Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis and causes a persistent infection that can leave cattle with no symptoms. Many countries have been able to successfully eradicate BLV through improved detection and management methods. However, with the increasing novel molecular detection methods there have been few efforts to standardize these results at global scale. This study aimed to determine the interlaboratory accuracy and agreement of 11 molecular tests in detecting BLV. Each qPCR/ddPCR method varied by target gene, primer design, DNA input and chemistries. DNA samples were extracted from blood of BLV-seropositive cattle and lyophilized to grant a better preservation during shipping to all participants around the globe. Twenty nine out of 44 samples were correctly identified by the 11 labs and all methods exhibited a diagnostic sensitivity between 74 and 100%. Agreement amongst different assays was linked to BLV copy numbers present in samples and the characteristics of each assay (i.e., BLV target sequence). Finally, the mean correlation value for all assays was within the range of strong correlation. This study highlights the importance of continuous need for standardization and harmonization amongst assays and the different participants. The results underscore the need of an international calibrator to estimate the efficiency (standard curve) of the different assays and improve quantitation accuracy. Additionally, this will inform future participants about the variability associated with emerging chemistries, methods, and technologies used to study BLV. Altogether, by improving tests performance worldwide it will positively aid in the eradication efforts.

The Yangtze River Estuary (YRE) is one of the areas in China most severely affected by harmful algal blooms (HABs). This study explored the distributive patterns of HABs in the YRE and how they are influenced by the El Niño-Southern Oscillation (ENSO) and other environmental factors. Quantitative real-time PCR (qPCR) was employed to detect and quantify the four predominant HAB species in the YRE, Karenia mikimotoi, Margalefidinium polykrikoides, Prorocentrum donghaiense, and Heterosigma akashiwo. Additionally, the study analyzed how turbidity, pH, salinity, and temperature influence these algae. Distribution of the four HAB species in the YRE area shows clear geographical variations: K. mikimotoi is predominantly found in the northwest and central sea areas, M. polykrikoides (East Asian Ribotype, EAR) is mainly distributed in the southeastern part, P. donghaiense is abundant in the northern regions, and H. akashiwo is especially prevalent at stations S26 and S27 in the northeastern part of the study area. HABs dominated by H. akashiwo and P. donghaiense were observed in the northeastern sea area of the YRE on July 22, 2020. Our study reveals that K. mikimotoi, M. polykrikoides (EAR), and P. donghaiense are mainly affected by turbidity, pH, and salinity, while temperature predominantly influences the blooms of H. akashiwo. Moreover, runoff in the YRE has a certain correlation with ENSO events, which may also impact the nutrient content of the region. The findings of this study illustrate the distributive patterns of the four HAB species under various ecological conditions in the YRE and emphasize the importance of establishing practical cases for future warning systems. To better understand how climate change affects HABs, exploring the link between ENSO and HABs is essential.

Bovine leukemia virus (BLV) is prevalent worldwide, causing serious problems in the cattle industry. The BLV proviral load (PVL) is a useful index for estimating disease progression and transmission risk. We previously developed a quantitative real-time PCR (qPCR) assay to measure the PVL using the coordination of common motif (CoCoMo) degenerate primers. Here, we constructed a novel duplex BLV-CoCoMo qPCR assay that can amplify two genes simultaneously using a FAM-labeled MGB probe for the BLV LTR gene and a VIC-labeled MGB probe for the BoLA-DRA gene. This liquid duplex assay maintained its original sensitivity and reproducibility in field samples. Furthermore, we developed a dry duplex assay composed of PCR reagents necessary for the optimized liquid duplex assay. We observed a strong positive correlation between the PVLs measured using the dry and liquid duplex assays. Validation analyses showed that the sensitivity of the dry duplex assay was slightly lower than that of the other methods for the detection of a BLV molecular clone, but it showed similar sensitivity to the singleplex assay and slightly higher sensitivity than the liquid duplex assay for the PVL quantification of 82 field samples. Thus, our liquid and dry duplex assays are useful for measuring the BLV PVL in field samples, similar to the original singleplex assay.

Grapevine varieties from \"Douro Superior\" (NE Portugal) experience high temperatures, solar radiation, and water deficit during the summer. This summer\'s stressful growing conditions induce nucleic acids, lipids, and protein oxidation, which cause cellular, physiological, molecular, and biochemical changes. Cell cycle anomalies, mitosis delay, or cell death may occur at the cellular level, leading to reduced plant productivity. However, the foliar application of kaolin (KL) can mitigate the impact of abiotic stress by decreasing leaf temperature and enhancing antioxidant defence. Hence, this study hypothesised that KL-treated grapevine plants growing in NE Portugal would reveal, under summer stressful growing conditions, higher progression and stability of the leaf mitotic cell cycle than the untreated (control) plants. KL was applied after veraison for two years. Leaves, sampled 3 and 5 weeks later, were cytogenetically, molecularly, and biochemically analysed. Globally, integrating these multidisciplinary data confirmed the decreased leaf temperature and enhanced antioxidant defence of the KL-treated plants, accompanied by an improved regularity and completion of the leaf cell cycle relative to the control plants. Nevertheless, the KL efficacy was significantly influenced by the sampling date and/or variety. In sum, the achieved results confirmed the hypothesis initially proposed.

Drug resistance surveillance is a major integral part of malaria control programs. Molecular methods play a pivotal role in drug resistance detection and related molecular research. This study aimed to develop a rapid and accurate detection method for drug resistance of Plasmodium falciparum (P. falciparum). A quantitative real-time PCR (qPCR) assay has been developed that identifies the mutation at locus A256T in the P.falciparum multi-drug resistance(pfmdr1) gene producing amino acid change at position 86. The results of 198 samples detected by qPCR were consistent with nested PCR and sequencing, giving an accuracy of 94.3%. The sensitivity, specificity, positive and negative predictive value of qPCR were 85.7%, 97.6%, 90.0% and 96.4%, respectively. The results of qPCR are basically consistent with the nested PCR, which is expected to replace the nested PCR as a new molecular biological method for drug resistance detection, providing reliable technical support for global malaria prevention and control.

Differences in functioning among various genotypes of arbuscular mycorrhizal (AM) fungi can determine their fitness under specific environmental conditions, although knowledge of the underlying mechanisms still is very fragmented. Here we compared seven homokaryotic isolates (genotypes) of Rhizophagus irregularis, aiming to characterize the range of intraspecific variability with respect to hyphal exploration of organic nitrogen (N) resources, and N supply to plants. To this end we established two experiments (one in vitro and one in open pots) and used 15N-chitin as the isotopically labeled organic N source. In Experiment 1 (in vitro), mycelium of all AM fungal genotypes transferred a higher amount of 15N to the plants than the passive transfer of 15N measured in the non-mycorrhizal (NM) controls. Noticeably, certain genotypes (e.g., LPA9) showed higher extraradical mycelium biomass production but not necessarily greater 15N acquisition than the others. Experiment 2 (in pots) highlighted that some of the AM fungal genotypes (e.g., MA2, STSI) exhibited higher rates of targeted hyphal exploration of chitin-enriched zones, indicative of distinct N exploration patterns from the other genotypes. Importantly, there was a high congruence of hyphal exploration patterns between the two experiments (isolate STSI always showing highest efficiency of hyphal exploration and isolate L23/1 being consistently the lowest), despite very different (micro) environmental conditions in the two experiments. This study suggests possible strategies that AM fungal genotypes employ for efficient N acquisition, and how to measure them. Implications of such traits for local mycorrhizal community assembly still need to be understood.

Canine circovirus (CanineCV), which is a new mammalian circovirus first reported in the United States in 2012, mainly causes diarrhea and vomiting in dogs. As CanineCV evolves and new subtypes emerge, there is an urgent need for new detection technologies to improve the sensitivity and detection rates of viruses in complex scenarios. A chip digital PCR(cdPCR) assay was established for the detection of CanineCV in this study. The results showed good reproducibility, specificity and a linear relationship; the minimum detection limit of CanineCV by cdPCR was 6.62 copies/μL, which is 10 times more sensitive than quantitative real-time PCR (qPCR). The qPCR-positive detection rate was 1 %, while CanineCV cdPCR (2.1 %) exhibited a greater positive detection rate. Fifteen complete genomes were sequenced and subdivided into CanineCV-1 and CanineCV-3. In conclusion, we developed a rapid, reliable, and specific cdPCR method for screening and monitoring canine CV.

In this study, we adopt an interdisciplinary approach, integrating agronomic field experiments with soil chemistry, molecular biology techniques, and statistics to investigate the impact of organic residue amendments, such as vinasse (a by-product of sugarcane ethanol production), on soil microbiome and greenhouse gas (GHG) production. The research investigates the effects of distinct disturbances, including organic residue application alone or combined with inorganic N fertilizer on the environment. The methods assess soil microbiome dynamics (composition and function), GHG emissions, and plant productivity. Detailed steps for field experimental setup, soil sampling, soil chemical analyses, determination of bacterial and fungal community diversity, quantification of genes related to nitrification and denitrification pathways, measurement and analysis of gas fluxes (N2O, CH4, and CO2), and determination of plant productivity are provided. The outcomes of the methods are detailed in our publications (Lourenço et al., 2018a; Lourenço et al., 2018b; Lourenço et al., 2019; Lourenço et al., 2020). Additionally, the statistical methods and scripts used for analyzing large datasets are outlined. The aim is to assist researchers by addressing common challenges in large-scale field experiments, offering practical recommendations to avoid common pitfalls, and proposing potential analyses, thereby encouraging collaboration among diverse research groups.•Interdisciplinary methods and scientific questions allow for exploring broader interconnected environmental problems.•The proposed method can serve as a model and protocol for evaluating the impact of soil amendments on soil microbiome, GHG emissions, and plant productivity, promoting more sustainable management practices.•Time-series data can offer detailed insights into specific ecosystems, particularly concerning soil microbiota (taxonomy and functions).

Environmental RNA (eRNA) analysis is conventionally expected to infer physiological information about organisms within their ecosystems, whereas environmental DNA (eDNA) analysis only infers their presence and abundance. Despite the promise of eRNA application, basic research on eRNA characteristics and dynamics is limited. The present study conducted aquarium experiments using zebrafish (Danio rerio) to estimate the particle size distribution (PSD) of eRNA in order to better understand the persistence state of eRNA particles. Rearing water samples were sequentially filtered using different pore-size filters, and the resulting size-fractioned mitochondrial cytochrome b (CytB) eDNA and eRNA data were modeled with the Weibull complementary cumulative distribution function (CCDF) to estimate the parameters characterizing the PSDs. It was revealed that the scale parameter (α) was significantly higher (i.e., the mean particle size was larger) for eRNA than eDNA, while the shape parameter (β) was not significantly different between them. This result supports the hypothesis that most eRNA particles are likely in a protected, intra-cellular state, which mitigates eRNA degradation in water. Moreover, these findings also imply the heterogeneous dispersion of eRNA relative to eDNA and suggest an efficient method of eRNA collection using a larger pore-size filter. Further studies on the characteristics and dynamics of eRNA particles should be pursued in the future.

Arbuscular mycorrhizal (AM) fungi are supposedly competing with ammonia-oxidizing microorganisms (AO) for soil nitrogen in form of ammonium. Despite a few studies directly addressing AM fungal and AO interactions, mostly in artificial cultivation substrates, it is not yet clear whether AM fungi can effectively suppress AO in field soils containing complex indigenous microbiomes. To fill this knowledge gap, we conducted compartmentalized pot experiments using four pairs of cropland and grassland soils with varying physicochemical properties. To exclude the interference of roots, a fine nylon mesh was used to separate the rhizosphere and mesh bags, with the latter being filled with unsterile field soils. Inoculation of plants with AM fungus Rhizophagus irregularis LPA9 suppressed AO bacteria (AOB) but not archaea (AOA) in the soils, indicating how soil nitrification could be suppressed by AM fungal presence/activity. In addition, in rhizosphere filled with artificial substrate, AM inoculation did suppress both AOB and AOA, implying more complex interactions between roots, AO, and AM fungi. Besides, we also observed that indigenous AM fungi contained in the field soils eventually did colonize the roots of plants behind the root barrier, and that the extent of such colonization was higher if the soil has previously been taken from cropland than from grassland. Despite this, the effect of experimental AM fungal inoculation on suppression of indigenous AOB in the unsterile field soils did not vanish. It seems that studying processes at a finer temporal scale, using larger buffer zones between rhizosphere and mesh bags, and/or detailed characterization of indigenous AM fungal and AO communities would be needed to uncover further details of the biotic interactions between the AM fungi and indigenous soil AO.