Cultivation-dependent Techniques Research Articles

Analysis of Microbial Diversity in South Shetland Islands and Antarctic Peninsula Soils Based on Illumina High-Throughput Sequencing and Cultivation-Dependent Techniques.

To assess the diversity of bacterial taxa in Antarctic soils and obtain novel microbial resources, 15 samples from 3 sampling sites (DIS5, GWS7, FPS10) of South Shetland Islands and 2 sampling sites (APS18, CIS17) of Antarctic Peninsula were collected. High-throughput sequencing (HTS) of 16S rRNA genes within these samples was conducted on an Illumina Miseq platform. A total of 140,303 16S rRNA gene reads comprising 802 operational taxonomic units (OTUs) were obtained. After taxonomic classification, 25 phyla, 196 genera, and a high proportion of unidentified taxa were detected, among which seven phyla and 99 genera were firstly detected in Antarctica. The bacterial communities were dominated by Actinomycetota (40.40%), Pseudomonadota (17.14%), Bacteroidota (10.55%) and Chloroflexota (10.26%). Based on the HTS analyses, cultivation-dependent techniques were optimized to identify the cultivable members. A total of 30 different genera including 91 strains were obtained, the majority of which has previously been reported from Antarctica. However, for the genera Microterricola, Dyadobacter, Filibacter, Duganella, Ensifer, Antarcticirhabdus and Microvirga, this is the first report in Antarctica. In addition, seven strains represented novel taxa, two of which were psychropoilic and could be valuable resources for further research of cold-adaptability and their ecological significance in Antarctica.

Diversity and spoilage potential of microbial communities associated with grape sour rot in eastern coastal areas of China.

As a polymicrobial disease, sour rot decreases grape berry yield and wine quality. The diversity of microbial communities in sour rot-affected grapes depends on the cultivation site, but the microbes responsible for this disease in eastern coastal China, has not been reported. To identify the microbes that cause sour grape rot in this important grape-producing region, the diversity and abundance of bacteria and fungi were assessed by metagenomic analysis and cultivation-dependent techniques. A total of 15 bacteria and 10 fungi were isolated from sour rot-affected grapes. High-throughput sequencing of PCR-amplicons generated from diseased grapes revealed 1343 OTUs of bacteria and 1038 OTUs of fungi. Proteobacteria and Firmicutes were dominant phyla among the 19 bacterial phyla identified. Ascomycota was the dominant fungal phylum and the fungi Issatchenkia terricola, Colletotrichum viniferum, Hanseniaspora vineae, Saprochaete gigas, and Candida diversa represented the vast majority ofmicrobial species associated with sour rot-affected grapes. An in vitro spoilage assay confirmed that four of the isolated bacteria strains (two Cronobacter species, Serratia marcescens and Lysinibacillus fusiformis) and five of the isolated fungi strains (three Aspergillus species, Alternaria tenuissima, and Fusarium proliferatum) spoiled grapes. These microorganisms, which appear responsible for spoiling grapes in eastern China, appear closely related to microbes that cause this plant disease around the world.

Comparison between cultivated and total bacterial communities associated with Cucurbita pepo using cultivation-dependent techniques and 454 pyrosequencing

Endophytic bacteria often have beneficial effects on their host plants that can be exploited for bioremediation applications but, according to the literature, only 0.001–1% of all endophytic microbes should be cultivable. This study compared the cultivated endophytic communities of the roots and shoots of Cucurbita pepo with the total endophytic communities as determined by cultivation-dependent techniques and 454 pyrosequencing. The ten most abundant taxa of the total communities aligned well with the cultivated taxa; however, the abundance of these taxa in the two communities differed greatly. Enterobacter showed very low presence in the total communities, whereas they were dominantly present in the cultivated communities. Although Rhizobium dominated in total root and shoot communities, it was poorly cultivable and even then only in growth media containing plant extract. Since endophytes likely contribute to plant-growth promotion, cultivated bacterial strains were tested for their plant-growth promoting capabilities, and the results were correlated with their abundance in the total community. Bacillus and Pseudomonas showed promising results when considering cultivability, abundance in the total community and plant-growth promoting capability.This study demonstrated that, although a limited number of bacterial genera were cultivable, current cultivation-dependent techniques may be sufficient for further isolation and inoculation experiments that aim to improve phytoremediation efficiency.

A PCR-based tool for cultivation-independent detection and quantification of Metarhizium clade 1

The entomopathogenic fungus Metarhizium anisopliae and sister species are some of the most widely used biological control agents for insects. Availability of specific monitoring and quantification tools are essential for the investigation of environmental factors influencing their environmental distribution. Naturally occurring as well as released Metarhizium strains in the environment traditionally are monitored with cultivation-dependent techniques. However, specific detection and quantification may be limited due to the lack of a defined and reliable detection range of such methods. Cultivation-independent PCR-based detection and quantification tools offer high throughput analyses of target taxa in various environments. In this study a cultivation-independent PCR-based method was developed, which allows for specific detection and quantification of the defined Metarhizium clade 1, which is formed by the species Metarhizium majus, Metarhizium guizhouense, Metarhizium pingshaense, Metarhizium anisopliae, Metarhizium robertsii and Metarhizium brunneum, formerly included in the M. anisopliae cryptic species complex. This method is based on the use of clade-specific primers, i.e. Ma 1763 and Ma 2097, that are positioned within the internal transcribed spacer regions 1 and 2 of the nuclear ribosomal RNA gene cluster, respectively. BLAST similarity searches and empirical specificity tests performed on target and non-target species, as well as on bulk soil DNA samples, demonstrated specificity of this diagnostic tool for the targeted Metarhizium clade 1. Testing of the primer pair in qPCR assays validated the diagnostic method for specific quantification of Metarhizium clade 1 in complex bulk soil DNA samples that significantly correlated with cultivation-dependent quantification. The new tool will allow for highly specific and rapid detection and quantification of the targeted Metarhizium clade 1 in the environment. Habitat with high Metarhizium clade 1 densities can then be analyzed for habitat preferences in greater detail using cultivation-dependent techniques and genetic typing of isolates.

Cultivation-Independent Detection of Autotrophic Hydrogen-Oxidizing Bacteria by DNA Stable-Isotope Probing

Knallgas bacteria are a physiologically defined group that is primarily studied using cultivation-dependent techniques. Given that current cultivation techniques fail to grow most bacteria, cultivation-independent techniques that selectively detect and identify knallgas bacteria will improve our ability to study their diversity and distribution. We used stable-isotope probing (SIP) to identify knallgas bacteria in rhizosphere soil of legumes and in a microbial mat from Obsidian Pool in Yellowstone National Park. When samples were incubated in the dark, incorporation of (13)CO(2) was H(2) dependent. SIP enabled the detection of knallgas bacteria that were not detected by cultivation, and the majority of bacteria identified in the rhizosphere soils were betaproteobacteria predominantly related to genera previously known to oxidize hydrogen. Bacteria in soil grew on hydrogen at concentrations as low as 100 ppm. A hydB homolog encoding a putative high-affinity NiFe hydrogenase was amplified from (13)C-labeled DNA from both vetch and clover rhizosphere soil. The results indicate that knallgas bacteria can be detected by SIP and populations that respond to different H(2) concentrations can be distinguished. The methods described here should be applicable to a variety of ecosystems and will enable the discovery of additional knallgas bacteria that are resistant to cultivation.

Effects of Deposition of Heavy-Metal-Polluted Harbor Mud on Microbial Diversity and Metal Resistance in Sandy Marine Sediments

Deposition of dredged harbor sediments in relatively undisturbed ecosystems is often considered a viable option for confinement of pollutants and possible natural attenuation. This study investigated the effects of deposition of heavy-metal-polluted sludge on the microbial diversity of sandy sediments during 12 months of mesocosm incubation. Geochemical analyses showed an initial increase in pore-water metal concentrations, which subsided after 3 months of incubation. No influence of the deposited sediment was observed in denaturing gradient gel electrophoresis (DGGE) profiles of bacterial 16S rRNA genes, whereas a minor, transient impact on the archaeal community was revealed. Phylogenetic analyses of bacterial 16S rRNA clone libraries showed an abundance of members of the Flavobacteriaceae, the alpha- and gamma-Proteobacteria, in both the muddy and the sandy sediments. Despite the finding that some groups of clones were shared between the metal-impacted sandy sediment and the harbor control, comparative analyses showed that the two sediments were significantly different in community composition. Consequences of redeposition of metal-polluted sediment were primarily underlined with cultivation-dependent techniques. Toxicity tests showed that the percentage of Cd- and Cu-tolerant aerobic heterotrophs was highest among isolates from the sandy sediment with metal-polluted mud on top.

Fungal diversity in agricultural soil under different farming management systems, with special reference to biocontrol strains of Trichoderma spp.

Fungal communities and their dynamics were investigated in relation to season, soil type and farming management practice. The research was done using soils from high- (H) and low-yield areas (L) of a field site cultivated with winter wheat under two different farming management systems (precision farming, P; conventional farming, C) over the vegetation period. Fungal diversity was analysed by cultivation-independent methods [direct extraction of DNA from soil followed by PCR amplification of a subunit of the 18S rDNA and fingerprinting (DGGE)] as well as cultivation-dependent techniques (isolation of pure cultures). The comparisons of the PCR amplicons by DGGE patterns showed no differences between the different sampling sites and no influence of the farming management systems. Only small differences were observed over the vegetation period. For cultivation purposes active hyphae were isolated using a soil-washing technique. The resulting isolates were subcultured and grouped by their morphology and genotype. In contrast to the cultivation independent approaches, clear site-specific and seasonal effects on the fungal community structure could be observed. However, minor effects of the different farming management techniques applied were visible for active populations. These results clearly indicate that the potential fungal community (including spores), investigated by the cultivation-independent approach, is almost entirely uninfluenced by the investigated factors, whereas active populations show a clear response to environmental changes. The most abundant group consisting of Trichoderma species was investigated in more detail using strain-specific genotype based fingerprinting techniques as well as a screening for potential biocontrol activity against the wheat pathogen Fusarium graminearum. The genotypic distribution as well as the potential biocontrol activity revealed clear site-specific patterns.