Miniprep Kit Research Articles

P-492. Quantification of HIV Shedding in Viremic Patients

Abstract Background Identifying pockets of undiagnosed HIV is a critical step in the U.S. Ending the HIV Epidemic (EHE). Wastewater-based epidemiology represents a novel way to build agnostic population-level data about disease distribution. We detected HIV virus in two sewersheds in Northern California. We are now conducting a study to measure how much HIV virus is shed into urine and stool, to better estimate cases represented by wastewater detection. HIV-1 RNA Detected in Concentrated Urine Methods We are recruiting participants from HIV clinics in San Mateo and Santa Clara Counties. Patients must be 18 years or older with HIV-1 viral load of >10,000 copies/ml at the time of recruitment. Subjects are asked to provide 5 weekly self-collected blood (Tasso), urine, and stool samples. The plasma is analyzed by established HIV-1 RNA quantitative PCR techniques. Nucleic acids from urine, concentrated (centrifuged) urine, and stool are suspended in DNA/RNA Shield, then extracted using the ZymoBIOMICS DNA/RNA Miniprep Kit (ZymoBIOMICS #R2002). HIV genome-specific primers target the Long Terminal Repeat (LTR) region of the HIV-1 genome. Droplets for digital droplet PCR are generated using the AutoDG Automated Droplet Generator. Reverse transcriptase PCR, which quantifies RNA and DNA combined, and direct PCR, which quantifies DNA, is then carried out with the Mastercycler Pro. Thresholding is performed using QX Manager Software. HIV-1 RNA Detected in Stool Results Two patients have been enrolled to date. Patient 1 was started on antiretroviral therapy after the first sample was obtained; Patient 2 was started on antiretroviral therapy 10 days before the first sample was collected. HIV-1 RNA was found in 87.5% (7/8) of concentrated urine samples, including 85.7% (6/7) of samples obtained while on ART (Table 1). Among stool samples, 25.0% (2/8) yielded HIV RNA, but only 14.3% (1/7) of those obtained on ART (Table 2). Nucleic acids were not detected in unconcentrated urine. Plasma viral load testing is pending, so only the initial clinical HIV-1 viral loads at enrollment are shown. Conclusion HIV-1 RNA is present in concentrated urine but also in stool samples of viremic patients, even up to five weeks after initiation of ART. When the study is complete, our results will bolster how to interpret HIV-1 detected in wastewater. Disclosures All Authors: No reported disclosures

Exploring animal food microbiomes and resistomes via 16S rRNA gene amplicon sequencing and shotgun metagenomics.

As a diverse and complex food matrix, the animal food microbiota and repertoire of antimicrobial resistance (AMR) genes remain to be better understood. In this study, 16S rRNA gene amplicon sequencing and shotgun metagenomics were applied to three types of animal food samples (cattle feed, dry dog food, and poultry feed). ZymoBIOMICS mock microbial community was used for workflow optimization including DNA extraction kits and bead-beating conditions. The four DNA extraction kits (AllPrep PowerViral DNA/RNA Kit, DNeasy Blood & Tissue Kit, DNeasy PowerSoil Kit, and ZymoBIOMICS DNA Miniprep Kit) were compared in animal food as well as the use of peptide nucleic acid blockers for 16S rRNA gene amplicon sequencing. Distinct microbial community profiles were generated, which varied by animal food type and DNA extraction kit. Employing peptide nucleic acid blockers prior to 16S rRNA gene amplicon sequencing was comparable with post-sequencing in silico filtering at removing chloroplast and mitochondrial sequences. There was a good agreement between 16S rRNA gene amplicon sequencing and shotgun metagenomics on community profiles in animal feed data sets; however, they differed in taxonomic resolution, with the latter superior at resolving at the species level. Although the overall prevalence of AMR genes was low, resistome analysis of animal feed data sets by shotgun metagenomics revealed 10 AMR gene/protein families, including beta-lactamases, erythromycin/lincomycin/pristinamycin/tylosin, fosfomycin, phenicol, and quinolone. Future expansion of microbiome and resistome profiling in animal food will help better understand the bacterial and AMR gene diversity in these commodities and help guide pathogen control and AMR prevention efforts.IMPORTANCEWith the growing interest and application of metagenomics in understanding the structure/composition and function of diverse microbial communities along the One Health continuum, this study represents one of the first attempts to employ these advanced sequencing technologies to characterize the microbiota and AMR genes in animal food. We unraveled the effects of DNA extraction kits on sample analysis by 16S rRNA gene amplicon sequencing and showed similar efficacies of two strategies at removing chloroplast and mitochondrial reads. The in-depth analysis using shotgun metagenomics shed light on the community compositions and the presence of an array of AMR genes in animal food. This exploration of microbiomes and resistomes in representative animal food samples by both sequencing approaches laid important groundwork for future metagenomic investigations to gain a better understanding of the baseline/core microbiomes and associated AMR functions in these diverse commodities and help guide pathogen control and AMR prevention efforts.

Comparative evaluation of commercial DNA isolation approaches for nanopore-only bacterial genome assembly and plasmid recovery

The advent of Oxford Nanopore Technologies has undergone significant improvements in terms of sequencing costs, accuracy, and sequencing read lengths, making it a cost-effective, and readily accessible approach for analyzing microbial genomes. A major challenge for bacterial whole genome sequencing by Nanopore technology is the requirement for a higher quality and quantity of high molecular weight DNA compared to short-read sequencing platforms. In this study, using eight pathogenic bacteria, we evaluated the quality, quantity, and fragmented size distribution of extracted DNA obtained from three different commercial DNA extraction kits, and one automated robotic platform. Our results demonstrated significant variation in DNA yield and purity among the extraction kits. The ZymoBIOMICS DNA Miniprep Kit (ZM) provided a higher purity of DNA compared to other kit-based extractions. All kit-based DNA extractions were successfully performed on all twenty-four samples using a single MinION flow cell, with the Nanobind CBB Big DNA kit (NB) yielding the longest raw reads. The Fire Monkey HMW-DNA Extraction Kit (FM) and the automated Roche MagNaPure 96 platform (RO) outperformed in genome assembly, particularly in gram-negative bacteria. Based on our finding, we recommend a minimum read coverage and raw read N50, obtained from the appropriate DNA extraction kit for each bacterial species, to optimize genome assembly and plasmid recovery. This approach will assist end-users in selecting the most effective kit-based extraction method for bacterial whole-genome assembly using only long-read nanopore sequences.

PSXII-18 Nitrogen balance and ruminal bacterial diversity indexes of Nellore cattle fed with amylolytic and fibrolytic enzymes in feedlot

Abstract This study was carried out to evaluate the use of dietary exogenous enzyme (ENZ) products with amylolytic and fibrolytic activity on nitrogen balance and ruminal alpha and beta diversity of Nellore cattle in feedlot. Rumen cannulated Nellore steers [n = 10; body weigh (BW) = 543 kg ± 28.6 kg] were distributed in a replicate 5 × 5 Latin-square design using individual pens. The treatments consisted of a negative control (CON; without ENZ); Amylase [AML, Amaize, Alltech; 0.5 g/kg of diet dry matter (DM); Xylanase (FBL, Fibrozyme, Alltech; 0.9 g/kg of diet DM]; Half dose (HD; AML 0.25 g/kg of diet DM and FBL 0.45 g/kg of diet DM); and Full dose (FD; AML 0.5 g/kg of diet DM and FBL 0.90 g/kg of diet DM; Table 1). The experimental period lasted 19 d (14 d of adaptation and 5 d of collection). Between d 15 and 18, total urine collection was performed to calculate the nitrogen balance. On d 19 a mixed ruminal sample (6 mL of solid and liquid content) was taken 4 h after feeding and stored at -80 ºC until DNA extraction. Total genomic DNA was extracted using the ZymoBIOMICS DNA Miniprep Kit. The library was prepared using primers for the region V3V4 of the16S rRNA gene; 341F (5’-CCTAYGGGRBGCASCAG-3’) x 806R (5’- GGACTACNNGGGTATCTAAT-3’). The amplicons (~ 470 bp) were sequenced with the Illumina NovaSeq 6000 PE 250 platform. The sequences were analyzed using qiime2 with the SILVA database for the taxonomic assignment. Alpha and beta diversities were calculated in Rstudio using phyloseq. All data were analyzed as orthogonal contrasts assuming treatment as a fixed effect: 1) CON vs. ENZ, 2) AML vs. FBL, 3) HD vs. FD, and 4) AML + FBL vs. HD + FD. Animals that received ENZ had reduced fecal nitrogen excretion (FNE, P = 0.014) and greater efficiency of nitrogen utilization (ENU, P = 0.031) than animals in the CON group. The FBL treatment resulted in greater CP/DOM ratio (P = 0.049), nitrogen balance (P = 0.089), and ENU (P = 0.015) compared with AML treatment. No differences were observed between HD and FD for any nitrogen balance variable (P > 0.10). Animals supplemented with both enzymes (HD + FD) had greater values of N intake (P = 0.018), greater CP/DOM ratio (P = 0.006), and greater FNE (P = 0.002) than animals fed either enzyme alone (AML + FBL). The ENZ supplementation did not impact alpha diversity index or beta diversity (P > 0.10). In conclusion, the addition of exogenous enzymes affected nitrogen balance but not ruminal alpha and beta diversity of Nellore cattle in feedlot.

145 Utilization of 16s sequencing data and physiological measurements to produce a comparative analysis of the equine microbiome

Abstract Rapid advancements in microbiome research, particularly in the technologies associated with next-generation sequencing have enabled unprecedented investigations into the composition of the equine microbiome. The large datasets produced from these studies often present challenges to researchers seeking to understand the applicability of these data to equine physiology. To that end, a multi-year project was conducted with the goal of determining the core bacterial population of the equine hindgut through analysis of rectal swabs collected from thousands of horses and applying logic to the association between these ecologies and equine health. Samples were obtained from horses that varied across a diverse range of considerations including location, breed, age, and diet, amongst other metadata points including disease state, health history, vaccination and deworming status, body condition, and pregnancy status. These data were used in the development of a comparative score, that authors defined as the Microbiome Quotient (MQ score). Rectal swabs were collected, and DNA was extracted using the Quick-DNA Fecal/Soil Microbe Miniprep kit (Zymo Research, Irvine, CA) and the V3-V4 hypervariable region of the 16S rRNA gene was sequenced by following the Illumina 16S Protocol (San Diego, CA). Sequences were denoised using DADA2 within the QIIME2 pipeline to obtain amplicon sequence variant composition. These data were filtered again in R (4.2.2) to remove samples with missing data and low sequencing depth. Sample metadata was used to classify samples as either typical or atypical. The MQ score was calculated by computing the Euclidean distance difference between a sample and the centroids representing typical and atypical microbial ecologies, and then subtracting these distances to obtain the individual score. This score quantifies how similar the microbiome in a sample is to those of typical and atypical horses. These scores were then categorized into tertiles, representing the status of the individual horse’s microbiome. These statuses indicate differences in bacterial ecology among samples and their subsequent relevance to the health of the horse. While a large portion of the equine fecal microbiome is conserved through homeostatic and other mechanisms, there remains a high degree of variability in the lesser abundant genera that may be responsible for the different physiological interactions between the microbes and their hosts. Utilizing this MQ score, feeding and management recommendations can be made, providing veterinarians and horse owners with a tool to support the health and performance of horses through modulation of the bacterial population and its functionality. Further research is necessary to fully elucidate the impact of the various metadata components on the bacterial ecology. Additionally, the function of the metagenome is a critical component of microbiome sciences. Prediction of metagenomic function is likely to provide insight into the regulation of physiological function by the microbial population residing within the host.

ISOLATION AND ANALYSIS OF NATIVE DNA AND RNA FROM ROOTS OF RUBBER-PRODUCING SPECIES SCORZONERA TAU-SAGHYZ LIPSCH. ET G.G. BOSSE

Scorzonera tau-saghyz Lipsch. et G.G.Bosse, is endemic species, growing in South Kazakhstan and alternative (to Hevea brasilians) source of natural rubber, related to the Asteraceae Family. Natural rubber (NR) is an indispensable raw material for the production of different products for medicine, automotive and aviation industries. The introduction of tau-saghyz into the culture will make it possible to obtain NC not only in Kazakhstan, but also on the territory of the countries of the Northern latitudes. At the growing phases of ontogenesis of tau-saghyz (April-June) latex is actively synthesized in the leaves and transported to the roots. In our experiments it was necessary to conduct molecular genetic studies of the selected forms of tau-saghyz with high rubber concentration. In this regard, it became necessary to isolate and analyze native DNA and RNA from dry roots. Samples with the highest rubber content were used for DNA extraction. The main problem in the isolation of DNA from the dry roots of tau-saghyz is the significant content of natural rubber. In this research the following commercial kits were used: 1) DNeasy Plant Mini Kit (Qiagene); 2) GenElute Plant Genomic DNA Miniprep Kit (Sigma); 3) NucleoSpin® Plant II / Midi / Maxi kit (Macherey-Nagel). Further DNA isolation was carried out in accordance with the kit manufacturer's protocol. Quantitative DNA analysis was performed using both UV spectrophotometry (Nanodrop) and fluorometric analysis (Qubit) method. DNA extracted by the 2 kit (Sigma) was further purified by precipitation with ethanol and demonstrates satisfactory quality. Functional analysis of isolated DNA samples was performed both by restriction analysis and by DNA amplification. It is worth to mention that for reaction primers that are characteristic of gene AACT (ACETOACETYL-COA TRANSFERASE) Cucumis melo were used. This enzyme catalyzes the first reaction of biosynthesis in the mevalonate pathway, which the final product is IDP (isopentenyl diphosphate). The cis-prenyltransferase (CPT) enzyme catalyzes the biosynthesis of rubber by the sequential addition of IDP to the terminal group of the initiating allylic pyrophosphate (APP). As a result of PCR analysis a putative 1200 bp AAST gene for tau-saghyz was identified. In this study two methods of RNA isolation were chosen: 1. A method widely used for plants of the Cucurbitaceae family; 2. A method approved for plants with a high content of polyphenols. The RNA isolation was carried out as described in the relevant protocols with minor modifications (Verwoerd TC, Dekker BMM, Hoekema A (1989) A small-scale procedure for the rapid isolation of plant RNAs. Nucleic Acids Res 17:2362). The final precipitates were dissolved in 50 µl of water pretreated with diethylpyrocarbonate. Quantitative analysis of RNA was carried out using UV spectrophotometry (Nanodrop). Functional analysis of extracted RNA samples was conducted by cDNA synthesis with usage of reverse transcriptase Superscript IV with the following amplification in PCR. In this reaction the primers specific for CPT gene of Taraxacum kok-saghyz were used. The prospective CPT gene was excised from the gel, purified and cloned into the pJET vector. In the further research it is planned to perform its sequencing and comparison with the CPT gene of Taraxacum kok-saghyz and Hevea brasiliensis.

GENETIC DISTANCE AND IDENTITY AT IGF-1 GENE LOCUS OF SOME INDIGENOUS CATTLE BREEDS IN NIGERIA

An experiment was conducted to determine the genetic distance and identity at insulin-like growth factor 1 (IGF-1) gene locus in some indigenous cattle breeds in Nigeria. One (1) Local Government Area (LGA) each from Adamawa, Taraba, Gombe and Sokoto States was purposively selected which include Mubi North, Yorro, Balanga, and Wurno Local Government Areas, respectively. The snowball method was used to sample (96) cattle consisting of (24) Adamawa Gudali, (24) Sokoto Gudali, (24) Bunaji and (24) Rahaji cattle from pastoralists in many communities of the LGAs. Blood samples were collected from the animals through jugular venepuncture. DNA extraction was done using Zymo Quick DNA™ Mini prep kit. NanoDrop 1000 spectrophotometer was used for the determination of DNA quality and quantity using DNA purification kit. Genetic distances among the breeds of cattle were calculated using distance measures with the aid of Gen AIEx software package. The highest level of genetic distance estimate was observed between Rahaji and Bunaji cattle breeds (0.085) while the closest genetic distance (0.001) was observed between Sokoto Gudali and Bunaji. The highest genetic identities were recorded between Sokoto Gudali and Bunaji (0.999) cattle breeds whereas the closest matrix of genetic identity (0.904) was recorded between Rahaji and Sokoto Gudali cattle. Cluster analysis using UPMGA revealed that Bunaji and Sokoto Gudali cattle formed the first cluster which is separate from Adamawa, which formed the second cluster. Rahaji cattle formed the third cluster, which is far and distant from the first and second clusters at IGF-1 gene locus. Rahaji cattle had the longest genetic distance with other three breeds of cattle. High genetic distance and identity existed among some indigenous breeds of cattle studied.

First report of two Pantoea species causing bacterial leaf blight on wheat in the United States.

Wheat (Triticum aestivum) is an important crop worldwide, contributing to about one third of the global caloric intake. In June 2021, leaves with bacterial blight symptoms, including yellow and necrotic lesions running parallel to veins, were found in several fields across five counties in eastern Colorado (Weld, Morgan, Sedgwick, Baca, and Kit Carson). Plants exhibiting these symptoms were scattered throughout fields, but symptoms appeared consistent across counties. To determine the causal agent and complete Koch's postulates, a 1 cm2 symptomatic leaf area was excised and macerated in 0.5 mL of sterilized water from four field samples. The lysate was spread on yeast extract dextrose calcium carbonate medium (YDC agar, 1% yeast extract, 2% dextrose, 2% calcium carbonate, 1.5% agar) to isolate bacteria. Single colonies of yellow, mucoid morphology were selected and streaked on new YDC plates. Isolate genomic DNA was extracted (Zymo Research Quick-DNA Fungal/Bacterial Miniprep Kit, #D6005), and ~30 ng of gDNA was used to amplify the 16S rRNA, gyrB, and rpoB genes of all four isolates (Barret et al., 2015; Delétoile et al., 2009; Krawczyk et al., 2020; Ogier et al., 2019). Amplified PCR products were cleaned (Zymo DNA Clean & Concentrator kit, #D4033) and Sanger sequenced, and all sequences have been deposited in NCBI (16S rRNA: OR707336, OR707337, OR707338, OR707339), (gyrB: PP407951, PP407952, PP407953, PP407954), (rpoB: PP407955, PP407956, PP407957, PP407958). A BLAST search against whole genomes identified one isolate from Kit Carson county (CO314) and two isolates from Baca county (CO316 and CO317) as Pantoea agglomerans with 100% identity for the 16S rRNA, gyrB, and rpoB genes, and one isolate from Weld county (CO315) was 100% identical to Pantoea allii for all three genes. To complete Koch's postulates and confirm Pantoea sp. as the causal disease agents, isolates were grown as lawns on DifcoTM Nutrient Agar (NA) medium (48h, 28℃), suspended in 10 mM MgCl2 using a final optical density of 0.1 (~109 colony forming units per milliliter (CFU/mL)), and syringe-infiltrated into the entire leaf area of 10-day-old wheat seedling leaves (var. Hatcher). Treatments of 10mM MgCl2 and a field isolate that does not cause symptoms, identified as Pseudomonas synxantha by 16S rRNA and gyrB sequencing, were negative controls. Inoculated wheat plants were transferred to a growth chamber (22℃, 90% relative humidity). Symptoms developed 14 days post inoculation (dpi), with the most severe appearing 21 dpi. Each of the four Pantoea isolates were re-isolated from symptomatic leaves by grinding them in a Tissue Lyser II (Qiagen) with two metal beads and diluting with 0.4 mL of sterile water. A 20 µL sample of each isolate was plated on NA (24h, 28℃). The colonies appeared phenotypically identical to the original isolates, and Sanger sequencing confirmed the identities of the isolates. To our knowledge, this is the first report of P. agglomerans causing disease in wheat in the United States, and the first report of P. allii as a wheat disease-causing agent. This report is consistent with previous communications showing P. agglomerans causing wheat disease in China (Gao et al., 2023), and P. ananatis in Poland (Krawczyk et al., 2020). The growing numbers of reports of Pantoea spp. as pathogens in recent years suggests increasing novel disease emergence on cereals worldwide.

Genetic Diversity of Panulirus Versicolor in the Waters of Barrang Caddi Island, Makassar Strait, South Sulawesi, Indonesia

Barong shrimp or lobster (Panulirus versicolor) is a fishery commodity with high economic value and great demand. This study aims to determine the genetic characteristics of Panulirus versicolor at the fishing location in the waters of Barrang Caddi Island, Makassar Strait, using primers LCO-1490F and HCO-2198R. Genomic DNA extraction was performed using a Quick-DNA Tissue Miniprep Kit for total isolation. For DNA purity, the elution buffer utilized in this research was specifically selected for its suitability for PCR amplification with KOD FX Neo, providing optimal conditions for efficient genetic analysis. The primary genes used were LCO-1490F with primers GGT CAA CAA ATC ATA AAG ATA TTG G, and HCO-2198R with TAA ACT TCA GGG TGA CCA AAA AAT CA. The study utilized a 1% agarose gel in a 1X TBE buffer at 50 voltage for 45 minutes to move the total DNA and PCR products. The analysis results at sampling stations 0.2-03 showed increasingly smaller genetic distances. Based on the results of BLASTn analysis, each station had the same type of Panulirus versicolor with a similarity of 99-100%. The morphological characteristics included a carapace with a black spot and black and white lines on each abdominal segment. The analysis of genetic variation in the lobster population on Barrang Caddi Island using primers LCO-1490F and HCO-2198R obtained a DNA fragment of 700 bp. The population of Panulirus versicolor lobster on Barrang Caddi Island showed a high level of similarity or low diversity.

The yield and purity of DNA extracts from seeds of eight six accessions of Treculia species using Zymo Research mini-prep DNA extraction kit

The yield and purity of DNA extracts from seeds of eight six accessions of Treculia species using Zymo Research mini-prep DNA extraction kit

Embryo Microinjection for Transgenesis in Drosophila.

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope.Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example andpresents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

First report of Candidatus Phytoplasma australasia (16SrII- subgroup D) associated with virescence of Chia (Salvia hispanica L.) from India.

Chia (Salvia hispanica L., Lamiaceae) is an important commercial and medicinal crop recently popularized in India and widely cultivated in Karnataka (Joy et al., 2022). During the field survey of chia crop diseases, characteristic virescence like symptoms were observed at Main Agricultural Research Station, UAS, Raichur as well as at Mysuru and HD Kote region. The incidence was ranged from 2 - 4 per cent in an area of 30 hectares. Typical symptoms associated with chia are malformed shoot and/or inflorescence axis with reduced floral parts with greenish florets. The stem axis become thick, flattened, leaves are reduced towards terminal region. A total of five phytoplasma suspected samples and five suspected healthy samples were used for identification purpose. The Plant Genomic DNA Miniprep Kit (Sigma Aldrich, USA) was used to extract the DNA from five symptomatic and five asymptomatic samples and the DNA was used as template to amplify the phytoplasma-specific 16S rDNA gene using P1/P7 primers (Deng and Hiruki, 1991; Schneider et al., 1995) followed by nested PCR using R16F2n/R16R2 primers (Gundersen and Lee 1996). The expected 1.25-kb amplicon was detected from the suspected symptomatic samples. Nested PCR products were purified and sequenced from both the directions using ABIX370 Genetic Analyzer (Applied Biosystems, Waltham, MA). The analysis revealed that all five sequences shared 100 per cent identity with Candidatus Phytoplasma aurantifolia (OM649850, ON975012) and Tomato big bud phytoplasma (EF193359). The in-silico RFLP pattern of F2n/R2 primed region of 16S rDNA gene analyzed by using iPhyClassifier (Zhao et al. 2009) revealed that the sequence shared 98.72 per cent nucleotide sequence similarity with coefficient value of 1.00 to the reference strain RFLP pattern of 16Sr group II, subgroup D (witches'-broom disease of lime; U15442). Based on 16SrDNA sequences and in-silico RFLP analysis, the phytoplasma associated with the chia virescence was identified as a member of 16SrII-D group. Further, SecA gene was also amplified from the samples using SecAfor1/SecArev3 primer pair (Hodgetts et al., 2008). All samples produced ~400 bp products and sequenced as detailed above. Sequence analysis by nBLAST revealed 100 per cent similarity to Ca. P. australasia (MW020545) and Ca. P. aurantifolia isolate Idukki Kerala 1 (MK726369) both representing 16SrII-D group phytoplasma. The representative sequence (16Sr: PP359693, PP359694; secA:PP386558, PP386559) were deposited in GenBank. Chia virescence phytoplasma belonging to Ca. phytoplasma australasia has not been reported anywhere. The phytopathological studies associated with chia crop are very limited. Joy et al. (2022) reported the occurrence of foot rot disease caused by Athelia rolfsii. Several hosts are recorded to be associated with 16SrII D phytoplasma which includes china aster, eggplant and crotalaria (Mahadevakumar et al., 2017, Yadav et al., 2016a, b). Now the wide occurrence of the phytoplasma in the area might have transmitted by vectors. The occurrence of virescence is of great importance as it affects the overall yield which reduces the market value. To our knowledge, this is the first report of a group 16SrII-D phytoplasma associated with chia virescence in India.

Availability of receptors for advanced glycation end-products (RAGE) influences differential transcriptome expression in mice exposed to chronic secondhand smoke

The receptor for advanced glycation end-products (RAGE) has a central function in orchestrating inflammatory responses in multiple disease states. RAGE is a transmembrane pattern recognition receptor with particular interest in lung disease due to its naturally abundant pulmonary expression. Our previous research demonstrated an inflammatory role for RAGE following acute exposure to secondhand smoke (SHS). However, chronic inflammatory mechanisms associated with RAGE remain unclear. In this study, we assessed transcriptional outcomes in mice exposed to chronic smoke in the context of RAGE expression. RAGE knockout (RKO) and wild type (WT) mice were exposed to SHS five times weekly via a nose-only delivery system (Scireq Scientific, Montreal, Canada) for six months and compared to mice exposed to room air (RA) only. Total lung RNA was isolated using the Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA) and mRNA was purified using poly-T oligo-attached magnetic beads. Synthesis of cDNA, library construction, and sequencing was performed using standard approaches. We specifically compared the phenotypic and environmental conditions from WT+RA, WT+SHS, and RKO+SHS mice. Preprocessing and analysis of RNA-sequencing gene expression data included read trimming, mapping and quantifying the reads to transcripts, and calculating significant differentially expressed genes. The results of these analyses were summarized and compared via Venn diagrams, volcano plots, and functional gene cluster enrichment analysis. Notable gene clusters were specific to cytoskeletal elements, inflammatory signaling, and ciliogenesis. Finally, gene ontologies (GO) demonstrated significant biological pathways that were differentially impacted by the presence of RAGE. These data collectively identify several opportunities to further dissect RAGE signaling in the context of SHS exposure and foreshadow possible therapeutic modalities. This work was supported by funding from the National Institutes of Health (NIH 1R15-HL152257). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Genetic Diversity of Mangrove and Nypa Palm Species in the Niger Delta, Nigeria

Mangroves and nypa palm are two dominant coastal vegetation in the Niger delta that are sources of numerous ecosystem services. We thus hypothesized that both species are monophyletic, which means they are identical by descent. We began the study by randomly collecting fresh leave samples of nypa palm in a mixed forest comprising nypa palm and mangrove at a coastal community called Eagle Island. We put the leaves samples in zip lock bags containing silica gel crystals and transported them to the laboratory, where the DNA of the leaves was extracted. A quick-DNA plant/seed miniprep kit (Zymo-research Lab, California, USA). A total of six primer pairs were used for the amplification, namely: three microsatellite markers and three universal primers. Fragments of the samples were sequenced using the Ninagen, Brilliant DyeTM Terminator Cycle Sequencing kit V3.1 BRD3-100/1000. All three samples of the nypa palm were amplified, and the BLAST prediction tool was used to produce the new species. Our results show that out of the three samples sequenced (NI, N2, and N3), the N1 sample was identified as Nypa fruticans with 100% identity (Accession Number is KT312925.1), while the others were the new strains identified in the study namely N2 identified as Astrocaryum aculeatum with 98.95% identity (Accession Number is NC 044482.1) and N3 identified as Astrocaryum aculeatum with 99.21% (Accession Number is 044482.1).

First Report of Shoot Blight of Grapevine Caused by Sclerotinia sclerotiorum in Illes Balears, Mallorca, Spain.

In 2021, grapevines (Vitis vinifera L.) cv. Callet growing in a commercial vineyard located at Pollença (northeast of the island of Majorca, Spain) showed severe symptoms of shoot blight during spring and early summer, with an incidence of 70%. Symptoms consisted of elongated cankered-like lesions, surrounded by water-soaked darker tissues, that developed at the base or around the middle nodes of the shoot. For fungal isolation, shoot samples with lesions were collected, surface disinfected with 2% NaCl for 90s, rinsed twice with deionized water and placed in Petri plates containing potato dextrose agar (PDA). The plates were incubated at 25°C under 12 h light-darkness for 6 days. Isolations consistently yielded on kind of fungal colonies that produced white mycelium and black spherical to elongated sclerotia (2 to 10 mm in diameter). Morphological characterization was consistent with the description of Sclerotinia sclerotiorum (Lib.) de Bary (Bolton et al. 2006). Three isolates (UIB 118-1, UIB 118-26, and UIB 129-41) were preserved and deposited in the Culture Collection of Microbiology-Faculty of Sciences, University of Balearic Islands, Spain. Genomic DNA was extracted from isolates UIB 118-26 and UIB 129-41 using the EZNA Miniprep Kit (Omega Bio-Tek, Norcross, GA). The internal transcribed spacer (ITS) region of ribosomal DNA, β-tubulin (BTUB) and calmodulin (CAL) gene regions were amplified using ITS1F-ITS4 (Gardes and Bruns, 1996; White et al. 1990), Bt-2a/Bt-2b (Glass and Donaldson 1995) and CAL228F/CAL737R (Carbone and Kohn 1999) primer sets, respectively. Amplicons were sequenced and deposited in GenBank with accession numbers MZ604647 and MZ604648 for ITS, OK634402 and OK634403 for BTUB and OK634404 and OK634405 for CAL. BLASTn search showed that isolates were >99 % (ITS, BTUB and CAL) identical to S. sclerotiorum GenBank accession no. KF859933, CP017815 and KF871381, respectively. Pathogenicity tests were conducted using eight one-year old grapevines cv. Cabernet Sauvignon. Old and new green shoots were inoculated by inserting a 6-mm plug of mycelium taken from actively growing cultures on PDA into cuts made at the base and at the distal part of each shoot with a sterile scalpel with a total of eight inoculation points per plant. Inoculated wounds were sealed with Parafilm tape to avoid rapid dehydration. Inoculated plants and an equal number of wounded but non-inoculated plants (negative controls) were maintained at 25 ± 1°C for 48 h in plastic containers to ensure a high relative humidity (>90%). After 5 days, the infection girdled and rotted the green new shoots, whereas the older partially lignified shoots developed a localized long brown lesion that reached 16 cm in length. Due to the rotting of the basal part of the petiole, leaves turned gray, wilted, and died, easily detaching from the stem. In advanced stages of the disease, 7 days after infection, branches died and fell with the leaves remained attached (Fig 1 A, B). Reisolations from diseased shoots were successfully performed on PDA to fulfill Koch's postulates. S. slerotiorum was previously reported on grapevine causing shoot blight in Chile (Latorre and Guerrero, 2001), Korea (Jong-Han et al. 2009), California-USA (Boland and Hall, 1994) and Australia (Hall et al. 2002). AlsoS. sclerotiorum was reported among the endophytic mycobiota associated with Vitis vinifera in the Iberian Peninsula (Gonzalez and Tello, 2011) but not as a pathogen causing visible symptoms on that crop. So, this is the first report of the occurrence of S. slerotiorum as a pathogen of grapevines in Spain causing symptoms of canker and shoot blight. This finding highlights a potential risk of this fungal disease for the wine industry in the Mediterranean region and specially for Spain, the country with the largest acreage devoted to grapevines. Although chemical and biological are suitable control strategies, disease management is difficult as sclerotia of Sclerotinia can remain in the soil for up to eight years (Adams and Ayears, 1979), and preventive surveys are greatly recommended as an important epidemiological tool to monitor the epidemiology of disease and identify potential outbreaks of this new pathogen on grapevine in Spain.

First report of Calonectria ilicicola causing red crown rot of soybean in Indiana.

Soybean (Glycine max [L.] Merr.) samples from commercial fields in Decatur and Spencer counties, Indiana were submitted to the Purdue Plant and Pest Diagnostic Lab in August to October 2022. Plants exhibited whole-leaf to interveinal chlorosis of the foliage, red to dark brown external lesions on the crown spreading from the soil-line upward, and severe root rot. In the fields, patches of diseased plants were observed, with greater than 50% of the plants affected and yield loss up to 50%. Orange to red perithecia were present on the exterior of symptomatic stem tissue and ranged in size from 329 to 433 × 232 to 306 µm (n = 10). Stems were surface sterilized in 10% Clorox (0.825% NaOCl) for 1 min, then rinsed with sterile distilled water and dried. In a laminar flow hood, sections of symptomatic stem tissue were plated on using quarter-strength potato dextrose agar (QPDA) and incubated under fluorescent lights on a 12-hr light/dark cycle at 20°C. After 6 days, fungal colonies with fluffy aerial hyphae, which were white near the colony margins and orange to burnt-red near their center, grew uniformly from the stem tissue plated. Elongate, cylindrical hyaline conidia with zero to three septations measuring 45.5 to 73.8 × 4.4 to 6.7 µm (n = 22) grew in clusters from symptomatic stem tissue within the plate. Perithecia developed after 14 days. Falcate, hyaline ascospores with one to two septa measuring 29.4 to 54.7 × 4.6 to 6.8 (n = 23) µm developed within the perithecia. Calonectria ilicicola Boedijn & Reitsma was confirmed based on morphological characteristics (Padgett et al. 2015). Isolate PPDL 22-01457B was used for DNA extraction using the ZR Fungal/Bacterial DNA Miniprep kit (Zymo Research, Irvine, CA). The internal transcriber region (ITS), actin (ACT) and β-tubulin (TUB2) genes were amplified (Carbone and Kohn 1999; Glass and Donaldson 1995; O'Donnell and Cigelnik 1997; White et al. 1990). Amplicons were sent for Sanger sequencing (Genewiz, Inc., South Plainfield, NJ), submitted to Genbank, and assigned accession numbers ITS: OQ932995, Actin: OR484986, and β-tubulin: OR546281. Sequences were analyzed using the NCBI BLASTn tool with results showing 99.5 to 100% identical to C. ilicicola (GenBank accessions LC500063, OQ303403, CP085825, respectively). To perform Koch's Postulates, 90 soybean seeds (CP3620E) were planted in potting media (Berger, Saint-Modeste, Quebec, Canada) in a seed flat with 45 of the plants used as controls and grown under grow lights for 16hr light/8hr dark at 20℃. Individual seedling crowns were inoculated 3 days post-emergence with a 5 to 10 ml spore and hyphal suspension that was scraped from the surface of a 14-day old QPDA culture after adding 300 mL deionized (DI) to each plate grown at 20 to 22°C. The control plants received sterile-DI water. Plants were covered in a plastic bag for 72 h. Plant stems were sprayed with sterile-DI water once a day for seven days. Symptoms were observed after four days, but significant crown rot and lesions developed after two weeks before wilting and dying. Calonectria iliciola was isolated uniformly from symptomatic plants and identified morphologically. Control plants showed no symptoms. Inoculations were repeated 3 times with similar results. As of fall 2023, red crown rot has been confirmed in Adams and Rush counties in Indiana. Red crown rot has been confirmed in several Midwest states (Kleczewski et al. 2019, Neves et al. 2023), but the extent of its distribution and disease management strategies are still limited.

Impact of artisanal refining activities on bacterial diversity in a Niger Delta fallow land

Hydrocarbon pollution is a major ecological problem facing oil-producing countries, especially in the Niger Delta region of Nigeria. In this study, a site that had been previously polluted by artisanal refining activity was investigated using 16S rRNA Illumina high-throughput sequencing technology and bioinformatics tools. These were used to investigate the bacterial diversity in soil with varying degrees of contamination, determined with a gas chromatography-flame ionization detector (GC-FID). Soil samples were collected from a heavily polluted (HP), mildly polluted (MP), and unpolluted (control sample, CS) portion of the study site. DNA was extracted using the Zymo Research (ZR) Fungi/Bacteria DNA MiniPrep kit, followed by PCR amplification and agarose gel electrophoresis. The microbiome was characterized based on the V3 and V4 hypervariable regions of the 16S rRNA gene. QIIME (Quantitative Insights Into Microbial Ecology) 2 software was used to analyse the sequence data. The final data set covered 20,640 demultiplexed high-quality reads and a total of 160 filtered bacterial OTUs. Proteobacteria dominated samples HP and CS, while Actinobacteria dominated sample MP. Denitratisoma, Pseudorhodoplanes, and Spirilospora were the leading genera in samples HP, CS, and MP respectively. Diversity analysis indicated that CS [with 25.98 ppm of total petroleum hydrocarbon (TPH)] is more diverse than HP (with 490,630 ppm of TPH) and MP (with 5398 ppm of TPH). A functional prediction study revealed that six functional modules dominated the dataset, with metabolism covering up to 70%, and 11 metabolic pathways. This study demonstrates that a higher hydrocarbon concentration in soil adversely impacts microbial diversity, creating a narrow bacterial diversity dominated by hydrocarbon-degrading species, in addition to the obvious land and ecosystem degradation caused by artisanal refining activities. Overall, the artisanal refining business is significantly driving ecosystem services losses in the Niger Delta, which calls for urgent intervention, with focus on bioremediation.

Mycobiota Associated with Dacryodes edulis H. J. Lam fruits sold In Rumokoro Market, Port Harcourt, Nigeria

Dacryodes edulis H. J. Lam is a common fruit in Nigeria used to eat roasted or boiled corn. It can also be eaten alone by softening with hot water, hot ash, or by roasting on low heat. It is called “ube” in the eastern part of Nigeria. D. edulis fruits are prone to infection by fungal pathogens in the field, in transit and during storage. Proper identification of these pathogens is important in disease prevention and control. This study was carried out to isolate and identify the fungal species associated with D. edulis fruits. Fruits were obtained from Rumokoro market, Rivers State, Nigeria in January 2020. Isolation of fungi was done by Potato Dextrose (PDA) method and identification was carried out by molecular method. Fungal DNA was extracted using the Zymo Fungal/Bacterial DNA Miniprep Kit. Polymerase chain reaction amplification of the internal transcribed spacer (ITS) region was carried out using the primer pair: ITS4 and ITS5. BLAST search was carried out on the National Centre for Biotechnology Information (NCBI) database and the fungi were identified as: Rhizopus delemar, Aspergillus oryzae and A. welwitschiae. The Phylogenetic tree was constructed to show the evolutionary relationship among the isolates and other fungal species retrieved from GenBank. The sequences of the isolates have been deposited in GenBank under the accession numbers: ON965489, ON965490 and ON965491 for Rhizopus delemar, Aspergillus oryzae and A. welwitschiae respectively. The molecular method used in this study enabled the isolates to be identified at the species levels.

First Report of Charcoal Rot on Soybean Caused by Macrophomina phaseolina in Manitoba, Canada.

First Report of Charcoal Rot on Soybean Caused by Macrophomina phaseolina in Manitoba, Canada.

Mycoplasmas detection in milk samples ¿are the techniques reliable?

Food microbiology is an important aspect that must be considered during food manufacturing, which is, considering the possible sources of contamination, including operators. Highlighting that the group of mycoplasmas are little studied and therefore their presence in foods is not considered. Different mollicutes, particularly mycoplasmas, have been associated with economic losses in the poultry and dairy industries. The objective of this work was to determine the presence of the mycoplasma genus in milk samples through specific microbiological culture and to complement the diagnosis with the PCR technique. Two hundred milk samples were analyzed in Puebla city-Mexico. The samples obtained were centrifuge and reseeded in triplicate on SP4 agar, incubating and analyzed by stereoscopic microscopy. From each milk sample, DNA extraction was carried out with Zymo Research Quick-DNA™ Mini prep kit and primers AR1 and AR2 were used. The microbiological study showed positivity for mycoplasmas of 17% and 39%, in the liquid and solid phase, respectively. With the PCR technique, 53% positivity for mycoplasmas was obtained, which allows us to consider the presence of factors associated with samples of animal origin and that may be interfering with the sensitivity of the technique. Analyzing the viability of techniques to detect microorganisms, particularly mycoplasma, in foods is a practice that is recommended for the well-being of consumers.