Culture-dependent Study Research Articles

Intestinal microbial diversity in domestic honey bees and evaluation of their probiotic properties

As populations of managed honey bees in various regions increase in comparison to wild colonies, there is a need to ensure that threats to bee health and survival in all environments are countered. One of the more direct methods of approaching bee health is through dietary means and as such, we investigated the intestinal microbial diversity in the gut of honey bees in order to identify strains with probiotic potential. Honey bees were fed with several feeds including Feedbee, Megabee, sugar syrup 60% (w/v), Yangbongnonghyup (YBNH), and Test A, to monitor the culture-dependent and culture-independent microbial diversity. In the culture-dependent study, most isolates belonged to Apilactobacillus (24.1%), Fructobacillus (21.8%), and Bacillus (15.4%) at the genus level. Culture-independent results showed that Bacillota and Pseudomonadota were the most abundant groups (99%) among the gut bacteria, while other bacteria showed a lower percentage than 0.1%. Next, the hydrolytic enzymes (α-amylase, protease, and cellulase) and antibacterial and antioxidant activities of the isolated strains were evaluated to examine their applicability as potential probiotics for honey bees. The selected strains showing three hydrolytic enzyme activities were used to determine their antibacterial activity against Paenibacillus larvae. Bacillus subtilis MBLB2796 showed the highest α-amylase (1.24 ± 0.06 U/mL) and cellulase (0.24 ± 0.01 U/mL) activities while B. tequilensis MBLB2834 exhibited the highest protease activity (0.15 ± 0.03 mU/mL) and the highest antioxidant activity (71.0 ± 3.8%). This study suggests that honey bee gut-originated isolates are prime candidates for use as probiotics in honey bees.

Operation of air conditioners affects the microbial pathogenic potential in bedrooms: A metagenomics and culture-dependent study

Air conditioning is central to indoor thermal regulation and its impact on indoor microorganisms and their health implications for occupants deserve extensive investigations. While previous research has largely focused on the composition and distribution of microbial communities, microbial functions, particularly the functional genes associated with human disease have been understudied. To address this gap, metagenomic sequencing was performed to analyze the microbial compositions and functionalities in residential bedrooms located in four cities in the Yangtze River basin. A laboratory culture-dependent experiment was conducted to verify the findings of the metagenomics analysis. Our results indicate that approximately 14% of the detected microbial genes were associated with human diseases. Moreover, the relative abundances of pathogenic bacteria and functional disease-related genes were higher during the cooling period compared to the non-cooling period, and this increased with the increase in air conditioner usage. This may be attributed to the indoor temperature during the cooling period, which promoted higher bacterial survivability. The laboratory culture-dependent exp eriment also supported the correlation between air conditioner usage and pathogenic potential. Indoor relative humidity, air changes per hour, geographical location, and outdoor air pollutants were also considered, but no significant correlations were found for these factors. Our study provides a fresh perspective on microbial risks to human health by focusing on functional genes and offers suggestions to create a healthier residential environment.

Antibiotic Resistance Profiles and ARG Detection from Isolated Bacteria in a Culture-Dependent Study at the Codfish Industry Level.

The antibiotic resistance phenomenon horizontally involves numerous bacteria cultured from fresh or processed seafood matrix microbiomes. In this study, the identified bacteria from food-producing processes and industrial environments were screened for phenotypic and genotypic resistance determinants. A total of 684 bacterial strains [537 from processed codfish (Gadus morhua and Gadus macrocephalus) products as salted and seasoned and soaked and 147 from environmental samples] were isolated. Antibiotic susceptibility tests showed resistance against tetracycline, oxacillin, and clindamycin in the Staphylococcus genus (both from food and environmental samples) and against beta-lactams (cefotaxime, carbapenems, etc.) and nitrofurans (nitrofurantoin) from E. coli and Salmonella enterica serovar. Enteritidis isolates. One-thousand and ten genetic determinants-tetracycline tetC (25.17%), tetK (21.06%), tetL (11.70%), clindamycin ermC (17.23%), ermB (7.60%), linezolid cfr (8.22%), optrA (3.62%), poxtA (2.05%), and oxacillin mecA (17.37%)-were amplified from Gram-positive resistant and phenotypically susceptible bacteria. Concerning Gram-negative bacteria, the beta-lactam-resistant genes (blaTEM, blaCIT, blaCTX-M, blaIMP, blaKPC, blaOXA-48-like) represented 57.30% of the amplified ARGs. This study found high antibiotic resistance genes in circulation in the fish food industry chain from the macro- to microenvironment. The obtained data confirmed the diffusion of the "antibiotic resistance phenomenon" and its repercussions on the One-health and food-producing systems.

Endophytic Fungal Community of Stellera chamaejasme L. and Its Possible Role in Improving Host Plants' Ecological Flexibility in Degraded Grasslands.

Stellera chamaejasme L. is a widely distributed poisonous plant in Chinese degraded grasslands. To investigate the role of endophytic fungi (EF) in S. chamaejasme's quick spread in grasslands, the endophytic fungal community of S. chamaejasme was studied through culture-dependent and culture-independent methods, and the plant-growth-promoting (PGP) traits of some culturable isolates were tested. Further, the growth-promoting effects of 8 isolates which showed better PGP traits were evaluated by pot experiments. The results showed that a total of 546 culturable EF were isolated from 1114 plant tissue segments, and the colonization rate (CR) of EF in roots (33.27%) was significantly higher than that in shoots (22.39%). Consistent with this, the number of specific types of EF was greater in roots (8 genera) than in shoots (1 genus). The same phenomenon was found in culture-independent study. There were 95 specific genera found in roots, while only 18 specific genera were found in shoots. In addition, the dominant EF were different between the two study methods. Cladosporium (18.13%) and Penicillium (15.93%) were the dominant EF in culture-dependent study, while Apiotrichum (13.21%) and Athelopsis (5.62%) were the dominant EF in culture-independent study. PGP trait tests indicated that 91.30% of the tested isolates (69) showed phosphorus solubilization, IAA production, or siderophores production activity. The benefit of 8 isolates on host plants' growth was further studied by pot experiments, and the results indicated that all of the isolates can improve host plants' growth. Among them, STL3G74 (Aspergillus niger) showed the best growth-promotion effect; it can increase the plant's shoot and root dry biomass by 68.44% and 74.50%, respectively, when compared with the controls. Our findings revealed that S. chamaejasme has a wide range of fungal endophytic assemblages, and most of them possess PGP activities, which may play a key role in its quick spread in degraded grasslands.

Bacterial Communities and Diversity of Western Ghats Soil: A Study of a Biodiversity Hotspot

The Western Ghats is one of India's mega-diversity hotspots and an ecologically and geologically important area for the diversity of endemic plants and animals. The present study provides insights into the aerobic bacterial diversity and composition of the soils of North Western Ghats located in Maharashtra state (NWGM), India. The samples for the culture-dependent study were collected from 6 different locations namely Malshej Ghat, Bhimashankar, Lonavala, Mulshi, Tail-Baila, and Mahabaleshwar. A total of 173 isolates were obtained from the different samples, which belonged to Proteobacteria (43%), Firmicutes (36%), and Actinobacteria (19%). Sequences of 15 strains shared ≤ 98.7% similarity (a species cut-off) which represent potential novel species. Metagenomic analysis revealed the presence of Actinobacteria and Proteobacteria as the most dominant phyla at both MB and MG. However, both sites showed variation in the composition of rare phyla and other dominant phyla. This difference in bacterial community composition could be due to differences in altitude or other physicochemical properties. The functional prediction from the amplicon sequencing showed the abundance of carbohydrate, protein, and lipid metabolism which was corroborated by screening the isolated bacterial strains for the same. The present study has a unique take on microbial diversity and defines the importance of community assembly processes such as drift, dispersal, and selection. Such processes are relatively important in controlling community diversity, distribution, as well as succession. This study has shown that the microbial community of NWGM is a rich source of polysaccharide degrading bacteria having biotechnological potential.

Khargia gen. nov., a new genus of simple trichal Cyanobacteria from the Persian Gulf

Information regarding the diversity of Cyanobacteria in many parts of the world is still minimal. One example of a region that has not yet been widely studied is south-western Asia, including the region of the Persian Gulf. A culture-dependent study of cyanobacterial diversity in a rainwater basin on Kharg Island enabled the isolation of a novel taxon, previously unnamed, from a simple trichal cyanobacterium. Further comparisons showed the existence of closely related strains/taxa from other parts of the world, namely the strains CCALA 945 isolated from South Italy and "<i>Leptolyngbya india" </i>from India. Herein, we have thus described the new genus <i>Khargia</i> and the new species <i>Khargia iranica. </i>Other strains, isolated by other authors, were included in the <i>Khargia</i> genus as additional species: <i>Khargia italica</i> and <i>Khargia indica</i>. The recognition of the new genus was based on morphological evaluations (identification by both light and electron microscopy), the phylogenetic analyses based on the 16S rRNA gene, and species delimitation based on Automatic Barcode Gap Definition (ABGD), Generalized Mixed Yule Coalescent (GMYC), bayesian version of Poisson Tree Processes (bPTP), and the secondary structure of 16S-23S rRNA ITS region of the studied strains.

A Culture-dependent Study of Bacteria Associated with the Roots of Organically and Inorganically Cultivated Rice Accessions

Rice is one of the most important staple foods consumed by the maximum human population in the world. India possesses a large number of traditionally maintained rice landraces which are cultivated using traditional practices or minimal fertilizer input. Such landraces may be associated with unique microbial flora adapted to various biotic and abiotic stresses. This work aimed to study the diversity of bacteria associated with roots of 18 randomly selected landraces from Jawhar, Maharashtra, grown under organic cultivation practices, and the cv. Indrayani from Mulshi, Maharashtra, grown using conventional cultivation practices. The cultivation-based approach yielded a total of 40 bacterial isolates. Isolates were purified and identified by sequencing the 16S rDNA region. Isolates were tested for plant growth-promoting characteristics such as nitrogen fixation, phosphate solubilization, IAA production, etc. 22 isolates tested positive for nitrogen fixation either by acetylene reduction or the presence of nifH gene, 27 had phosphate solubilization and 39 had IAA production ability. Bacterial diversity demonstrated the dominance of phylum Proteobacteria and isolates belonging to 11 genera and 23 species e.g., Stenotrophomonas sp., Bradyrhizobium sp. etc. PCA showed a clear difference between bacterial diversity associated with landraces and cv. Indrayani. Our findings provide a basis for further investigations into the role and impact of microbial communities in plant growth promotion. Rice is one of the most important staple foods consumed by the maximum human population in the world. India possesses a large number of traditionally maintained rice landraces which are cultivated using traditional practices or minimal fertilizer input. Such landraces may be associated with unique microbial flora adapted to various biotic and abiotic stresses. This work aimed to study the diversity of bacteria associated with roots of 18 randomly selected landraces from Jawhar, Maharashtra, grown under organic cultivation practices, and the cv. Indrayani from Mulshi, Maharashtra, grown using conventional cultivation practices. The cultivation-based approach yielded a total of 40 bacterial isolates. Isolates were purified and identified by sequencing the 16S rDNA region. Isolates were tested for plant growth-promoting characteristics such as nitrogen fixation, phosphate solubilization, IAA production, etc. 22 isolates tested positive for nitrogen fixation either by acetylene reduction or the presence of nifH gene, 27 had phosphate solubilization and 39 had IAA production ability. Bacterial diversity demonstrated the dominance of phylum Proteobacteria and isolates belonging to 11 genera and 23 species e.g., Stenotrophomonas sp., Bradyrhizobium sp. etc. PCA showed a clear difference between bacterial diversity associated with landraces and cv. Indrayani. Our findings provide a basis for further investigations into the role and impact of microbial communities in plant growth promotion.

Culturomics of Bacteria from Radon-Saturated Water of the World's Oldest Radium Mine.

ABSTRACTBalneotherapeutic water springs, such as those with thermal, saline, sulfur, or any other characteristics, have recently been the subject of phylogenetic studies with a closer focus on the description and/or isolation of phylogenetically novel or biotechnologically interesting microorganisms. Generally, however, most such microorganisms are rarely obtained in pure culture or are even, for now, unculturable under laboratory conditions. In this culture-dependent study of radioactive water springs of Jáchymov (Joachimstahl), Czech Republic, we investigated a combination of classical cultivation approaches with those imitating sampling source conditions. Using these environmentally relevant cultivation approaches, over 1,000 pure cultures were successfully isolated from 4 radioactive springs. Subsequent dereplication yielded 121 unique taxonomic units spanning 44 genera and 9 taxonomic classes, ~10% of which were identified as hitherto undescribed taxa. Genomes of the latter were sequenced and analyzed, with a special focus on endogenous defense systems to withstand oxidative stress and aid in radiotolerance. Due to their origin from radioactive waters, we determined the resistance of the isolates to oxidative stress. Most of the isolates were more resistant to menadione than the model strain Deinococcus radiodurans DSM 20539T. Moreover, isolates of the Deinococcacecae, Micrococcaceae, Bacillaceae, Moraxellaceae, and Pseudomonadaceae families even exhibited higher resistance in the presence of hydrogen peroxide. In summary, our culturomic analysis shows that subsurface water springs contain diverse bacterial populations, including as-yet-undescribed taxa and strains with promising biotechnological potential. Furthermore, this study suggests that environmentally relevant cultivation techniques increase the efficiency of cultivation, thus enhancing the chance of isolating hitherto uncultured microorganisms.IMPORTANCE The mine Svornost in Jáchymov (Joachimstahl), Czech Republic is a former silver-uranium mine and the world’s first and for a long time only radium mine, nowadays the deepest mine devoted to the extraction of water which is saturated with radon and has therapeutic benefits given its chemical properties. This healing water, which is approximately 13 thousand years old, is used under medical supervision for the treatment of patients with neurological and rheumatic disorders. Our culturomic approach using low concentrations of growth substrates or the environmental matrix itself (i.e., water filtrate) in culturing media combined with prolonged cultivation time resulted in the isolation of a broad spectrum of microorganisms from 4 radioactive springs of Jáchymov which are phylogenetically novel and/or bear various adaptive or coping mechanisms to thrive under selective pressure and can thus provide a wide spectrum of capabilities potentially exploitable in diverse scientific, biotechnological, or medical disciplines.

Cultivable microbial diversity in speleothems using MALDI-TOF spectrometry and DNA sequencing from Krem Soitan, Krem Lawbah, Krem Mawpun, Khasi Hills, Meghalaya, India

The microbial diversity in the Indian caves is inadequately characterized. This study reports on the culturable microbial communities in caves from the Indian sub-continent. This study aims to expand the current understanding of bacterial diversity in the speleothems and wall deposits from Krem Soitan, Krem Lawbah, Krem Mawpun in Khasi Hills, Meghalaya, India. A culture-dependent approach was employed for elucidating the community structure in the caves using MALDI-TOF spectrometry and 16S rRNA gene sequencing. A high bacterial diversity and a greater bacterial taxonomic diversity is reported using MALDI-TOF spectrometry and 16S rRNA gene sequencing. High microbial enumerations were observed on dilute nutrient agar (5.3 × 103 to 8.8 × 105) followed by M9 minimal medium (4 × 104 to 1.7 × 105) and R2A medium (1.0 × 104 to 5.7 × 105). A total of 826 bacterial isolates were selected and preserved for the study. 295 bacterial isolates were identified using MALDI-TOF spectrometry and the isolates which showed no reliable peaks were further identified by 16S rRNA gene sequencing. A total 91% of the bacterial diversity was dominated by Proteobacteria (61%) and Actinobacteria (30%). In addition, bacterial phyla include Firmicutes (7.45%), Deinococcus-Thermus (0.33%) and Bacteroidetes (0.67%) were found in the samples. At the genus level, Pseudomonas (55%) and Arthrobacter (23%) were ubiquitous followed by Acinetobacter, Bacillus, Brevundimonas, Deinococcus, Flavobacterium, Paenibacillus, Pseudarthrobacter. Multivariate statistical analysis indicated that the bacterial genera formed separate clusters depending on the geochemical constituents in the spring waters suitable for their growth and metabolism. To the best of our knowledge, there are no previous geomicrobiological investigations in these caves and this study is a pioneering culture dependent study of the microbial community with many cultured isolates.

Biofilm formation, antimicrobial assay, and toxin-genotypes of Clostridium perfringens type C isolates cultured from a neonatal Yangtze finless porpoise.

This is a culture-dependent study with the objective of pure culturing and characterizing pathogenic bacteria from the blowhole, lung, stomach and fecal samples of a neonatal crucially endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) that died 27days after birth. Bacteria were inoculated using a swab onto blood and MacConkey agar plates and representative isolates were identified through 16S rRNA gene sequence analysis. A total of three Clostridium perfringens type C strains from the fecal samples were isolated. Toxin genes, including cpa, cpb and cpb2, were detected by PCR amplification, whereas the etx, iap and cpe genes were not detected. Biofilm formation of the three strains was then examined. Only one strain was capable of biofilm formation. In addition, isolates showed strong resistance against the antibiotics amikacin (3/3), erythromycin (1/3), gentamicin (3/3), streptomycin (3/3), and trimethoprim (3/3), while sensitivity to ampicillin (3/3), bacitracin (3/3), erythromycin (2/3), penicillin G (3/3), and tetracycline (3/3). The results suggested C. perfringens type C could have contributed to the death of this neonatal porpoise.

Endophytic bacterial community of Stellera chamaejasme L. and its role in improving host plants' competitiveness in grasslands.

Stellera chamaejasme has become a problematic weed in northern and south-western grasslands of China. To evaluate a possible role of endophytes in its strong competitive capacity, the endophytic bacterial community of S. chamaejasme was investigated by culture-dependent and independent methods, and the growth-promoting traits of some culturable isolates as well as the benefit of endophyte ST3CS3 (Brevundimonas sp.) on host plants growth were studied. The results showed that 823 OTUs were generated with a 97% similarity level in the culture-independent study. They were classified into 29 phyla, 61 classes, 147 orders, 237 families and 440 genera. Among them, Pseudomonas and Ralstonia were the most dominant genera in belowground parts (G) (64.25%) and aboveground parts (S) (26.54%) respectively. The diversity and species richness of endophytes in S were significantly higher than that of G (P< 0.001, t-test). Contrary to this, the number of culturable bacteria in S was a little lower than that of G (P> 0.05, t-test). Totally, 176 isolates belonging to 30 morphotypes were obtained in the culture-dependent study. Among them, Acinetobacter was the most dominant genus in G (51.30%), then followed by Pseudomonas (6.09%) and Brevundimonas (6.09%), while Lysinibacillus (21.31%) was the most dominant genus in S, followed by Pseudomonas (11.48%). Growth-promoting trait tests indicated that 93.65% of the tested isolates (63) exhibited nitrogen-fixing, IAA-synthesizing, phosphorus or potassium solubilizing capacity, in which 77.97% belonged to Proteobacteria, a phylum found to contain more active isolates. Pot experiments demonstrated that endophyte ST3CS3 can significantly improve host plants growth and increase its nitrogen and chlorophyll content (P< 0.01, t-test). Therefore, we suggest that strong competitiveness of S. chamaejasme may in part be due to possession of high ratios of plant growth-promoting proteobacterial endophytes such as Pseudomonas, Acinetobacter and Brevundimonas.

Culture-dependent study of arsenic-reducing bacteria in deep aquatic sediments of Bengal Delta.

Biogeochemical release of soil-bound arsenic (As) governs mobilization of the toxic metalloid into the groundwater. The present study has examined AsV-reduction ability of bacteria from anoxic aquatic sediments that might contribute to arsenic mobilization in the Bengal Delta. Arsenic-reducing bacteria from deep layers of pond sediment were enriched and isolated in anaerobic environments and AsV reduction was assessed in culture medium. The pond sediment enrichments harboured AsV-reducing bacteria belonging to the phyla Firmicutes and Proteobacteria with dominance of Paraclostridium benzoelyticum and P. bifermentans. Among total 17 isolates, the respiratory reductase genes were not detected by the most common primers and only 3 strains had arsenic reductase ArsC gene suggesting involvement of resistance and some unknown mechanisms in AsV reduction. Presence of high levels of organic matter, As, and As-reducing bacteria might make deep aquatic sediments a hot spot of As mobilization and aquifer contamination.

Investigation of culturable human gut mycobiota from the segamat community in Johor, Malaysia.

Although several studies have already been carried out in investigating the general profile of the gut mycobiome across several countries, there has yet to be an officially established baseline of a healthy human gut mycobiome, to the best of our knowledge. Microbial composition within the gastrointestinal tract differ across individuals worldwide, and most human gut fungi studies concentrate specifically on individuals from developed countries or diseased cohorts. The present study is the first culture-dependent community study assessing the prevalence and diversity of gut fungi among different ethnic groups from South East Asia. Samples were obtained from a multi-ethnic semi-rural community from Segamat in southern Malaysia. Faecal samples were screened for culturable fungi and questionnaire data analysis was performed. Culturable fungi were present in 45% of the participants' stool samples. Ethnicity had an impact on fungal prevalence and density in stool samples. The prevalence of resistance to fluconazole, itraconazole, voriconazole and 5-fluorocytosine, from the Segamat community, were 14%, 14%, 11% and 7% respectively. It was found that Jakun individuals had lower levels of antifungal resistance irrespective of the drug tested, and male participants had more fluconazole resistant yeast in their stool samples. Two novel point mutations were identified in the ERG11 gene from one azole resistant Candida glabrata, suggesting a possible cause of the occurrence of antifungal resistant isolates in the participant's faecal sample.

Bacterial communities of sponges from the wetland ecosystem of Little Rann of Kutch, India with particular reference to Planctomycetes.

Five sponge specimens belonging to the generaSpongillaandCiocalyptawere collected from Little Rann of Kutch (in Gujarat, India) and analysed for associated microbiomes. Critical analysis was done with respect to members of the phylum Planctomycetes, using two different strategies; 1. Culture-independent metagenomic approach and 2. culture-dependent anaerobic enrichment for anammox-planctomycetes. The 16S rRNA gene (V1-V3 region) amplicon metagenome analysis revealed significant divergence in bacterial diversity, including Planctomycetes among the sponges analysed. Community metagenomics revealed a total of 376 Operational Taxonomic Units (OTUs) belonging to 41 different phyla. OTUs belonging to Proteobacteria was the most abundant (38%) among the sponge analysed. The KEGG annotation predicted a total of 6909 KEGG orthologs (KOs); most of the KOs are associated with membrane transport, xenobiotic degradation, production of secondary metabolites, amino acid metabolism and carbohydrate metabolism. In the culture-dependent study, FISH analysis confirmed the association of anammox-planctomycetes with sponges. Partial 16S rRNA gene sequences of two planctomycetes (JC545, JC543) were cladding with those of unculturedPhycisphaeraeclass. The other three putative anammox bacteria (JC541, JC542, JC544) formed a different clade with "CandidatusBrocadia anammoxidans". These three putative bacteria believably represent new species/genus related to "CandidatusBrocadia".

Community structure, taxonomic diversity and spatio-temporal variation of sediment and water bacteria in Bhitarkanika mangrove ecosystem, India

Bacterial diversity of Bhitarkanika mangrove was studied by both the culture-dependent and culture-independent methods. The variation of culture-dependent bacterial population with respect to different physico-chemical parameters was also observed seasonally (summer and monsoon) from five stations—Dangmal, Ekakula, Gupti, Habalikhati and Kalibhanjadiha. Overall, culture-dependent total heterotrophic bacteria varied from 2.16 ± 0.5 to 168.25 ± 1.73 × 103 cfu/g in sediment and 1.53 ± 0.915 to 38.33 ± 0.108 × 103 cfu/ml in water. Major regulatory factors for bacterial abundance of water were nitrate phosphate, conductivity, dissolved oxygen in different seasons while no such clear influence was observed on sediment bacterial abundance. Culture-dependent study revealed that Bacilli, Gammaproteobacteria and Betaproteobacteria were dominant bacterial classes. Culture-independent study revealed that the operational taxonomic unit varied from 5077 to 21,207 in sediment and 14,213 to 17,501 in water. Majority of the bacterial community were found to be under the class Gammaproteobacteria followed by Alphaproteobacteria and Bacilli. In sediment, highest and lowest species richness as well as evenness was recorded at Dangmal and Ekakula, respectively, while in water, both the parameters were more or less similar in all the sampling sites. Maximum species diversity was observed at Dangmal sediment (Shannon diversity index: 10.43) and Habalikhati water (Shannon diversity index: 7.45). Principal Coordinate Analysis (PCoA) revealed that Ekakula, Kalibhanjadiha and Gupti, Habalikhati sediment had similar species composition in culture-dependent and independent analysis, respectively. Dangmal sediment had the unique species composition, whereas bacterial species composition in water varied greatly within different sites in both the cases. This study depicts the highly rich bacterial diversity of Bhitarkanika.

Assessing the potential of culture-independent 16S rRNA microbiome analysis in disease diagnostics: the example of Dianthus gratianopolitanus and Robbsia andropogonis

The goal of this study was to determine if culture-independent 16S rRNA sequencing of plant-associated microbiomes could facilitate disease diagnosis of cheddar pinks (Dianthus gratianopolitanus) with symptoms of leaf spotting at a Virginia nursery. The microbiome composition of cheddar pinks at the same nursery and at a second nursery in the absence of any disease outbreak was determined as well. After the pathogen was identified as Burkholderia andropogonis (synonym: Robbsia andropogonis) in a parallel culture-dependent study, the microbiome of plants artificially inoculated with R. andropogonis was also analyzed. The genus Robbsia was found to be ubiquitously present on all Dianthus gratianopolitanus nursery plants. However, because of the low resolution of 16S rRNA sequencing, it was not possible to determine the presence or absence of the pathogen at the species level. While relative abundance of Robbsia sequences had slightly increased during the disease outbreak, symptomatic plants did not have a significantly higher abundance of Robbsia than asymptomatic plants. Only microbiomes of artificially inoculated plants were dominated by Robbsia. We conclude that culture-independent microbiome analysis using 16S rRNA sequencing was unable to aid disease diagnosis in this specific case. Limitations and potential of the approach in disease diagnosis in general are discussed.

Cyanobacterial diversity in mat sample obtained from hypersaline desert, Rann of Kachchh.

Rann of Kachchh (RoK) is a unique geoformation, which is exposed to dynamic environmental changes such as salinity, temperature, and nutrients throughout the year. In this study, the pooled mat sample was examined for the cyanobacterial community structure using culture-dependent and culture-independent approaches. Taxonomic profiling was studied using amplicon sequencing that revealed the enrichment of Pseudanabaenales and Oscillatoriales by QIIME and MG-RAST, respectively. Other abundant orders were represented by Chroococcales, Nostocales, and unclassified cyanobacteria by both approaches. Nine cyanobacterial cultures were isolated from mat samples showing 90-98% similarities with available sequences in GenBank. The culture-dependent study suggested that mat was dominated by cyanobacterial orders such as Oscillatoriales-filamentous and Chroococcales-unicellular. Our results from the culture-dependent approach also indicated that despite high similarities in gene sequences, six cyanobacteria fall into the separate clade in the phylogenetic analysis that could be signs of evolution due to an extreme environment. Cultured isolates are correlated well with abundant taxa from amplicon sequencing. Further, protein profiling was done specifically for phycobiliproteins which will be helpful to elucidate their roles in light harvesting and energy transfer mechanism in the unique environment of RoK.

Molecular identification and phylogenetic analysis of Bothrops insularis bacterial and fungal microbiota

ABSTRACTBothrops insularis, known as the golden lancehead snake, has its natural habitat restricted to Queimada Grande Island on the southern coast of Brazil. This culture-dependent study aimed to identify microorganisms obtained from the mouth, eyes, and cloaca of this species. Swabs from 20 snakes were collected for fungal and bacterial isolation. DNA was extracted from all samples, and identification was performed by amplifying the ITS1-5.8S-ITS2 regions and the 16S rDNA gene, respectively. All strains were identified and deposited in the GenBank nucleotide database. MEGA v6.0 software was utilized to construct phylogenetic trees. In total, 100 strains were isolated and characterized, from which 42 fungi were distributed into 23 species and 58 bacteria into 13 species. The genus Fusarium was predominant since 11 strains and probably a new species was isolated from this fungus. Pseudomonas aeruginosa and Enterococcus faecalis were the predominant groups of aerobic bacteria isolated. Phylogenetic analyses between bacterial and fungal sequences suggest a similarity between the microorganisms found on the island and on the continent. These findings may be attributed to anthropic actions resulting from both expeditions to the island and actions of migratory birds, which are the main sources of food for snakes.

Genetic diversity and antagonistic activity of plant growth promoting bacteria, isolated from tea-rhizosphere: a culture dependent study

Microbial activity in root environment is responsible for plant nutrition, growth and defence. The objective of this study is to analyse the plant growth promoting and antagonistic activity rendered by the rhizospheric bacteria of tea plant. In this study, 292 bacterial isolates were screened for indole acetic acid (IAA) production, phosphate solubilisation, ammonia production, chitinase production and protease production. Among all the bacterial isolates, 58 isolates were able to elicit minimum four plant growth promoting rhizobacteria (PGPR) traits, which were further analysed quantitatively for hydrogen cyanide (HCN), siderophore production and antagonistic activity. Based on the plant growth promoting potential scores and principle component analysis, 12 samples were further screened for the study of salt tolerance and antifungal activity profile against tea fungal pathogens (Rhizoctonia solani, Fomes lamenensis, Corticium invisum). The identity the isolates was revealed by 16s rDNA sequence analysis. Six isolates (n63, k32, n61, 31k, n57, n56) showing efficient PGPR traits were evaluated for growth promotion studies on rice seedlings. Isolate n61 (Bacillus cereus) induced significant increase in root length (9.85 mm), shoot length (7.86 mm), germination percentage (92.44) and vigour index (1566.42) in rice seedlings. Fresh bio mass was significantly higher in 31k (Pseudomonas putida) (81.33 mg) followed by n61 (Bacillus cereus) (80.84 mg) and dry mass was higher in n63 (Bacillus pseudomycoides) (15.84 mg) followed by n61 (Bacillus cereus ) (15.82 mg) while compared with control and the other isolates selected for in vitro growth experiments. ANOVA analysis showed significant (P<0.05) increase compared with control, indicating that, bacterial isolates are potent for plant growth promotion and productivity.

PCR-DGGE assessment of the bacterial diversity in Spanish-style green table-olive fermentations

The bacterial ecology associated to Spanish-style green olive fermentations has been studied, attending to its dynamics along the time and its distribution, by a culture-independent approach based on PCR-DGGE. Forty-three 10-tonne fermenters were selected from the fermentation yards (patios) of two large table-olive manufacturing companies in southern Spain. The fermenting brines of 20 of these fermenters were previously analyzed through culture-dependent methods, allowing comparisons of both methodologies. A statistical analysis of DGGE banding profiles obtained using bacteria universal primers demonstrated significant evidences of discrimination of bacterial communities by location (patio) and fermentation stage. Specific microbial “fingerprints” could be established for these variables. At least 17 bacterial species were detected, most of them previously isolated from the same fermenters. Most of these species belonged to the lactic acid bacteria (LAB) group. Dominance of species within the Lactobacillus plantarum group was confirmed. Marinilactibacillus sp. and Propionibacterium olivae, which were not isolated in the previous culture-dependent study, were detected. Alkalibacterium sp. and Halolactibacillus sp. were detected for the first time in table olive fermentations. Using Lactobacillus-group specific primers, significant clustering within the DGGE banding profiles was observed, allowing discrimination regarding the actual fermentation stage. These results corroborated the previous culture-dependent study, and added the detection of Alkalibacterium sp. and Pediococcus acidilactici. The species Alkalibacterium sp., Marinilactibacillus sp. and Halolactibacillus sp. are characterized by their ability to carry out lactic acid fermentation under alkaline conditions and thus ascribed within the halophilic and alkaliphilic lactic acid bacteria (HALAB). Their ubiquitous presence suggests that they could play an important role in Spanish-style olive fermentations, especially at the initial fermentation stage. Thus, they could contribute to brine conditioning before L. plantarum group-driven lactic acid fermentation takes place.