Computational Analysis of T-Cell Receptor Repertoire Workflow: From T-Cell Isolation to Bioinformatics Analysis.

The T-cell receptor (TCR) is the key molecule involved in the adaptive immune response. It is generated by the V(D)J recombination, responsible of the enormous diversity of the TCR repertoire, a crucial feature determining the individual capability to response to antigens and to build immunological memory. A pivotal role in the recognition of antigen is played by the hypervariable complementarity-determining region 3 (CDR3) of the V-beta chain of TCR. Investigating the CDR3 supports the understanding of the adaptive immune system dynamics in physiological processes, such as immune aging, and in disease, especially autoimmune disorders in which T cells are main actors. High-throughput sequencing (HTS) paved the way for a great progress in the investigation of TCR repertoire, enhancing the read depth in the process of library generation of sequencing and the number of samples that can be analyzed simultaneously. Therefore, the leverage of big datasets stressed the need to develop computational approach, by bioinformatics, to unravel the characteristics of the TCR repertoire.

Changes in Soil Bacterial and Nematode Communities during Long-term Continuous Cotton Cropping in an Arid Region.

larifying the effects of continuous cotton cropping (CC) on soil biological communities is essential for maintaining agricultural productivity. In this study, high-throughput sequencing was used to study the effects of different CC durations (0-yr, 5-yr, 10-yr, 15-yr, 20-yr, and 25-yr CC treatments) on soil microbial and nematode communities. The results showed that the dominant bacterial phyla were Actinobacteria and Proteobacteria, and the dominant nematode genus was Helicotylenchus in all CC treatments. The richness indexes (ACE and Chao1 index) and diversity index (Shannon index) of bacterial and nematode communities were the highest in the 15-yr and 10-yr CC treatments, respectively. Bacterial community was significantly correlated with soil pH and available potassium (AK), and nematode abundance was significantly correlated with microbial biomass carbon (MBC). Soil bacterial PICRUSt analysis results showed that carbon metabolism and amino acid metabolism were the main metabolic functions of bacteria in the CC treatments. The composition and diversity of soil nematode communities were significantly related to the structure of soil bacterial communities, and the niche breadth of soil bacteria was negatively correlated with that of nematodes. Panagrolaimus and Acrobeles were the main genera of bacterialfeeding nematodes affecting bacterial communities, and their relative abundances were significantly positively correlated with the relative abundance of bacterial communities. Overall, long-term (10-15 years) continuous cotton cropping negatively impacts soil biota and the microecological environment of cotton fields in arid regions.

Insights into cytomegalovirus-associated T cell receptors in recipients following allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Cytomegalovirus (CMV) reactivation is a serious problem in recipients of allogeneic hematopoietic stem cell transplantation. Long-term latency depends on specific T cell immune reconstitution, which identifies various pathogens by T cell receptors (TCRs). However, the mechanisms underlying the selection of CMV-specific TCRs in recipients after transplantation remain unclear. METHODS: Using high-throughput sequencing and bioinformatics analysis, the T cell immune repertoire of seven CMV reactivated recipients (CRRs) were analyzed and compared to those of seven CMV non-activated recipients (CNRs) at an early stage after transplant. RESULTS: The counts of unique complementarity-determining region 3 (CDR3) were significantly higher in CNRs than in CRRs. The CDR3 clones in the CNRs exhibit higher homogeneity compared to the CRRs. With regard to T cell receptor ß-chain variable region (TRBV) and joint region (TRBJ) genotypes, significant differences were observed in the frequencies of TRBV6, BV23, and BV7-8 between the two groups. In addition to TRBV29-1/BJ1-2, TRBV2/BJ2-2, and TRBV12-4/BJ1-5, 11 V-J combinations had significantly different expression levels between CRRs and CNRs. CONCLUSIONS: The differences in TCR diversity, TRBV segments, and TRBV-BJ combinations observed between CNRs and CRRs might be associated with post-transplant CMV reactivation and could serve as a foundation for further research.

Dothistroma septosporum and Dothistroma pini, the causal agents of Dothistroma Needle Blight, are infected by multiple viruses.

Dothistroma septosporum and Dothistroma pini are severe foliar pathogens of conifers. They infect a broad spectrum of hosts (mainly Pinus spp.), causing chlorosis, defoliation of needles, and eventually the death of pine trees in extreme cases. Mycoviruses represent a novel and innovative avenue for controlling pathogens. To search for possible viruses hosted by Dothistroma spp. we screened a subset of isolates (20 strains of D. septosporum and one D. pini) originating from the Czech Republic, Slovenia, Italy and Ireland for viral dsRNA segments. Only five of them showed the presence of dsRNA segments. A total of 21 fungal isolates were prepared for total RNA extractions. RNA samples were pooled, and two separate RNA libraries were constructed for stranded total RNA sequencing. RNA-Seq data processing, pairwise sequence comparisons (PASC) and phylogenetic analyses revealed the presence of thirteen novel putative viruses with varying genome types: seven negative-sense single-stranded RNA viruses, including six bunya-like viruses and one new member of the order Mononegavirales; three positive-sense single-stranded RNA viruses, two of which are similar to those of the family Narnaviridae, while the genome of the third correspond to those of the family Gammaflexiviridae; and three double-stranded RNA viruses, comprising two novel members of the family Chrysoviridae and a potentially new species of gammapartitivirus. The results were confirmed with RT-PCR screening that the fungal pathogens hosted all the viruses and showed that particular fungal strains harbour multiple virus infections and that they are transmitted vertically. In this study, we described the narnavirus infecting D. pini. To our knowledge, this is the first virus discovered in D. pini.

Elucidating the endophytic bacterial and fungal community composition and diversity in the tree fern Alsophila spinulosa through meta-amplicon sequencing.

Plant tissues harbor abundant endophytes, which are crucial for plant growth. Endophytes present in Alsophila spinulosa, which is enriched with medicinal components, have not been isolated and characterized yet. Here we employed meta-amplicon sequencing to identify endophytic species and examined their diversity in the leaves, petioles, roots and stems of A. spinulosa. Our findings revealed 1,247 operational taxonomic units (OTUs) for endophytic bacteria across 210 species and 476 OTUs for endophytic fungi across 222 species. Alpha diversity analysis showed the highest endophytic bacterial diversity in A. spinulosa roots, whereas fungal diversity was similar across the leaf, petiole and root tissues. Fungal diversity in the leaves and petioles was markedly higher than that in the stems. Furthermore, beta diversity analysis revealed similarities in the endophytic bacterial and fungal compositions between the leaves and petioles, whereas the compositions in roots and stems considerably differed from those in the leaves and petioles. At the genus level, the predominant endophytic bacteria were Methylobacterium-Methylorubrum and Pseudomonas, whereas the predominant endophytic fungi were Cutaneotrichosporon and Pseudofabraea. Linear discriminant analysis effect size revealed characteristic endophytic bacterial genera specific to each tissue type and characteristic endophytic fungal genera specifically in the leaves, petioles and roots. The co-occurrence network analysis indicated that the complexity of endophyte networks was the highest in the leaves and the lowest in the stems of A. spinulosa. Overall, this study elucidates the distribution patterns of endophytes in A. spinulosa across various tissues, offering valuable microbial resources for the development of natural products for medicinal application.

Decoding the genomic enigma: Approaches to studying extrachromosomal circular DNA.

Extrachromosomal circular DNA (eccDNA), a pervasive yet enigmatic component of the eukaryotic genome, exists autonomously from its chromosomal counterparts. Ubiquitous in eukaryotes, eccDNA plays a critical role in the orchestration of cellular processes and the etiology of diseases, particularly cancers. However, the full scope of its influence on health and disease remains elusive, presenting a rich vein of research yet to be mined. Unraveling the complexities of eccDNA necessitates a distillation of methodologies - from biogenesis to functional analysis - a landscape we overview in this study with precision and clarity. Here, we systematically outline cutting-edge methodologies from high-throughput sequencing and bioinformatics to experimental validations, showcasing the intricate world of eccDNAs. We combed through a treasure trove of auxiliary research resources and analytical tools. Moreover, we chart a course for future inquiry, illuminating the horizon with potential groundbreaking strategies for designing eccDNA research projects and pioneering new methodological frontiers.

Achieving rapid start-up and efficient nitrogen removal of partial-denitrification/anammox process using organic matter in brewery wastewater as carbon source.

To find a cost-efficient carbon source for the partial denitrification/anaerobic ammonium oxidation (anammox) (PD/A) process, the practicability of using the organic matter contained in brewery wastewater as carbon source was investigated. Quick self-enrichment of denitrifying bacteria was achieved by supplying brewery wastewater as organic carbon source and using the mature anammox sludge as the seeding sludge. The PD/A process was successfully established after 33-day operation and then the average total nitrogen removal efficiency reached 92.29% when the influent CODCr: NO3--N: NH4+-N ratio was around 2.5: 1.0: 0.67. The relative abundance of Thauera increased from 0.03% in the seeding sludge to 54.29% on day 110, whereas Candidatus brocadia decreased from 30.66% to 2.08%. The metagenomic analysis indicated that the sludge on day 110 contained more nar and napA (total of 41.24%) than nirK and nirS (total of 11.93%). Thus NO2--N was accumulated efficiently in the process of denitrification and sufficient NO2--N was supplied for anammox bacteria in the PD/A process. Using brewery wastewater as carbon source not only saved the cost of nitrogen removal but also converted waste into resource and reduced the treatment expense of brewery wastewater.

Microbial community analysis of sand filters used to treat mine water from a closed uranium mine.

Rapid sand filters (RSFs) are employed in a drinking water treatment to remove undesirable elements such as suspended solids and dissolved metal ions. At a closed uranium (U) mine site, two sets of tandemly linked paired RSF systems (RSF1-RSF2 and RSF1-RSF3) were utilized to remove iron and manganese from mine water. In this study, a 16S rRNA-based amplicon sequencing survey was conducted to investigate the core microbes within the RSF system treating the mine water. In RSF1, two operational taxonomic units (OTUs) related to methanotrophic bacteria, Methylobacter tundripaludum (relative abundance: 18.1%) and Methylovulum psychrotolerans (11.5%), were the most and second most dominant species, respectively, alongside iron-oxidizing bacteria. The presence of these OUTs in RSF1 can be attributed to the microbial community in the inlet mine water, as the three most abundant OTUs in the mine water also dominated RSF1. Conversely, in both RSF2 and RSF3, Nevskia sp., previously isolated from the Ytterby mine manganese oxide producing ecosystem, became dominant, although known manganese-oxidizing bacterial OTUs were not detected. In contrast, a unique OTU related to Rhodanobacter sp. was the third most abundant (8.0%) in RSF1, possibly due to selective pressure from the radionuclide-contaminated environment during RSF operation, as this genus is known to be abundant at nuclear legacy waste sites. Understanding the key bacterial taxa in RSF system for mine water treatment could enhance the effectiveness of RSF processes in treating mine water from closed U mines.

Barriers and facilitators to next-generation sequencing use in United States oncology settings: a systematic review.

Aim: Next-generation sequencing (NGS) of solid tumors can inform treatment decisions; however, uptake remains low. This objective of this systematic review was to identify barriers to and facilitators of NGS in US oncology settings.Materials & methods: Embase and MEDLINE were searched in March 2023 for articles published from 2012 to 2023 on barriers and facilitators of NGS adoption for solid tumors. Surveys, interviews and observational studies were eligible. Studies on genetic testing for hereditary cancers and non-US studies were excluded. The Motheral scale, Joanna Briggs Institute critical appraisal checklist and McGill Mixed Methods Appraisal Tool were used to assess study quality. Data were synthesized narratively.Results: Twenty-one studies were included. Study participants were clinicians, payers and administrators. Key barriers included complex reimbursement processes and uncertainties around clinical utility. Including recommendations for NGS in clinical practice guidelines was a key facilitator, although insurance policies were often more restrictive than guideline recommendations.Conclusion: Uptake of NGS is increasing but barriers remain. Changes to the current reimbursement frameworks are needed to increase access to NGS. The impact of implementing the 2018 National Coverage Determination, which allows access to NGS for all Medicare beneficiaries with advanced cancer, is not yet evident in the published literature.

[Box: see text].

Potential prebiotic properties and proliferation mechanism of fermented milk-derived polypeptides.

The purpose of this paper is to investigate the potential prebiotic properties and proliferation mechanism of fermented milk-derived peptides. In this study, fermented milk-derived polypeptides were obtained by extraction, separation, and purification. The purified peptides were used to culture fecal flora in vitro, and the relative abundance and composition of the flora were analyzed by high-throughput 16S rRNA sequencing technology. The results showed that peptides can promote the proliferation of beneficial bacteria Lactococcus in the intestine and inhibit the proliferation of harmful bacteria Escherichia coli-Shigella. The amino acid sequence of polypeptide components was determined and synthesized in vitro to verify the proliferation of intestinal flora; the proliferation mechanism of peptides on Lactococcus lactis was studied using non-targeted LC-MS metabolomics technology. Five important peptides with molecular weights of 1000-2000 Da were identified by LC-MS: GRP1 (LTEEEK), GRP2 (ENDAPSPVM*K), GRP3 (ITVDDK), GRP4 (EAM*APK) and GRP5 (LPPPEK). The results showed that the peptides could affect the arginine biosynthesis pathway and the amino sugar and nucleotide sugar metabolism of Lactococcus lactis. In addition, the peptides increased the expression of organic acids and their derivatives in Lactococcus lactis. This study provides a research basis for expanding the potential sources of new prebiotics and also opens up a new idea for discovering new prebiotics in vitro.

Impact of floods and landslides on rhizosphere bacterial communities: a high-throughput 16S rRNA gene sequencing study.

This study investigates the diversity and composition of soil bacterial communities in the rhizosphere of Attapadi and Nelliyampathy, prominent hill stations in Palakkad district, Kerala, India. The persistent flooding and landslides in 2018 and 2019 significantly impacted agricultural productivity in these regions. Utilizing high-throughput 16S rRNA gene sequencing (Illumina MiSeq), we conducted a comprehensive analysis of soil samples. Correlative assessments between soil parameters and microbial relative abundance at the phylum level revealed noteworthy positive associations. Notably, nitrogen (N) exhibited a positive relation with Crenarchaeota, Chloroflexi, Actinobacteriota, and Acidobacteriota; pH correlated with Firmicutes; organic carbon (OC) with WPS-2; and phosphorous with Proteobacteria. A total of 31,402 operational taxonomic units (OTUs) were identified, with the highest feature counts observed in undisturbed soils from Attapadi (AUD) and Nelliyampathy (NUD) (13,007 and 12,915, respectively). Disturbed soils in Nelliyampathy (ND) and Attapadi (AD) displayed a substantial decline in microbial diversity and composition, harbouring 1409 and 4071 OTUs, respectively. Alpha and beta diversity indices further underscored the more severe impairment of ND soils compared to AD soils. Interestingly, a majority of ND samples were landslide-affected (four out of five), while flood-affected soils accounted for four out of six AD samples. This indicates that landslides exert a more pronounced impact on microbial diversity and composition than floods. The observed decline in microbial count, composition, and diversity, even after 2 years of the disaster, raises concerns about potential threats to agricultural output. The findings emphasize the need for corrective measures, including the incorporation of microbial inoculum, to restore soil fertility in post-disaster landscapes.

Identification and function of microRNAs in hemipteran pests: A review.

Hemiptera is one of the most significant orders of insect pests, including whiteflies, true bugs, aphids, planthoppers, psyllids, and so forth, which have led to substantial economic losses in agricultural industries and have significantly affected food yields through their ability to suck the phloem sap of crops and transmit numerous bacterial and viral pathogens. Therefore, explorations of pest-specific, eco-friendly and easy-to-adopt technologies for hemipteran pest control are urgently needed. To the best of our knowledge, microRNAs (miRNAs), which are endogenous non-coding small RNAs approximately 22 nucleotides in length, are involved in regulating gene expression via the direct recognition and binding of the 3'-untranslated region (3'-UTR) of target messenger RNAs (mRNAs) or by acting as a center of a competitive endogenous RNA (ceRNA) network at the post-transcriptional level. This review systematically outlines the characterization and functional investigation of the miRNA biogenesis pathway in hemipteran pests, such as whiteflies, true bugs, aphids and planthoppers. In addition, we explored the results of small RNA sequencing and functional observations of miRNAs in these pests, and the results suggest that the numerous miRNAs obtained and annotated via high-throughput sequencing technology and bioinformatic analyses contribute to molting development, fitness, wing polyphenism, symbiont interactions and insecticide resistance in hemipteran pests. Finally, we summarize current advances and propose a framework for future research to extend the current data and address potential limitations in the investigation and application of hemipteran miRNAs.

Taxanorm: a novel taxa-specific normalization approach for microbiome data.

BACKGROUND: In high-throughput sequencing studies, sequencing depth, which quantifies the total number of reads, varies across samples. Unequal sequencing depth can obscure true biological signals of interest and prevent direct comparisons between samples. To remove variability due to differential sequencing depth, taxa counts are usually normalized before downstream analysis. However, most existing normalization methods scale counts using size factors that are sample specific but not taxa specific, which can result in over- or under-correction for some taxa. RESULTS: We developed TaxaNorm, a novel normalization method based on a zero-inflated negative binomial model. This method assumes the effects of sequencing depth on mean and dispersion vary across taxa. Incorporating the zero-inflation part can better capture the nature of microbiome data. We also propose two corresponding diagnosis tests on the varying sequencing depth effect for validation. We find that TaxaNorm achieves comparable performance to existing methods in most simulation scenarios in downstream analysis and reaches a higher power for some cases. Specifically, it balances power and false discovery control well. When applying the method in a real dataset, TaxaNorm has improved performance when correcting technical bias. CONCLUSION: TaxaNorm both sample- and taxon- specific bias by introducing an appropriate regression framework in the microbiome data, which aids in data interpretation and visualization. The 'TaxaNorm' R package is freely available through the CRAN repository https://CRAN.R-project.org/package=TaxaNorm and the source code can be downloaded at https://github.com/wangziyue57/TaxaNorm .

Microbial Community Structure and Metabolic Function in the Venom Glands of the Predatory Stink Bug, Picromerus lewisi (Hemiptera: Pentatomidae).

The predatory stink bug, Picromerus lewisi (Hemiptera: Pentatomidae), is an important and valuable natural enemy of insect pests in their ecosystems. While insects are known to harbor symbiotic microorganisms, and these microbial symbionts play a crucial role in various aspects of the host's biology, there is a paucity of knowledge regarding the microbiota present in the venom glands of P. lewisi. This study investigated the venom glands of adult bugs using both traditional in vitro isolation and cultural methods, as well as Illumina high-throughput sequencing technology. Additionally, the carbon metabolism of the venom gland's microorganisms was analyzed using Biolog ECO metabolic phenotyping technology. The results showed 10 different culturable bacteria where the dominant ones were Enterococcus spp. and Lactococcus lactis. With high-throughput sequencing, the main bacterial phyla in the microbial community of the venom glands of P. lewisi were Proteobacteria (78.1%) and Firmicutes (20.3%), with the dominant bacterial genera being Wolbachia, Enterococcus, Serratia, and Lactococcus. At the fungal community level, Ascomycota accounted for the largest proportion (64.1%), followed by Basidiomycota (27.6%), with Vishniacozyma, Cladosporium, Papiliotrema, Penicillium, Fusarium, and Aspergillus as the most highly represented fungal genera. The bacterial and fungal community structure of the venom glands of P. lewisi exhibited high species richness and diversity, along with a strong metabolism of 22 carbon sources. Functional prediction indicated that the primary dominant function of P. lewisi venom-gland bacteria was metabolism. The dominant eco-functional groups of the fungal community included undefined saprotroph, fungal parasite-undefined saprotroph, unassigned, endophyte-plant pathogen, plant pathogen-soil saprotroph-wood saprotroph, animal pathogen-endophyte-plant pathogen-wood saprotroph, plant pathogen, and animal pathogen-endophyte-epiphyte-plant pathogen-undefined saprotroph. These results provide a comprehensive characterization of the venom-gland microbiota of P. lewisi and demonstrate the stability (over one week) of the microbial community within the venom glands. This study represents the first report on the characterization of microbial composition from the venom glands of captive-reared P. lewisi individuals. The insights gained from this study are invaluable for future investigations into P. lewisi's development and the possible interactions between P. lewisi's microbiota and some Lepidopteran pests.

Influence of herbaceous litter thickness on bacterial community structure and physicochemical properties of aeolian sand.

The change and mechanism of soil and soil bacterial diversity during the change of herbaceous litter thickness in desert areas is crucial to understand. In the study, the dominant herbaceous litter mixture in Baijitan National Nature Reserve was selected as the research material, and an experiment was established by adjusting the litter depth. The results showed that the measured values of soil physicochemical factors (total nitrogen, total protein, total potassium, available phosphorus, available potassium, pH, and soil water content) increased with the increase of herbaceous litter mixture thickness in 0-5 cm soil layer. Actinobacteria, Proteobacteria, Acidobacteria, Chloroflexi, and Gemmatimonadetes were the dominant bacterial phyla under different thicknesses of herbaceous litter in 0-5 and 5-10 cm soil layers. Balneimonas, Rubrobacter, and Geodermatophilus were the dominant bacterial genera under different thicknesses of herbaceous litter in 0-5 and 5-10 cm soil layers. There was no obvious change in the α-diversity index of bacterial community the same soil layer, but the α-diversity index in the 0-5 cm soil layer was lower compared to the 5-10 cm soil layer. The results of this study revealed that the change of herbaceous litter thickness had no significant effect on soil bacterial community structure in desert areas.

Sediment grain size regulates the biogeochemical processes of nitrate in the riparian zone by influencing nutrient concentrations and microbial abundance.

Riparian zones play a crucial role in reducing nitrate pollution in both terrestrial and aquatic environments. Complex deposition action and dynamic hydrological processes will change the grain size distribution of riparian sediments, affect the residence time of substances, and have a cascade effect on the biogeochemical process of nitrate nitrogen (NO3--N). However, simultaneous studies on NO3--N transformation and the potential drivers in riparian zones are still lacking, especially neglecting the effect of sediment grain size (SGS). To fill this knowledge gap, we first systematically identified and quantified NO3--N biogeochemical processes in the riparian zone by integrating molecular biotechnology, 15N stable isotope tracing, and microcosmic incubation experiments. We then evaluated the combined effects of environmental variables (including pH, dissolved organic carbon (DOC), oxidation reduction potential, SGS, etc.) on NO3--N transformation through Random Forest and Structural Equation Models. The results demonstrated that NO3--N underwent five microbial-mediated processes, with denitrification, dissimilatory nitrate reduction to ammonium (DNRA) dominated the NO3--N attenuation (69.4 % and 20.1 %, respectively), followed by anaerobic ammonia oxidation (anammox) and nitrate-dependent ferric oxidation (NDFO) (8.4 % and 2.1 %, respectively), while nitrification dominated the NO3--N production. SGS emerged as the most critical factor influencing NO3--N transformation (24.96 %, p < 0.01), followed by functional genes (nirS, nrfA) abundance, DOC, and ammonia concentrations (14.12 %, 16.40 %, 13.08 %, p < 0.01). SGS influenced NO3--N transformation by regulating microbial abundance and nutrient concentrations. RF predicted that a 5 % increase in the proportion of fine grains (diameter < 50 µm) may increase the NO3--N transformation rate by 3.8 %. This work highlights the significance of integrating machine learning and geochemical analysis for a comprehensive understanding of nitrate biogeochemical processes in riparian zones, contributing valuable references for future nitrogen management strategies.

The Effects of Mixed Robinia pseudoacacia and Quercus variabilis Plantation on Soil Bacterial Community Structure and Nitrogen-Cycling Gene Abundance in the Southern Taihang Mountain Foothills.

Mixed forests often increase their stability and species richness in comparison to pure stands. However, a comprehensive understanding of the effects of mixed forests on soil properties, bacterial community diversity, and soil nitrogen cycling remains elusive. This study investigated soil samples from pure Robinia pseudoacacia stands, pure Quercus variabilis stands, and mixed stands of both species in the southern foothills of the Taihang Mountains. Utilizing high-throughput sequencing and real-time fluorescence quantitative PCR, this study analyzed the bacterial community structure and the abundance of nitrogen-cycling functional genes within soils from different stands. The results demonstrated that Proteobacteria, Acidobacteria, and Actinobacteria were the dominant bacterial groups across all three forest soil types. The mixed-forest soil exhibited a higher relative abundance of Firmicutes and Bacteroidetes, while Nitrospirae and Crenarchaeota were most abundant in the pure R. pseudoacacia stand soils. Employing FAPROTAX for predictive bacterial function analysis in various soil layers, this study found that nitrogen-cycling processes such as nitrification and denitrification were most prominent in pure R. pseudoacacia soils. Whether in surface or deeper soil layers, the abundance of AOB amoA, nirS, and nirK genes was typically highest in pure R. pseudoacacia stand soils. In conclusion, the mixed forest of R. pseudoacacia and Q. variabilis can moderate the intensity of nitrification and denitrification processes, consequently reducing soil nitrogen loss.

Involvement of MicroRNAs in the Hypersensitive Response of Capsicum Plants to the Capsicum Chlorosis Virus at Elevated Temperatures.

The orthotospovirus capsicum chlorosis virus (CaCV) is an important pathogen affecting capsicum plants. Elevated temperatures may affect disease progression and pose a potential challenge to capsicum production. To date, CaCV-resistant capsicum breeding lines have been established; however, the impact of an elevated temperature of 35 °C on this genetic resistance remains unexplored. Thus, this study aimed to investigate how high temperature (HT) influences the response of CaCV-resistant capsicum to the virus. Phenotypic analysis revealed a compromised resistance in capsicum plants grown at HT, with systemic necrotic spots appearing in 8 out of 14 CaCV-infected plants. Molecular analysis through next-generation sequencing identified 105 known and 83 novel microRNAs (miRNAs) in CaCV-resistant capsicum plants. Gene ontology revealed that phenylpropanoid and lignin metabolic processes, regulated by Can-miR408a and Can- miR397, are likely involved in elevated-temperature-mediated resistance-breaking responses. Additionally, real-time PCR validated an upregulation of Can-miR408a and Can-miR397 by CaCV infection at HT; however, only the Laccase 4 transcript, targeted by Can-miR397, showed a tendency of negative correlation with this miRNA. Overall, this study provides the first molecular insights into how elevated temperature affects CaCV resistance in capsicum plants and reveals the potential role of miRNA in temperature-sensitive tospovirus resistance.

Characterization of Genetic Diversity in the Capsid Protein Gene of Grapevine Fleck Virus and Development of a New Real-Time RT-PCR Assay.

The grapevine fleck virus (GFkV) is a ubiquitous grapevine-infecting virus found worldwide, is associated with the grapevine fleck complex, and is often found in mixed infections with viruses of the grapevine leafroll complex and/or vitiviruses. Although GFkV has been studied for a long time, limited sequence information is available in the public databases. In this study, the GFkV sequence data available in GenBank and data generated at the Foundation Plant Services, University of California, Davis, were used to perform nucleotide sequence comparisons, construct a phylogenetic tree, and develop a new RT-qPCR assay. Sequence comparisons showed high genetic diversity among the GFkV isolates, and the phylogenetic analyses revealed a new group comprised of GFkV isolates identified in the present study. A new assay, referred to as GFkV-CP, was designed and validated using an existing GFkV positive control together with 11 samples known to be infected with combinations of different marafiviruses and maculaviruses but not GFkV. In addition, the newly designed assay was used in a field survey to screen grapevines from diverse geographical locations that are maintained at the United States Department of Agriculture (USDA) National Clonal Germplasm Repository (NCGR) in Winters, CA.