that we isolated were representative of the major bacterial OTUs recovered by 454-pyrosequencing. Isolation of fungi was moderately more effective at capturing the dominant OTUs observed by culture-independent analysis, as 3 of 31 cultured fungal strains ranked among the 20 most abundant fungal OTUs in the 454-pyrosequencing dataset. This study is one of the most comprehensive comparisons of microbial communities from hydrocarbon-contaminated soils using both isolation and high-throughput sequencing methods.
| S-EPMC4460130 | biostudies-literature
Project description:Traditional fermented cereals are a rich source of naturally derived, diverse microorganisms. Illumina MiSeq high-throughput sequencing was used to investigate thoroughly fungal microflora in Western Inner Mongolian acidic gruel. A total of 589,495 sequences were obtained from 16 acidic gruel samples. Ascomycota was found to be the predominant phylum with a relatively abundance of 97.54%, followed by Basidiomycota (2.26%) and Chytridiomycota (0.1%). The dominant genera obtained from the acidic gruel were Candida, Galactomyces, Hanseniaspora, Guehomyces, Zygosaccharomyces, Trichosporon, Rhodosporidium, Penicillium, and Blastobotrys. Candida and Galactomyces were predominant genera, and their relative abundances were 57.59% and 34.95%, respectively. A total of 50 yeast strains were isolated and identified. Statistical analysis indicated that P kudriavzevii and Geo. silvicola affiliated with Ascomycota were the dominant yeasts in acidic gruel, accounting for 28% and 22%, respectively. This study provides an unequivocal theoretical basis for the study of fungal diversity and the identification and preservation of yeasts in traditional fermented cereals. It also provides validated strain resources for further exploration of the effect of yeasts on acidic gruel quality.
| S-EPMC7684608 | biostudies-literature
Project description:Bacterial diversity from McMurdo Dry Valleys in Antarctica, the coldest desert on earth, has become more easily assessed with the development of High Throughput Sequencing (HTS) techniques. However, some of the diversity remains inaccessible by the power of sequencing. In this study, we combine cultivation and HTS techniques to survey actinobacteria and cyanobacteria diversity along different soil and endolithic micro-environments of Victoria Valley in McMurdo Dry Valleys. Our results demonstrate that the Dry Valleys actinobacteria and cyanobacteria distribution is driven by environmental forces, in particular the effect of water availability and endolithic environments clearly conditioned the distribution of those communities. Data derived from HTS show that the percentage of cyanobacteria decreases from about 20% in the sample closest to the water source to negligible values on the last three samples of the transect with less water availability. Inversely, actinobacteria relative abundance increases from about 20% in wet soils to over 50% in the driest samples. Over 30% of the total HTS data set was composed of actinobacterial strains, mainly distributed by 5 families: Sporichthyaceae, Euzebyaceae, Patulibacteraceae, Nocardioidaceae, and Rubrobacteraceae. However, the 11 actinobacterial strains isolated in this study, belonged to Micrococcaceae and Dermacoccaceae families that were underrepresented in the HTS data set. A total of 10 cyanobacterial strains from the order Synechococcales were also isolated, distributed by 4 different genera (Nodosilinea, Leptolyngbya, Pectolyngbya, and Acaryochloris-like). In agreement with the cultivation results, Leptolyngbya was identified as dominant genus in the HTS data set. Acaryochloris-like cyanobacteria were found exclusively in the endolithic sample and represented 44% of the total 16S rRNA sequences, although despite our efforts we were not able to properly isolate any strain from this Acaryochloris-related group. The importance of combining cultivation and sequencing techniques is highlighted, as we have shown that culture-dependent methods employed in this study were able to retrieve actinobacteria and cyanobacteria taxa that were not detected in HTS data set, suggesting that the combination of both strategies can be usefull to recover both abundant and rare members of the communities.
| S-EPMC6555387 | biostudies-literature