MiSeq Sequencing Research Articles

Symbiotic diazotrophs in response to yak grazing and Tibetan sheep grazing in Qinghai-Tibetan plateau grassland soils.

Grazing by local livestock is the traditional human practice in Qinghai-Tibetan Plateau grassland, and moderate intensity grazing can maintain high productivity and diversity of alpine grassland. Grazing ecosystems are often nitrogen-limited, but N2-fixing communities in response to yak grazing and Tibetan sheep grazing in Qinghai-Tibetan Plateau grassland have remained underexplored. In this study, we applied quantitative PCR quantitation and MiSeq sequencing of nifH under yak grazing and Tibetan grazing through a manipulated grazing experiment on an alpine grassland. The results showed that the grazing treatments significantly increased the soil ammonium nitrogen (AN) and total phosphorus (TP), but reduced the diazotrophs abundance. Compared with no grazing treatment, the composition of diazotrophs could be maximally maintained when the ratio of yak and Tibetan sheep were 1:2. The foraging strategies of grazing livestock reduced the legumes biomass, and thus reduced the diazotrophs abundance. Data analysis suggested that the direct key factors in regulating diazotrophs are AN and TP, and the changes of these two soil chemical properties were affected by the dung and urine of herbivore assemblages. Overall, these results indicated that the mixed grazing with a ratio of yak to Tibetan sheep as 1:2 can stabilize the soil diazotrophsic community, suggesting that MG12 are more reasonable grazing regimes in this region.

Dynamics of Bacterial Diversity and Functions with Physicochemical Properties in Different Phases of Pig Manure Composting.

Bacteria are key drivers in regulating ecosystem functions, and understanding the diversity and dynamic changes in bacteria in composting is very important for optimizing compost. This study investigated the structure, composition, and function of bacterial communities in alkaline pig manure compost using Miseq sequencing, PICRUSt2. The ACE and Chao1 indices of the bacterial communities in various phases were significantly different. Bacterial communities of alkaline pig compost were different from neutral and acidic swine manure compost, and there were 438 genera of common bacteria in various stages. The main bacterium was the phylum Firmicutes. There were six genera, including Romboutsia, Clostridium, Terrisporobacter, norank_f_Marinococcaceae, Saccharomonospora, and unclassified_f_Bacillaceae, that were significantly correlated (p < 0.05), or even extremely significantly correlated (p < 0.001), with the physicochemical properties. TOC, moisture, C/N, and Tem were the key factors that caused changes in bacterial communities in composting. PICRUSt2 analysis showed that there were seven functional groups: metabolism (45.02-48.07%), environmental information processing (15.25-16.00%), genetic information processing (16.97-20.02%), cellular processes (3.63-4.37%), human diseases (0.71-0.82%), organismal systems (0.66-0.77%), and unclassified (13.93-14.36%). This study will provide a reference for improving bacteria growth and reproduction conditions in pig manure composting, optimizing the process, and improving the efficiency of composting.

Irreversible community difference between bacterioplankton generalists and specialists in response to lake dredging.

Understanding response of bacterioplankton community responsible for maintaining ecological functions of aquatic ecosystems to environmental disturbance is an important subject. However, it remains largely unclear how bacterioplankton generalists and specialists respond to dredging disturbance. Illumina MiSeq sequencing and statistical analyses were used to evaluate landscape patterns, evolutionary potentials, environmental adaptability, and community assembly processes of generalists and specialists in response to dredging in eutrophic Lake Nanhu. The Proteobacteria and Actinobacteria dominated bacterioplankton communities of generalists and specialists, and abundances of Proteobacteria decreased and Actinobacteria increased after dredging. The generalists displayed higher phylogenetic distance, richness difference, speciation rate, extinction rate, and diversification rate as well as stronger environmental adaptation than that of specialists. In contrast, the specialists rather than generalists showed higher community diversity, taxonomic distance, and species replacement as well as closer phylogenetic clustering. Stochastic processes dominated community assemblies of generalists and specialists, and stochasticity exhibited a larger effect on community assembly of generalists rather than specialists. Our results emphasized that lake dredging could change landscape patterns of bacterioplankton generalists and specialists, whereas the short-term dredging conducted within one year was unable to reverse community difference between generalists and specialists. Our findings extend our understanding of how bacterioplankton generalists and specialists responding to dredging disturbance, and these findings might in turn call on long-term dredging for better ecological restoration of eutrophic lakes.

Genetic resources of macroalgae: Development of an efficient method using microsatellite markers in non-model organisms

Red and brown seaweeds are species with high ecological and economic importance. Here we report the feasibility of cost-effective molecular marker development in 6 species from different phyla. Microsatellite markers of two brown seaweed species Alaria esculenta and Pylaiella littoralis, and of four red seaweed species Calliblepharis jubata, Gracilaria gracilis, Gracilaria dura and Palmaria palmata were identified and characterized in genomic sequences obtained using Double-Digest Restriction site Associated DNA (ddRAD). A total of 64,623,186 reads were generated from two runs of multiplexed Illumina Miseq sequencing, from which 30,636 reads containing microsatellite motifs including 15,443 where primer pairs could be designed. Five hundred seventy-six primers pairs were selected for amplification trials to determine levels of polymorphism. Of these 576 loci, 338 could be amplified and 142 of the 338 loci were polymorphic. A total of 28 usable polymorphic markers were developed in A. esculenta, 18 in P. littoralis, 11 in C. jubata, 14 in G. gracilis, 21 in G. dura and 13 in P. palmata. The overall number of alleles per locus ranged from 2 to 22. These 105 new microsatellite markers will be useful for further studies of population genetics, breeding programs and conservation genetics of these species. Compared with traditional approaches, our study yielded thousands of microsatellite loci for six different species in a short time and with affordable costs. This study, based on the use of ddRADseq, provided preliminary data about the genetic structure and reproduction mode of these six non-model species based on a small number of individuals from two geographically-distant populations and on the genetic structure and reproduction mode of two non-model species, i.e. detection of clonality for two red algae, C. jubata and G. dura and detection of highly genetically divergent populations corresponding probably to different cryptic species under the name P. littoralis.

Comparative evaluation of 16S rRNA metagenomic sequencing in the diagnosis and understanding of bacterial endophthalmitis

ObjectiveTo evaluate the usefulness of metagenomic analysis in the search for causative organisms of bacterial endophthalmitis.Methods and analysisTwenty-one consecutive treatment-naïve patients (13 men and 8 women; mean age, 60.8±19.8 years)...

Precision metagenomics sequencing for food safety: hybrid assembly of Shiga toxin-producing Escherichia coli in enriched agricultural water.

Culture-independent metagenomic sequencing of enriched agricultural water could expedite the detection and virulotyping of Shiga toxin-producing Escherichia coli (STEC). We previously determined the limits of a complete, closed metagenome-assembled genome (MAG) assembly and of a complete, fragmented MAG assembly for O157:H7 in enriched agricultural water using long reads (Oxford Nanopore Technologies, Oxford), which were 107 and 105 CFU/ml, respectively. However, the nanopore assemblies did not have enough accuracy to be used in Single Nucleotide Polymorphism (SNP) phylogenies and cannot be used for the precise identification of an outbreak STEC strain. The present study aimed to determine the limits of detection and assembly for STECs in enriched agricultural water by Illumina MiSeq sequencing technology alone, followed by establishing the limit of hybrid assembly with nanopore long-read sequencing using three different hybrid assemblers (SPAdes, Unicycler, and OPERA-MS). We also aimed to generate a genome with enough accuracy to be used in a SNP phylogeny. The classification of MiSeq and nanopore sequencing identified the same highly abundant species. Using the totality of the MiSeq output and a precision metagenomics approach in which the E. coli reads are binned before assembly, the limit of detection and assembly of STECs by MiSeq were determined to be 105 and 107 CFU/ml, respectively. While a complete, closed MAG could not be generated at any concentration, a complete, fragmented MAG was produced using the SPAdes assembler with an STEC concentration of at least 107 CFU/ml. At this concentration, hybrid assembled contigs aligned to the nanopore-assembled genome could be accurately placed in a neighbor-joining tree. The MiSeq limit of detection and assembly was less sensitive than nanopore sequencing, which was likely due to factors including the small starting material (50 vs. 1 μg) and the dilution of the library loaded on the cartridge. This pilot study demonstrates that MiSeq sequencing requires higher coverage in precision metagenomic samples; however, with sufficient concentration, STECs can be characterized and phylogeny can be accurately determined.

Effects of host species on microbiota composition in Phlebotomus and Lutzomyia sand flies

BackgroundBlood-sucking phlebotomine sand flies are vectors of the protozoan parasites Leishmania spp. Although the intestinal microbiota is involved in a wide range of biological and physiological processes and has the potential to alter vector competence, little is known about the factors that modify the gut microbiota composition of sand flies. As a key step toward addressing this issue, we investigated the impact of host species on the gut bacterial composition in Phlebotomus and Lutzomyia sand flies reared under the same conditions.MethodsBacterial 16S rRNA gene amplification and Illumina MiSeq sequencing were used to characterize the overall bacterial composition of three laboratory-reared sandflies: Phlebotomus papatasi, Ph. duboscqi, and Lutzomyia longipalpis.ResultsOur results showed that the larvae of the three sand fly species harbored almost the same microbes but had different relative abundances. Adult Ph. papatasi and Ph. duboscqi revealed similar microbiome compositions, which were distinct from that of adult Lu. longipalpis. Furthermore, we showed that Ph. papatasi and Ph. duboscqi are hosts for different bacterial genera. The experiment was repeated twice to improve accuracy and increase reliability of the data, and the same results were obtained even when a distinct composition of the microbiome among the same species was identified probably because of the use of different larvae food batch.ConclusionsThe present study provides key insights into the role of host species in the gut microbial content of different sand fly species reared under the same conditions, which may influence their susceptibility to Leishmania infection.Graphical

Microbiota-derived short chain fatty acids in fermented Kidachi Aloe promote antimicrobial, anticancer, and immunomodulatory activities

BackgroundFermented Aloe leaf juice is a commonly used food supplement in Japan. In a previous study, fermentation of A. arborescence juice was performed and the presence of short-chain fatty acids (SCFAs) was confirmed and quantified. Samples were collected before and after the fermentation process to be subjected, in the present study, to DNA extraction, 16S rRNA gene (V3–V4 regions) amplification, and sequencing by the next-generation Illumina MiSeq sequencer. Our work aims to analyze the sequences to assess the bacterial diversity in the juice before and after fermentation, identify the beneficial microbes responsible for the production of SCFAs, and evaluate some of the biological activities of the fermented juice.ResultsData revealed the richness and diversity of the bacterial community in the fermented juice compared to the unfermented control. Relative abundance of bacterial phyla showed that the majority of the microbial community in the test samples corresponded to Pseudomonadota (unfermented; 10.4%, fermented; 76.36%), followed by Bacillota (unfermented; 4.71%, fermented; 17.13%) and then Bacteroidota (unfermented; 0.57%, fermented; 1.64%). For the fermented sample, 84% of Bacillota were lactobacilli. A hierarchically clustered heatmap revealed that Lactobacillus was the most abundant genus in both samples suggesting its involvement in the production of SCFAs.To assess potential health benefits, the anticancer efficacy of the fermented product of A. arborescens was investigated against colorectal cancer (IC50 = 3.5 µg/ml) and liver cancer (IC50 = 6.367 µg/ml) compared to the normal peripheral blood mononuclear cells (PBMCs). Flow cytometric analysis of the cell cycle pattern revealed remarkable population arrest in G0 and G1, however, the highest percentages were mainly in the G1 phase for Hep-G2 (40.1%) and HCT-116 (53.2%) cell lines. This effect was accompanied by early apoptotic profiles of HCT-116 (36.9%) and late apoptosis for Hep-G2 (17.3%). Furthermore, immunomodulatory properties demonstrated a significantly (p < 0.001) reduced percentage of induced TNF-α while enhancing IFN-γ dramatically. For antimicrobial activities, marked broad-spectrum activities were recorded against some bacterial and fungal pathogens (17–37 mm inhibition zone diameter range).ConclusionTherefore, this study affords the basis of bacterial community composition in fermented A. arborescens juice as well as its potential biological benefits.

Effects of Canopy Damage and Litterfall Input on CO2-Fixing Bacterial Communities

Extreme weather events often cause canopy disturbance and litter deposition. To study the CO2-fixing bacterial response to forest damage, we simulated the canopy damage caused by extreme weather with four different treatments: control (CN), canopy trimming + removal of branches and leaves debris (TR), canopy trimming + retaining of branches and leaves debris (TD), and undamaged + transplantation of branches and leaves debris (UD). We used the cbbL gene, which encodes ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO), for Miseq sequencing to analyze the dynamics of community composition of soil CO2-fixing bacteria for five consecutive years after canopy damage. Double treatments of canopy damage and litterfall inputs (TD) facilitate forest restoration better than single treatments (TR or UD). Most soil CO2-fixing bacteria are facultative autotrophic bacteria, and Nitrosospira, Streptomyces, and Saccharomonospora are the main carbon-fixing microorganisms, which have significant differences during the restoration of damaged forest canopy. The forest ecosystem restoration after canopy damage lasted 4–5 years. Rainfall and pH showed a significant negative correlation with most soil CO2-fixing bacteria communities. This study provides a theoretical basis for improving the carbon sequestration capacity of forest soil CO2-fixing bacteria after extreme weather and also provides guidance for forest ecosystem management.

Variations in bacterial diversity and community structure in the sediments of an alkaline lake in Inner Mongolia plateau, China.

Alkaline lakes are a special aquatic ecosystem that act as important water and alkali resource in the arid-semiarid regions. The primary aim of the study is to explore how environmental factors affect community diversity and structure, and to find whether there are key microbes that can indicate changes in environmental factors in alkaline lakes. Therefore, four sediment samples (S1, S2, S3, and S4) were collected from Hamatai Lake which is an important alkali resource in Ordos' desert plateau of Inner Mongolia. Samples were collected along the salinity and alkalinity gradients and bacterial community compositions were investigated by Illumina Miseq sequencing. The results revealed that the diversity and richness of bacterial community decreased with increasing alkalinity (pH) and salinity, and bacterial community structure was obviously different for the relatively light alkaline and hyposaline samples (LAHO; pH < 8.5; salinity < 20‰) and high alkaline and hypersaline samples (HAHR; pH > 8.5; salinity > 20‰). Firmicutes, Proteobacteria and Bacteriodetes were observed to be the dominant phyla. Furthermore, Acidobacteria, Actinobacteria, and low salt-tolerant alkaliphilic nitrifying taxa were mainly distributed in S1 with LAHO characteristic. Firmicutes, Clostridia, Gammaproteobacteria, salt-tolerant alkaliphilic denitrifying taxa, haloalkaliphilic sulfur cycling taxa were mainly distributed in S2, S3 and S4, and were well adapted to haloalkaline conditions. Correlation analysis revealed that the community diversity (operational taxonomic unit numbers and/or Shannon index) and richness (Chao1) were significantly positively correlated with ammonium nitrogen (r = 0.654, p < 0.05; r = 0.680, p < 0.05) and negatively correlated with pH (r = -0.924, p < 0.01; r = -0.800, p < 0.01; r = -0.933, p < 0.01) and salinity (r = -0.615, p < 0.05; r = -0.647, p < 0.05). A redundancy analysis and variation partitioning analysis revealed that pH (explanation degrees of 53.5%, pseudo-F = 11.5, p < 0.01), TOC/TN (24.8%, pseudo-F = 10.3, p < 0.05) and salinity (9.2%, pseudo-F = 9.5, p < 0.05) were the most significant factors that caused the variations in bacterial community structure. The results suggested that alkalinity, nutrient salt and salinity jointly affect bacterial diversity and community structure, in which one taxon (Acidobacteria), six taxa (Cyanobacteria, Nitrosomonadaceae, Nitrospira, Bacillus, Lactococcus and Halomonas) and five taxa (Desulfonatronobacter, Dethiobacter, Desulfurivibrio, Thioalkalivibrio and Halorhodospira) are related to carbon, nitrogen and sulfur cycles, respectively. Classes Clostridia and Gammaproteobacteria might indicate changes of saline-alkali conditions in the sediments of alkaline lakes in desert plateau.

The Potential of Gut Microbiota in Prediction of Stroke-Associated Pneumonia.

Stroke-associated pneumonia (SAP) is a common stroke complication, and the changes in the gut microbiota composition may play a role. Our study aimed to evaluate the predictive ability of gut microbiota for SAP. Acute ischemic stroke patients were prospectively enrolled and divided into two groups based on the presence or absence of SAP. The composition of gut microbiota was characterized by the 16S RNA Miseq sequencing. The gut microbiota that differed significantly between groups were incorporated into the conventional risk scores, the Acute Ischemic Stroke-Associated Pneumonia Score (AIS-APS), and the Age, Atrial fibrillation, Dysphagia, Sex, Stroke Severity Score (A2DS2). The predictive performances were assessed in terms of the area under the curve (AUC), the Net Reclassification Improvement (NRI), and the Integrated Discrimination Improvement (IDI) indices. A total of 135 patients were enrolled, of whom 43 had SAP (31%). The short-chain fatty acids (SCFAs)-producing bacteria, such as Bacteroides, Fusicatenibacter, and Butyricicoccus, were decreased in the SAP group. The integrated models showed better predictive ability for SAP (AUC = 0.813, NRI = 0.333, p = 0.052, IDI = 0.038, p = 0.018, for AIS-APS; AUC = 0.816, NRI = 0.575, p < 0.001, IDI = 0.043, p = 0.007, for A2DS2) in comparison to the differential genera (AUC = 0.699) and each predictive score (AUCAISAPS = 0.777; AUCA2DS2 = 0.777). The lower abundance of SCFAs-producing gut microbiota after acute ischemic stroke was associated with SAP and may play a role in SAP prediction.

Endophytic bacterium Bacillus cereus affects host litter decomposition by regulating soil microbial structure and phosphate mineralization

Background and aimsEndophytic bacteria (EB) commonly exist in plants and are recognized to increase plant growth especially under adverse environmental conditions. We here demonstrate that EB Bacillus cereus can accelerate the decomposition of plant litter and enhance nutrient availability for plant growth. MethodsFirst, we obtained plant litter with and without EB inoculation using a model plant Arabidopsis thaliana in a microcosmic experiment. After that we conducted a litter decomposition experiment to investigate the effect of EB on mass loss, phosphorus availability, and soil microbial community composition. We further evaluated wheat (Triticum aestivum) growth using soils amended with either EB-inoculated litter or with litter alone. ResultsInoculation of EB significantly increased the mass loss of A. thaliana litter in the second to fourth month of decomposition, elevated the activity of alkaline phosphatase in the first month of decomposition, and increased soil available phosphorus at the end of decomposition. Illumina MiSeq sequencing and structural equation models also showed that EB inoculation had pronounced impact on the bacterial diversity and composition in soil. Finally, the wheat growth was significantly promoted in the litter with EB decomposition system. ConclusionEB affected host after life effect likely through accelerating the release of P from plant litter and regulating the composition of soil microorganisms, improving nutrient utilization efficiency in a terrestrial ecosystem.

The Microbiome and Antibiotic Resistome in Soil under Biodegradable Composite Carbon Source Amendment.

The decomposition of biodegradable composite carbon sources generates a large amount of biodegradable microplastics, which may not only furnish microbial denitrification, but might also pose potential environmental risks. In the present study, the effects of different dosages of a biodegradable composite carbon source on the microbial communities, the nitrogen metabolic pathways and the antibiotic resistome were explored through Illumina MiSeq sequencing analysis and metagenomic analysis. The results of partial least-square discriminant analysis (PLS-DA) and analysis of similarity (ANOSIM) demonstrated that the response of the bacterial community to a biodegradable composite carbon source was more obvious than the fungal community. The application of biodegradable microplastics diminished the complexity of the microbial communities to some extent and obviously stimulated denitrification. Antibiotics resistance gene (ARG) dispersal was not evidently accelerated after the addition of biodegradable composite carbon source. Lysobacter, Methylobacillus, Phyllobacterium, Sinorhizobium, Sphingomonas from Proteobacteria and Actinomadura, Agromyces, Gaiella and Micromonospora from Actinobacteria were the major ARG hosts. Overall, the addition of a biodegradable composite carbon source shaped microbial communities and their antibiotic resistance profiles in this study.

Microbial genome (Illumina MiSeq) sequencing of drinking water treatment residuals to evaluate compatibility with environmental applications

The clarification of drinking water leads to the production of large quantities of water treatment residuals (WTRs). DNA was extracted from six WTR samples collected from water treatment plants within the UK to compare their bacterial communities and examine whether factors such as coagulant usage (aluminium versus iron salt), the type of water source (reservoir or river), or leachable chemical composition influence these communities. Bacterial 16S variable region 4 (V4) was amplified and sequenced using Illumina MiSeq sequencing. The most abundant phyla in WTR samples were Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, and Firmicutes, collectively representing 92.77–97.8% of the total bacterial sequences. Statistical analysis of microbial profiles indicated that water source played a significant role in microbial community structure, diversity, and richness, however coagulant type did not. PERMANOVA analysis showed that no single chemical variable (pH, organic matter, or extractable element concentration) influenced microbial composition significantly; however, canonical correspondence analysis of WTR microbiomes yielded a model using all these variables that could be used to explain variations in microbial community structures of WTRs (p < 0.05). No common, potentially toxic cyanobacteria, or related pathogens of concern were found. Analysis with PICRUSt showed that WTRs all had similar predicted microbial functional profiles. Overall, the results indicate that WTRs analysed in this study are unlikely to pose any threat to soil microbial community structure when applied to land as a soil conditioner or enhancer and may help to enhance the soil microbial community.

Remediation Effect and Mechanism of Biochar in Combination with Nitrogen Fertilizer on Cd-contaminated Paddy Soil

Cadmium (Cd) heavy metal pollution has posed serious threats to soil health and the safe production utilization of agricultural products. A pot experiment was conducted to study the effects of biochar (BC) and nitrogen fertilizer with three levels, namely 2.6 g·pot-1 (N1), 3.5 g·pot-1 (N2), 4.4 g·pot-1 (N3) biochar combined with nitrogen fertilizer (BCN1, BCN2, and BCN3), on soil Cd fractions, Cd enrichment, the transport of rice, and soil enzyme activity, as well as the changes in microbial community composition and complex interactions between microorganisms through high-throughput sequencing. The results showed that biochar combined with nitrogen fertilizer led to the transformation of Cd from the exchangeable state to the residue state, and the proportion of the exchangeable state was significantly reduced by 6.2%-14.7%; by contrast, the proportion of the residue state increased by 18.6%-26.4% relative to that in CK. In addition, singular treatments of nitrogen fertilizer enhanced the accumulation capacities of Cd in roots, which increased by 22%-33.5% compared with that in CK. By contrast, the BC and BCN treatments reduced Cd accumulation in roots and the transfer capacity from stems to rice husks and husk to rice. Furthermore, the BCN treatments promoted soil enzyme activities (urease, acid phosphatase, invertase, and catalase). MiSeq sequencing showed that BCN treatments increased the abundance of the main species of soil bacterial microbes (such as Acidobacteriales, Solibacterales, Pedosphaerales, and Nitrospirales). Moreover, co-occurrence network analysis showed that the complexity of the soil bacterial network was enhanced under the N, BC, and BCN treatments. Overall, biochar combined with nitrogen fertilizer reduced soil Cd availability, inhibited the capacity of Cd accumulation and the transport of rice, and improved the soil eco-environmental quality. Thus, using BCN could be a feasible practice for the remediation of Cd-polluted agricultural soil.

Short-term effects of Brevibacillus laterosporus supplemented diet on worker honey bee microbiome: a pilot study

In the current study, honey bees’ diet was supplemented with spores of Brevibacillus laterosporus BGSP11 at concentration of 108 CFU/ml in sucrose solution and its short-term effects on their micro- and mycobiota have been analyzed using Illumina MiSeq sequencing. Obtained results indicate that this treatment does not lead to potentially harmful changes in the bacterial microbiome of worker bees, slightly affecting the composition of core microbiota. Moreover, several potentially beneficial changes have been observed. The treatment has led to a significant increase in the abundance of Snodgrassella alvi, and species from Lactobacillus and Bifidobacterium genera which play important roles in protection against several honey bee pathogens. Simultaneously, B. laterosporus enriched diet have led to almost complete eradication of Enterobacteriaceae family, the taxon that contains several putative pathogen species. On the other hand, the treatment affected mycobiota more profoundly, which was expected considering the greater instability compared to microbiota. Although the observed changes in honey bee mycobiome cannot be considered a priori beneficial or harmful, since the interaction between the bee and its mycobiome has not been sufficiently studied, certain beneficial consequences of the treatment have been observed. They are primarily reflected in the reduction of phytopathogenic fungi that can affect the organoleptic and techno-functional characteristics of honey. In addition, before introducing B. laterosporus in beekeeping practice as a biological agent for pathogen control it is necessary to perform more thorough studies of the impact on the honey bee microbiome, immune system, physiology and economic characteristics of honey bee colonies.

Conservation tillage practices affect soil microbial diversity and composition in experimental fields.

Conservation tillage is a widely used technique worldwide, but the effects of conservation tillage on bacterial community structure are poorly understood. We explored proportional alterations in the bacterial community under different tillage treatments. Hence, this study utilized high-throughput sequencing technique to investigate the structure and assembly processes of microbial communities in different tillage treatments. Tillage treatments included tillage no-straw retention (CntWt), no-tillage with straw retention (CntWntS), tillage with straw retention (CntWtS), no-tillage and no-straw retention (CntWnt). The influence of tillage practices on soil bacterial communities was investigated using Illumina MiSeq sequencing. Different tillage methods and straw retention systems significantly influenced soil parameters such as total potassium and pH were not affected by tillage practices, while straw retention significantly affected soil parameters including nitrogen content, available phosphorus and available potassium. Straw retention decreased bacterial diversity while increased bacterial richness. The effect of straw retention and tillage on bacterial communities was greater than with no tillage. Phylogenetic β-diversity analysis showed that deterministic homogeneous selection processes were dominated, while stochastic processes were more pronounced in tillage without straw retention. Ecological network analysis showed that microbial community correlation was increased in CntWntS and CntWnt. Straw retention treatment significantly increased the relative abundance of bacterial taxa Proteobacteria, Bacteroidetes, and OD1, while Nitrospirae, Actinobacteria, and Verrucomicrobia significantly decreased. The conservation tillage practices significantly affect soil properties, bacterial composition, and assembly processes; however, further studies are required to investigate the impact of different crops, tillage practices and physiological characteristics on bacterial community structure and functions.

Exploration of the candidate beneficial bacteria for P. vannamei culture by core microbiome analysis using amplicon sequencing.

Globally, P. vannamei is the vital species in aquaculture production. Beneficial bacterial exploration of gut, sediment and water were investigated in P. vannamei culture using Illumina Miseq sequencing of 16S RNA V3-V4 hypervariable regions. Predominant phyla identified were Proteobacteria, Tenericutes, Bacteroidetes in gut; Proteobacteria, Bacteroidetes, Planctomycetes in sediment and Cyanobacteria, Proteobacteria and Planctomycetes in water. In total, 46 phyla, 509 families and 902 genera; 70 phyla, 735 families and 1255 genera; 55 phyla, 580 families and 996 genera were observed in gut, sediment and water, respectively. Diversity of microbial communities in respect of observed Operational Taxonomic Units, diversity indices (Shannon and Simpson), richness index (Chao1) were significantly high P(<0.05) in 60 DoC in gut and 30 DoC in sediment. Beta diversity indicated separate clusters for bacterial communities in gut, sediment and water samples and formation of distinct community profiles. Core microbiome in P. vannamei rearing ponds over a time consisted of 9, 21 and 20 OTUs in gut, rearing water and sediment, respectively. This study helps to intervene with suitable beneficial microbes to establish aquaculture system thereby contributes to enhance the productivity, improve water quality and pond bottom condition and control the pathogenic agents at each stage of the culture.

First report of Alstroemeria necrotic streak virus infecting greenhouse bell pepper (Capsicum annuum) in Canada.

Alstroemeria necrotic streak virus (ANSV) is an Orthotospovirus that has been isolated from symptomatic Alstroemeria plant in 2010 (Hassani-Mehraban et al. 2010). It has been shown to infect crops of bell pepper (Capsicum annuum) and tomato (Solanum lycopersicum) (Olaya et al. 2017) which are two of the three biggest greenhouse crops in Canada in terms of production volume and value (Statistic Canada. 2020). In July of 2022, the entire production of bell pepper (all plants) from a greenhouse in Québec was presenting necrotic rings and discoloration in fruit and seemingly healthy leaves. Samples from these infected bell pepper were found to be negative for twenty two common viruses infecting bell pepper by ELISA immunoassay by the Laboratoire d'expertise et de diagnostic en phytoprotection (LEDP) (Québec, Canada). To identify the causal agent, double-stranded RNA was extracted from leaf and fruit of one plant to form two separate samples (leaf and fruit) and used for cDNA library preparations with Nextera XT DNA Sample Prep kit (Illumina, USA). The libraries were sequenced using Illumina Miseq (Fall et al. 2020). The same dsRNA were also sequenced with MinION nanopore sequencing method as described previously (Javaran et al. 2021; Javaran et al. 2023). The obtained raw FASTQ data were processed following the methodology described in Fall et al. 2020 and Javaran et al. 2023. The Miseq sequencing yielded over 2 million reads per sample with a percentage of mapped viral reads ranging from 26.92 to 47.29% of the total number of reads. The leaf samples were positive to Bell pepper endornavirus (BPEV) with the full genome covered 16713 times and Alstroemeria necrotic streak virus (ANSV) with 98% of the genome covered 4929 times. The MinION sequencing yielded 1,028,460 reads and the same viruses were detected with 1288 long reads (mean length of 745bp) assigned to ANSV genome. Both viruses were detected in the leaf and fruit samples. The complete ANSV genome comprising three segments (L, M, and S) was assembled and deposited in GenBank: (OQ261731-OQ261733). These L, M and S segments shown 99% nt identity with an isolate from the Columbia (GenBank: MF469036, MF469037, MF469038). It is interesting that read coverage at near the 2000th position of the S segment, was very low. This phenomenon may suggest a cleavage site nearby by a viral or host factor. ANSV was mainly found in leaf samples and very low numbers of reads in fruit samples. The presence of ANSV was confirmed by RT-PCR using the primers specific to the ANSV nucleocapsid gene Tospo_S_F (5'- CAG AAT CAG GCT GCA TTT AAT TTC C-3') and Tospo_S_R (5'-CAA CGC TTC CTT TAG CAT TAG G-3') (Gallo et al. 2019). The sequences of ∼600 bp amplicons were determined using Sanger sequencing and showed 100% nt identity with Miseq-derived sequences of ANSV. The virus has previously been detected in Colombia (Hassani-Mehraban et al. 2010) and then in California in 2018 (Tian et al. 2020). This is to our knowledge the first detection of ANSV in Canada. Bell pepper is one of the most important crops in Canada and the ANSV vector, the western flower thrips (Frankliniella occidentalis), known to spread the tomato spotted wilt virus (TSWV) is established in Canada (Allen et al. 1986). The detection of ANSV in Canada is line with the hypothesis of an international spread of this virus (Tian et al. 2020) as is it not known to spread through seeds.

Exploring the bacterial genetic diversity and community structure of crude oil contaminated soils using microbiomics

The impact of environmental pollution in air and water is reflected mainly in the soil ecosystem as it impairs soil functions. Also, since the soil is the habitat for billions of organisms, the biodiversity is in turn altered. Microbes are precise sensors of ecological contamination, and bacteria have a key and important function in terms of bioremediation of the contaminated soil. Hence in the current work, we aimed at assessing the unidentified bacterial population through Illumina MiSeq sequencing technology and their community structural changes in different levels of petroleum-contaminated soil and sludge samples (aged, sludge, and leakage soil) to identify unique bacteria for their potential application in remediation. The studies showed that major bacterial consortiums namely, Proteobacteria (57%), Alphaproteobacteria (31%), and Moraxellaceae (23%) were present in aged soil, whereas Proteobacteria (52%), Alphaproteobacteria (33%), and Rhodobacteraceae (28%) were dominantly found in sludge soil. In leakage soil, Proteobacteria (59%), Alphaproteobacteria (33%), and Rhodobacteraceae (29%) were abundantly present. The Venn diagrams are used to analyze the distribution of abundances in individual operational taxonomic units (OTUs) within three soil samples. After data filtering, they were grouped into OTU clusters and 329 OTUs were identified from the three soil samples. Among the 329, 160 OTUs were common in the three soil samples. The bacterial diversity is estimated using alpha diversity indices and Shanon index and was found to be 4.490, 4.073 and 4.631 in aged soil, sludge soil and leakage soil, respectively and similarly richness was found to be 618, 417 and 418. The heat map was generated by QIIME software and from the top 50 enriched genera few microbes such as Pseudomonas, Bacillus, Mycobacterium, Sphingomonas and Paracoccus, were shown across all the samples. In addition, we also analyzed various physicochemical properties of soil including pH, temperature, salinity, electrical conductivity, alkalinity, total carbon, total organic matter, nitrogen, phosphorus and potassium to calculate the soil quality index (SQI). The SQI of aged, sludge and leakage soil samples were 0.73, 0.64, and 0.89, respectively. These findings show the presence of unexplored bacterial species which could be applied for hydrocarbon remediation and further they can be exploited for the same.