DNA Extraction Kit Research Articles

The Phenotypic and Molecular Identification of Phyllospheric Bacteria Possessing Antimicrobial Activity from Funtumia elastica (Preuss) Stapf.

Unlike plant phytochemicals, little has been done to explore the metabolites from phyllosphere bacterial flora, some of which enabled them to survive interspecific competition through amensalism. This study evaluated the antimicrobial activity of metabolites from Phyllospheric Bacteria (PB) isolated from Funtumia elastica (FE), against selected bacterial and fungal pathogens. Phenotypic and molecular methods were used to identify the isolated phyllo-microbiota. The PB were aseptically isolated by sonication. Their metabolites were obtained from the fresh overnight culture of the organisms. The cell-free supernatants containing the metabolites were used for antimicrobial assays against the pathogens. The DNA of the bacterial isolates were isolated using a NIMR-BIOTECH DNA extraction kit, while their 16S rRNA was amplified with the primer: 799F 5'-AACACGGATTA GATACC-3', 1193R 5'- ACGTCATCCCCACCTTCC-3', using SolisFast* Master Mix, (Solis Biodyne-Estonia). The BLAST of the sequence was done from the NCBI Gen-bank. The PB strains identified were submitted to NCBI and accession numbers were assigned to them. The phyllosphere of FE yielded 21 bacterial isolates: 7 Gram-positives and 14 Gram-negatives. The metabolites from these isolates showed varying degrees of bioactivity against Staphylococcus aureus (ATCC29213), Escherichia coli (ATCC 25922) Klebsiella pneumoniae (ATCC 35659); Trychophyton rubrum, Candida albicans and Microsporum canis. Fifteen bioactive isolates sequenced yielded four genera, Enterobacter (E. hormaechei 98.44%), Bacillus (B. cereus 100%), Pontoea (P. dispersa 99.72%), Staphylococcus (S. arlettae 99.72%). Bacteria from FE phyllosphere, produced metabolites antagonistic (cidal) to some human pathogens. This has great potential for possible drug discovery.

A pilot study of childhood-onset Takayasu arteritis using whole exome sequencing suggests oligogenic inheritance involving classical complement, collagen, and autoinflammatory pathways.

Takayasu arteritis (TA) is a chronic granulomatous inflammatory disease affecting the aorta and its branches. Paediatric TA (pTA) may present from 6months after birth till the adolescent age group. Genetics and pathogenesis of pTA are not fully understood. Earlier studies reported monogenic mutation in NOD2, XIAP, and STAT1 genes in patients with pTA. TA, a relatively rare disease, is more common in geographical pockets, including India. We hypothesized that South Asian patients with pTA, namely, those of Indian subcontinent origin, may have clinically relevant and unique pathogenic variants involving one or more genes, especially those linked to genetically driven vasculitic illnesses, including autoinflammatory pathologies. Children with pTA fulfilling EULAR/PRINTO/PReS classification criteria and presenting with clinical symptoms to the Paediatric Rheumatology clinic of Christian Medical College, Vellore, were included. Blood samples were collected after getting informed consent from parents or guardians and assent forms from children. DNA was extracted from whole blood using the Qiagen DNA extraction kit. Initially, the common variant in Indian population, namely, ADA2 c.139G > A; p.Gly47Arg, was screened, followed by whole exome sequencing. Fourteen children were recruited for the study. Median age of patients was 11years (4months-14years) with a male-to-female ratio of 4:10. Distribution of angiographic subsets by Numano's classification of included children were as follows: type 5 (n = 7), type 4 (n = 5), and type 3 (n = 2). We identified novel variants in ten different genes. This include variants in genes of classical complement pathway, namely, C2, C3, C6, C7, and C9, and other genes, namely, CYBA, SH3BP2, GUCY2C, CTC1, COL5A1, and NLPR3. Two of 14 patients have heterozygous pathogenic variants; this implies that combination of heterozygous variants in C3 and COL5A1 might lead to disease development, suggesting digenic inheritance. One patient has a homozygous variant in CYBA. None of the patients were identified to have ADA2 variants. Whole exome sequencing reveals combination of rare variants in genes C3, COL5A1, and CYBA associated with disease development in children with Takayasu Arteritis. Key Points • We identified novel variants in genes of classical complement pathway, namely, C2, C3, C6, C7, and C9, and other genes, namely, CYBA, SH3BP2, GUCY2C, CTC1, COL5A1, and NLPR3. • Two of 14 patients have heterozygous pathogenic variants in C3 and COL5A1; this may have implications in disease development, suggesting digenic inheritance. • One patient has homozygous variant in CYBA. • None of the patients were identified to have ADA2 variants.

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

Microbiome analyses of poultry feeds: Part I. Comparison of five different DNA extraction methods

Given extensive variability in feed composition, the absence of a dedicated DNA extraction kit for poultry feed underscores the need for an optimized extraction technique for reliable downstream sequencing analyses. This study investigates the impact of five DNA extraction techniques: Qiagen QIAamp DNA Stool Mini Kit (Qiagen), modified Qiagen with Lysing Matrix B (MQ), modified Qiagen with celite purification (MQC), polyethylene glycol (PEG), and 1-Day Direct. Genomic DNA amplification and Illumina MiSeq sequencing were conducted. QIIME2-2021.4 facilitated data analysis, revealing significant diversity and compositional differences influenced by extraction methods. Qiagen exhibited lower evenness and richness compared to other methods. 1-Day Direct and PEG enhanced bacterial diversities by employing bead beating and lysozyme. Despite similar taxonomic resolution, the Qiagen kit provides a rapid, consistent method for assessing poultry feed microbiomes. Modified techniques (MQ and MQC) improve DNA purification, reducing bias in commercial poultry feed samples. PEG and 1-Day Direct methods were effective but may require standardization. Overall, this study underscores the importance of optimized extraction techniques in poultry feed analysis, with potential implications for future standardization of effective methods.

First Report of Alternaria alternata Causing Postharvest Fruit Rot of Indian Jujube (Ziziphus mauritiana) in China.

Ziziphus mauritiana Lam., commonly known as Indian jujube or ber, is a popular fruit crops grown in tropical and sub-tropical regions of China. It is commonly stored at 4℃, relative humidity of about 90%, combined with waxing or sealing with film bag. In January 2023, a postharvest fruit rot was observed on Indian jujube in three markets located in Nanchang city of Jiangxi province, China, with a disease incidence of 4 to 10%. Initially, brown spots appeared on the surface or base of thefruit, which gradually expanded into irregular brown lesions. Gray-white hyphae developed in the center of the lesions, and ultimately thefruitrotted. To isolate the pathogen, small pieces (5 × 5 mm) of ten infected fruits were surface-sterilized in 75% ethanol for 15 s and then 1% sodium hypochlorite for 30 s, rinsed three times in sterile water, plated onto potato dextrose agar (PDA), and incubated at 25°C for 3 days. Eight strains with similar morphological characteristics were isolated, and one representative isolate (JXAA-1) was used for morphological and molecular characterization. The colonies on PDA were initially olive green with white margins, and later turned dark olive or black with profuse sporulation. Conidia were borne singly or in a chain, brown, with 1 to 5 transverse septa and 0 to 3 longitudinal septa, obclavate to obpyriform, and measured 12.9 to 33.7 × 7.5 to 12.9 μm (n = 30). On the basis of morphological characteristics, the isolates were tentatively identified as Alternaria spp. (Simmons 2007). To confirm the identification, genomic DNA was extracted from the isolate JXAA-1 with the Fungi Genomic DNA Extraction Kit (Solarbio Biotech, China). The 18S nrDNA (SSU), 28S nrDNA (LSU), internal transcribed spacer of the rDNA (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1-alpha (TEF1), Alternaria major allergen gene (Alt a 1), endopolygalacturonase (EndoPG) and an anonymous gene regions (OPA 10-2) were amplified and sequenced using primers NS1/NS4, LR7/LR0R, ITS5/ITS4, gpd1/gpd2, EF1-728F/EF1-986R, Alt-for/Alt-rev, PG3/PG2b, OPA10-2L/OPA10-2R, respectively (Woudenberg et al. 2015). The obtained DNA sequences (SSU: PP190241; LSU: PP190242; ITS: PP189927; GAPDH: PP196557; TEF1: PP196558; Alt a 1: PP196559; EndoPG: PP196560; and OPA 10-2: PP196561) showed 100% homology with those of A. alternata (GenBank accession nos. MT000349 [1020/1020 bp]; KP940477 [1312/1312 bp]; MK972909 [583/583 bp]; MN615421 [593/593 bp]; MN046379 [280/280 bp]; MN304714 [490/490 bp]; MN698284 [458/458 bp] and MH975214 [701/701 bp]). A maximum likelihood phylogenetic tree was constructed by combining all sequenced loci in IQTREE web servers. The isolate JXAA-1 clustered with Alternaria alternata (CBS 121336). The fungus associated with postharvest fruit rot on Z. mauritiana was thus identified as A. alternata. To evaluate the pathogenicity, six surface sterilized fruits were wounded by a sterile scalpel and inoculated with a 10 μl drop of spore suspension (1 × 105 conidia/ml) of isolate JXAA-1. Another six fruits were inoculated with sterilized ddH2O as control and the experiment was repeated three times. All fruits were incubated at 25°C and 80% relative humidity. After 5 days, all the wounded fruit inoculated with A. alternata showed similar symptoms to those observed previously, while the control fruits remained healthy. A. alternata was consistently reisolated from infected fruit and confirmed by morphological and molecular data, fulfilling Koch's postulates. A.alternata has previously been reported causing leaf spot and fruit rot on Chinese jujube (Ziziphus jujuba) in China (Bai et al. 2015; Li et al. 2021). But to our knowledge, this is the first report of A. alternata causing postharvest fruit rot on Indian jujube (Z. mauritiana) in China. Therefore, managers should pay more attention to postharvest fruit rot of jujube caused by A. alternata, the foam bag is put on after the membrane bag is sealed, the broken or infected fruit is picked out in time to reduce the spread of pathogenic fungus.

The impact of kit, environment, and sampling contamination on the observed microbiome of bovine milk.

In low-microbial biomass samples such as bovine milk, contaminants can outnumber endogenous bacteria. Because of this, milk microbiome research suffers from a critical knowledge gap, namely, does non-mastitis bovine milk contain a native microbiome? In this study, we sampled external and internal mammary epithelia and stripped and cisternal milk and used numerous negative controls, including air and sampling controls and extraction and library preparation blanks, to identify the potential sources of contamination. Two algorithms were used to mathematically remove contaminants and track the potential movement of microbes among samples. Results suggest that the majority (i.e., >75%) of sequence data generated from bovine milk and mammary epithelium samples represents contaminating DNA. Contaminants in milk samples were primarily sourced from DNA extraction kits and the internal and external skin of the teat, while teat canal and apex samples were mainly contaminated during the sampling process. After decontamination, the milk microbiome displayed a more dispersed, less diverse, and compositionally distinct bacterial profile compared with epithelial samples. Similar microbial compositions were observed between cisternal and stripped milk samples, as well as between teat apex and canal samples. Staphylococcus and Acinetobacter were the predominant genera detected in milk sample sequences, and bacterial culture showed growth of Staphylococcus and Corynebacterium spp. in 50% (7/14) of stripped milk samples and growth of Staphylococcus spp. in 7% (1/14) of cisternal milk samples. Our study suggests that microbiome data generated from milk samples obtained from clinically healthy bovine udders may be heavily biased by contaminants that enter the sample during sample collection and processing workflows.IMPORTANCEObtaining a non-contaminated sample of bovine milk is challenging due to the nature of the sampling environment and the route by which milk is typically extracted from the mammary gland. Furthermore, the very low bacterial biomass of bovine milk exacerbates the impacts of contaminant sequences in downstream analyses, which can lead to severe biases. Our finding showed that bovine milk contains very low bacterial biomass and each contamination event (including sampling procedure and DNA extraction process) introduces bacteria and/or DNA fragments that easily outnumber the native bacterial cells. This finding has important implications for our ability to draw robust conclusions from milk microbiome data, especially if the data have not been subjected to rigorous decontamination procedures. Based on these findings, we strongly urge researchers to include numerous negative controls into their sampling and sample processing workflows and to utilize several complementary methods for identifying potential contaminants within the resulting sequence data. These measures will improve the accuracy, reliability, reproducibility, and interpretability of milk microbiome data and research.

First report of anthracnose of Lithocarpus polystachyus caused by Colletotrichum fructicola in China.

Lithocarpus polystachyus (Wall. ex A. DC.), an economically valuable plant species belonging to the Fagaceae family, has been used as herbal tea to prevent diabetes because of the high content of flavonoids and dihydrochalcones in the leaves (Shang et al. 2022). In July 2022, the severe leaf lesion on L. polystachyus was first observed in Yongshun County, Xiangxi autonomous prefecture (28°45'34''N, 109°40'11''E), Hunan province, China. Yongshun County is characterized by hills and mountains, situated in a subtropical region with a mild and humid climate. A second outbreak in July 2023 was observed in the same area. The observed incident rates in the past two years were 87.3% and 90.6%, respectively. Once infected, almost all plant leaves will be infected, leading to a substantial reduction in the yield of L. polystachyus. The disease presented symptoms characterized by round or irregularly shaped lesions that initially manifested as brown spots. These lesions frequently merged into larger, dark-brown areas along the leaf margins before eventually wilting. To ascertain the pathogenic species responsible for this disease, fungal isolation was conducted using a tissue separation method (Xu et al. 2023). The infected leaf tissues were surface-disinfected with 75% ethanol and 0.1% HgCl then small pieces (1×1 cm), were placed onto potato dextrose agar (PDA) medium (Sigma-Aldrich, 70139) and incubated at 28°C for 6-9 days. Colonies were villiform and initially white, becoming gray after 6 days. Sterilized dissecting needles were used to pick single hyphal tips from the edge of the colonies and placed onto PDA for strain purification. After 15 days, the purified colonies grew fluffy white hyphae with abundant conidia. The conidia were cylindrical, had round ends, and ranged from 5.75 to 14.83 μm long and 1.75 to 2.38 μm wide (n=50). According to morphological and cultural characteristics, these isolates were preliminarily identified as Colletotrichum fructicola Prihast., L. Cai & K.D. Hyde (Damm et al. 2012). To further affirm the identity of the pathogen, DNA was extracted from mycelia using a DNA extraction kit (Sigma-Aldrich, G2N70). The internal transcribed spacer (ITS) region, the transcription elongation factor (TEF), and the actin (ACT) gene were then amplified from genomic DNA extracted from three isolates (Cof1, Cof2, and Cof3) using specific primers. The primers utilized were ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R and ACT-512F/ACT-783R (Carbone and Kohn 1999) for ITS region, transcription elongation factor gene and actin gene amplification, respectively. Sequence identity indicated that these isolates were highly homologous to C. fructicola. The ITS (Genbank No. PP002156, OR880553 and OR880554), TEF (No. PP061421, PP061422 and PP061423), and ACT (No. PP061418, PP061419 and PP061420) sequences of the isolates Cof1, Cof2, and Cof3 shared 99 to 100% identity with their counterparts (No. OR083309, MF627961, and OQ427895) in C. fructicola, respectively. A neighbor-joining phylogenetic tree constructed using MEGA11 (Tamura et al. 2021) also indicated that these isolates were C. fructicola. Both morphological and molecular characteristics confirmed the identification of this pathogen as C. fructicola. Colletotrichum species are known to cause anthracnose disease in a variety of economically important crops (Sharma and Kulshrestha 2015). To further validate the ability of the isolated C. fructicola to induce the same symptoms as observed in the field, the pathogenicity assay was assessed following Koch's postulates (Gradmann, 2014). Conidial suspensions (1×105 conidia per mL) from three isolates were individually inoculated onto artificially wounded leaves of 3-year-old L. polystachyus. Negative controls were established by inoculating leaf wounds with sterile distilled water. The plants were incubated in a greenhouse at 28°C and 90% humidity with a 12-h photoperiod. The experiment was replicated three times. Necrotic lesions were observed on all pathogen-inoculated wounds within 6 days after inoculation, whereas controls showed no observable symptoms. Morphological and molecular characterization of re-isolated pathogens from infected leaves indicated that the pathogens were identical. To our knowledge, this is the first report of anthracnose of L. polystachyus caused by C. fructicola in China. Farmers in the local mountainous areas are economically reliant on L. polystachyus production, while anthracnose has caused over half of the trees to lose their commercial value, resulting in significant economic losses. Our findings hold great promise for advancing strategies in the prevention and treatment of anthracnose in L. polystachyus.

Rapid on-site authentication of three cockle species using real-time loop-mediated isothermal amplification (LAMP) assay

Cockles, crucial for commercial fishing and aquaculture, emerge as highly marketable shellfish. However, the morphological similarities among various cockle species are frequently exploited for shellfish fraud. In this study, we developed LAMP assays (end-point LAMP and real-time LAMP) to detect three commercially important cockle species: Tegillarca granosa, Anadara broughtonii, and Anadara kagoshimensis. The primers were designed based on mitochondrial DNA and used to develop LAMP assays. Exclusivity was assessed using 17 shellfish species, and no cross-reactivity was confirmed. The estimate of the detection limit was determined to be 0.0001 ng. The established assays were further refined to create an on-site real-time LAMP assay by combining fast DNA isolation with a real-time fluorometer for field application. The effectiveness of the on-site real-time LAMP assay was successfully verified using 38 commercial samples, with analysis accuracy confirmed using a DNA extraction kit. Consequently, the on-site real-time LAMP assay can complete the whole procedure, including DNA extraction, real-time LAMP reaction, and data analysis within a 45 min timeframe, maintaining the accuracy of detecting three cockle species from processed cockle samples in the field. Our method proves valuable for detecting shellfish fraud by identifying cockle species using samples of both raw and processed products in the field.

Swarm and UNOISE outperform DADA2 and Deblur for denoising high-diversity marine seafloor samples.

The performance of sequence variant resolution analytic tools for metabarcoding has not yet been adequately benchmarked for high-diversity environmental samples. We therefore evaluated the sequence variant tools DADA2, Deblur, Swarm, and UNOISE, using high-diversity seafloor samples, resulting in comparisons of 1800 sequence variant tables. The evaluation was based on 30 sediment grab samples, for which 3 replica samples were collected. Each replica sample was extracted using 5 common DNA extraction kits, resulting in 450 DNA extracts which were 16S rRNA gene sequenced (V3-V4), using Illumina. Assessments included variation across replica samples, extraction kits, and denoising methods, in addition to applying prior knowledge about alpha diversity correlations toward the cosmopolitan marine archaeon Nitrosopumilus with high diversity and the sulfide oxidizing Sulfurovum with low diversity. DADA2 displayed the highest variance between replicates (Manhattan distance 1.14), while Swarm showed the lowest variance (Manhattan distance 0.93). For the analysis based on prior biological knowledge, UNOISE displayed the highest alpha diversity (Simpson's D) correlation toward Nitrosopumilus (Spearman rho = 0.85), while DADA2 showed the lowest (Spearman rho = 0.10). Deblur completely eliminated Nitrosopumilus from the dataset. For Sulfurovum, on the other hand, all the methods showed comparable results. In conclusion, our evaluations show that Swarm and UNOISE performed better than DADA2 and Deblur for high-diversity seafloor samples.

DNA Extraction and Comparison between Old and Fresh Necrophilic Fly Samples.

A total of five samples of Chrysomya megacephala samples - three fresh samples, one sample stored in alcohol for 2 years, and one sample stored in dry sealed storage for 2 years protected from light only - were selected to investigate whether a blood DNA extraction kit could extract DNA from necrophilous flies and to determine whether alcohol could prolong the preservation of necrophilous flies' DNA. First, the blood DNA extraction kit was used to extract DNA from their thorax tissues. Then, the DNA purity and concentration were examined using a microplate reader and a fluorometer. Finally, PCR amplification and electrophoresis of the extracted DNA were done with necrophilic fly-specific primers located in the mitochondrial CO I gene sequence. The results showed that the DNA purity of all samples was greater than 2.0. The DNA concentration was observed to be of the following order: fresh samples > alcohol-preserved old samples > untreated, old samples. All samples had specific electrophoretic bands after PCR amplification. In conclusion, a blood DNA extraction kit can be used to extract DNA from necrophilic flies successfully, and the DNA concentration of fresh fly samples is greater than that of old fly samples. The flies can be stored in alcohol for a long time.

Prolactin gene (C576A) polymorphism is associated with milk production performance in crossbred Anglo-Nubian dairy goats

Crossbred Anglo-Nubian goats (Anglo-Nubian × native goat breeds), are valued for exceptional milk production and adaptability. In-depth research on these goats is imperative to propel advancements in dairy goat breeds and enhance milk production efficiency within the region. The present study aims to assess polymorphism in prolactin (C576A) gene and determine the influence of different prolactin (PRL) genotypes to milk yield performance in crossbred Anglo-Nubian dairy goats raised from Awang, Opol, Misamis Oriental and Talay, Dumaguete City, Negros Oriental. Genomic DNA was extracted from hair follicles using commercial DNA extraction kit and PCR-RFLP was performed for the genotyping of the C576A polymorphism located in exon 5 of goats’ prolactin gene using Eco241 restriction enzyme. Genotypic frequencies of 0.56 for AA, 0.44 for AB, while allelic frequencies of 0.78 for A, and 0.22 for B were recorded. All populations followed the Hardy–Weinberg principle, except for dairy goats from Farm A located in Opol, Misamis Oriental. A two-way factorial (2 × 4) in a Randomized Complete Block Design was used to evaluate the relationship between genotypes and milk yield performance. AB genotype goats produced significantly higher milk yield traits (average daily milk yield and total milk production) than AA genotype, an indication that the polymorphism in the caprine PRL (C576A) gene influenced milk yield performance in the population of studied crossbred Anglo-Nubian goats. These results have to be validated in other dairy goat breeds.

A polymerase chain reaction experiment using Escherichia coli and Mars sand simulant for detection and analysis of extraterrestrial life

In this study, we conducted polymerase chain reaction (PCR) experiments using Escherichia coli (E. coli) and a Mars sand simulant (Mars Global Simulant MGS-1, Exolith Lab) to detect and analyze potential extraterrestrial life. The targeted DNA sequence is common among the bacterial kingdom on Earth. PCR experiments conducted after alkaline heat extraction, wherein samples with varying amounts of Mars sand simulant were compared, revealed that the simulant interfered with DNA detection. We then conducted PCR experiments following treatment with a sand DNA extraction kit on samples with various E. coli densities. DNA bands for a minimum E. coli density of 900 cells/(g sand) were confirmed, while no DNA bands were visible in the 90 cells/(g sand) sample with and without the Mars sand simulant. The total DNA mass contained in 900 cells was calculated to be 15.3 pg (i.e., 1.53 pg in 0.1 g sand sample we evaluated). We tested and compared the influence of the eluate of Mars sand simulant and DNA adsorption onto Mars sand simulant based on optical absorbance measurements. Our findings suggest that the mechanism by which the Mars sand simulant prevents PCR is through the adsorption of DNA onto the Mars sand simulant.

Whole genome sequence of Lactiplantibacillus plantarum strain HA9 isolated from conventional Iraqi cheese: First report

Background: Lactiplantibacillus plantarum is a renowned probiotic known for its ability to enhance health when ingested in sufficient quantities. While L. plantarum typically exhibits unique characteristics, variations in strains and environments can occur. Therefore, it is crucial to conduct genetic identification of the strain before incorporating it into products or administering it for health benefits.Methods: After being isolated from classic artisanal milk cheeses, the bacterial DNA extraction kit from GENEAID, Korea, was used, and strain HA9's genomic DNA was isolated. Then, the highly conserved section of ribosomal RNA was amplified with universal primers. The results were confirmed by a blast analysis of the NCBI database. The genome of HA9 was sequenced using the Illumina and PacBio Sequel II technologies (HN00194138; Psomagen/USA). The Prokaryotic Dynamic Programming Gene-Finding Algorithm software was applied to forecast coding genes. The Pathosystems Resource Integration Center constructed the L. plantarum genome.Result: According to the findings, the genome of the L. plantarum HA9 strain has a length of 3,321,986 bp and a GC percentage of 44.40. This assembled genome contained a total of 1,420 coding proteins and 82 contigs, as discovered by the Comprehensive Genome Analysis. The isolate had been submitted to the NCBI and received the HA9 genome sequence (accession ID: JAUTDL000000000.1).Conclusion Microbes' whole-genome sequencing plays a crucial role in clarifying their probiotic properties and enables accurate identification down to the strain level. The authors argue that further research should be done on probiotics' whole genomes, with a special emphasis on second-generation strains.Keywords: Lactiplantibacillus plantarum; Probiotics; Whole genome sequencing

Comparison of DNA Extraction Methods for Processed Foods Containing Soybean or Maize

Processed foods containing soybean or maize are subject to labeling regulations pertinent to genetically modified (GM) foods in Japan. To confirm the reliability of the labeling procedure of GM foods, the Japanese standard analytical methods (standard methods) using real-time PCR technique have been established. Although certain DNA extraction protocols are stipulated as standard in these methods, the use of other protocols confirmed to be equivalent to the existing ones was permitted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or corn was conducted. In this study, the equivalence testing of the techniques employed for DNA extraction from processed foods containing soybean or maize was conducted. The silica membrane-based DNA extraction kits, GM quicker 4 and DNeasy Plant Maxi Kit (Maxi Kit), as an existing method were compared. GM quicker 4 was considered to be equivalent to or better than Maxi Kit.

First report of Cryptostroma corticale causing sooty bark disease in California and first worldwide report of silver maple as a host.

In November 2022 and February 2023, CAL FIRE tree health experts examined four maples (Acer spp.) planted decades earlier in a residential setting in Elk Grove, Sacramento Co., California (USA). Three of the trees were silver maples (Acer saccharinum ) and one was a Norway maple (A. platanoides); both species are exotic to California. The trees were in an irreversible state of decline, with the canopy substantially thinned and browning. Extensive bark cankers elongating longitudinally along the stem were visible on all trees (Fig. S1). Cankers were filled by fungal stromata protruding through the bark and producing masses of elliptical dark brown conidia (Fig. S2), approximately 5.5 x 3.7 um in size, giving the cankers a sooty appearance. The cankered bark could be peeled off easily, revealing dark and discrete lesions in the phloem and xylem. Samples from the three trees were shipped to the U.C. Berkeley Forest Pathology and Mycology Laboratory and to the CDFA PPDC in Sacramento, CA. In the laboratories, small wood chips were taken from the margins of the lesions, surface sterilized by dipping them for 30 seconds in 70% Ethanol, rinsed for 30 seconds in sterile water and plated onto 2.5% Malt Extract Agar amended with 0.3g/L Streptomycin or onto one-half strength acidified potato dextrose agar (APDA). Two morphologically identical cultures were obtained, one (T2) from a silver maple and one (T6) from the Norway maple. Cultures were then grown in liquid 2.5% malt extract broth and, after one week, DNA was extracted using the Qiagen Plant DNeasy DNA extraction kit. The ITS sequences are diagnostic for this fungus (Li et al. 2021) and those of the two cultures (GB OR064033 and OR933565) were 100% homologous to GenBank sequences of Cryptostroma corticale ( e.g. GB OP474010-11). The RPB2 sequence of T2 ( GB OR992132) was 100% homologous to that of C. corticale (GB HG934116.1). The isolate obtained from silver maple was inoculated in four potted silver maples by removing a bark disk 50 mm in diameter with a cork borer in three spots staggered at different heights and sides on the stem, placing a colonized agar plug of C. corticale in contact with the phloem, replacing the bark disk and wrapping with parafilm. Two control trees were mock inoculated using sterile agar plugs. Trees were in 57 L pots, had an average stem caliper of 2.7 cm, an average height of 3.5 m and were kept in a lath house at average high temperatures of 18-24 degrees C. After ten weeks, average lesion length was 15.4 cm (SE= 4.6) and 4.3 cm (SE=2.3) in the fungus-inoculated and control trees, respectively. An ANOVA test, nesting lesions sizes within tree, determined lesions lengths were different between inoculated and mock trees (P= 0.04). The fungus was reisolated from all points in all inoculated trees but never from control trees. C. corticale was first described in the UK from sycamore maple (Acer pseudoplatanus) (Gregory et al. 1949) and is an emerging problem in Europe (Muller et al. 2023). In North America, it has been reported from A. negundo, A. campestre, A. macrophyllum and Cornus nuttallii (Worral 2020), and it appears to be present in the Pacific Northwest (Brooks et al. 2023, Goree 1969). Norway maple is included in the European Plant Protection Organization list of hosts for C. corticale (EPPO 2023), however our finding of C. corticale on silver maple is a first report of this host worldwide and of this pathogen in California. This report is noteworthy, given that C. corticale is also a human pathogen infecting the respiratory system (Braun et al. 2021).

Comparison of DNA extraction methods on different sample matrices within the same terrestrial ecosystem

Metataxonomic studies of ecosystem microbiotas require the simultaneous processing of samples with contrasting physical and biochemical traits. However, there are no published studies of comparisons of different DNA extraction kits to characterize the microbiotas of the main components of terrestrial ecosystems. Here, and to our knowledge for the first time, five DNA extraction kits were used to investigate the composition and diversity of the microbiota of a subset of samples typically studied in terrestrial ecosystems such as bulk soil, rhizosphere soil, invertebrate taxa and mammalian feces. DNA extraction kit was associated with changes in the relative abundance of hundreds of ASVs, in the same samples, resulting in significant differences in alpha and beta diversity estimates of their microbiotas. Importantly, the impact of DNA extraction kit on sample diversity varies according to sample type, with mammalian feces and soil samples showing the most and least consistent diversity estimates across DNA extraction kits, respectively. We show that the MACHEREY–NAGEL NucleoSpin® Soil kit was associated with the highest alpha diversity estimates, providing the highest contribution to the overall sample diversity, as indicated by comparisons with computationally assembled reference communities, and is recommended to be used for any large-scale microbiota study of terrestrial ecosystems.

First Detection of Atractylodes Mild Mottle Virus in Atractylodes lancea (Thunb.) DC. in Hubei Province of China.

The cultivated aromatic medicinal herb Atractylodes lancea (Thunb.) DC. is widely used in the pharmaceuticals, nutraceuticals, and cosmetics industries (Na-Bangchang et al. 2014; Zhan et al. 2023). Huanggang in Hubei Province is a major production area for A. lancea (Huang et al. 2022; Wang et al. 2023). In April 2023, more than two-thirds of the surveyed plant leaves in this region exhibited virus-like symptoms, such as curling and mosaic patterns. To identify the underlying cause, 80 symptomatic plant leaf samples were collected from four fields (20 leaves per field) in this region and pooled for virome analysis. Total RNA, including ribosomal RNA, was extracted from the pooled samples using the Plant RNA Extraction Mini Kit (Onrew Biotech, Guangdong, China), for sequencing library construction. The Illumina NovaSeq 6000 platform was used to sequence the library and generate 150 bp paired-end reads. After processing the raw data with Trimmomatic software, a total of 44,354,650 high-quality clean reads were obtained. The clean reads were aligned against ribosomal RNA using BWA software (v0.7.17) to avoid interference and eliminate corresponding sequences. After removing potential contamination, contig assembly of the clean reads was performed using Megahit software (v1.2.9). The resulting contigs were compared with the virus NT database using the BLASTn program. Sequence pairwise comparison revealed 8 contigs (574 nt to 2243 nt) with identities ranging from 81.88% to 90.77% with Atractylodes mild mottle virus (AMMV, NC_027924.1, Lim et al., 2015). Additionally, contigs mapped to Carlavirus, Pelarspovirus, and other plant viruses in our virome dataset had low coverage and pairwise identity (less than 70%), which need to be further investigated. The presence of AMMV was confirmed by aligning the clean reads to the reference sequence (NC_027924.1) using BWA and SAMtools software, resulting in a consensus sequence (8024 nt) with gaps. DNA extraction from the pooled samples was performed using the Rapid Universal Genomic DNA Extraction Kit (Simgen, Zhejiang, China). Two pairs of specific primers, 3399F (5'-AAAGAAGAACCTCCTGATACGG-3')/5924R (5'-TGAACCTGATTCTCTTGGC-3') and 1830F (5'- CTCAGGAAATCCCAATGC -3')/3640R(5'-TTTCCCAATGTTCTTCGGG-3'), were designed to amplify the complete gene sequences of polymerase and coat protein (CP), based on the consensus sequence. The PCR products with the lengths of 2521 bp and 1814 bp were cloned into the pMD18-T vector (Takara Biotech, Dalian, China) for sequencing. The BLASTn analysis showed that the polymerase and CP gene sequences shared an identity of 94.51% (1929/2041 nt) and 88.41% (1419/1605 nt) with the AMMV isolate (NC_027924.1), respectively. The sequences have been deposited in GenBank under the accession numbers OR544810 and OR544811. We collected leaves from 32 A. lancea plants (16 symptomatic and 16 asymptomatic) in the fields. RT-PCR was conducted using CPF (5'-CTGCGAATATGAAAGTGC-3') and CPR (5'-GGTGAGCTTGTCTGTTAGG-3') primers, which were designed targeting a 527bp fragment of the CP gene (OR544811). Amplicons of the expected size (527bp) were detected in 24 plants (11 symptomatic and 13 asymptomatic), three of which were sequenced by Sanger sequencing, showing a 100% match to OR544811. These findings indicate that AMMV is prevalent in the major production area of A. lancea. Further research is needed to better characterize the potential risks of AMMV to A. lancea cultivation in China as well as other countries.

Strong association between angiotensin I-converting enzyme insertion/deletion polymorphism and unexplained recurrent spontaneous abortion of Sudanese women: a case-control study

This study investigated the link between angiotensin I-converting enzyme insertion/deletion (ACE I/D) polymorphism and unexplained spontaneous abortion. This retrospective analytical case-control was conducted at the Omdurman Maternity Hospital in Sudan. The current study contained 230 individuals, including 119 cases (women who had at least three abortions) of unknown cause and 119 controls (healthy women who had at least two full-term deliveries without spontaneous abortion). Patients and controls were provided five ml of ethylenediaminetetraacetic acid blood and answered questionnaires about their demographics, personal lives, and family histories. ACE I/D polymorphisms were assessed using a conventional polymerase chain reaction approach after total genomic DNA was isolated from blood leukocytes using the GF-1 blood DNA extraction kit. Data was analyzed using the Statistical Package for the Social Sciences version 24. ACE I/D polymorphism is strongly linked to unexplained spontaneous abortion, and women with the I/D and D/D genotypes are more likely to have it than those with the I/I genotype. The current study reveals that ACEI/D polymorphism increases pregnancy problems. Sudanese women may have spontaneous abortions due to the ACE I/D polymorphism.

A meta‐analysis revealing the technical, environmental, and host‐associated factors that shape the gut microbiota of Atlantic salmon and rainbow trout

AbstractSalmonids, specifically Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), are commonly farmed and their gut microbiota plays important roles for optimal growth, health, and physiology. However, differences in experimental design, technical factors and bioinformatics make it challenging to compare the results from different studies and draw general conclusions about their influence on the fish gut microbiota. For a more comprehensive understanding of the gut microbiota, we collected all the publicly accessible 16S rRNA gene sequencing data with clearly stated sample metadata from freshwater Atlantic salmon and rainbow trout intestinal contents and mucosa sequenced on the Illumina MiSeq platform. A total of 783 samples from 19 published studies were included in this meta‐analysis to test the impact of the technical, environmental, and host‐accociated factors. This meta‐analysis revealed that all the tested factors significantly influenced the alpha and beta diversities of the gut microbiota of salmon and trout. Technical factors, especially target region and DNA extraction kit, affected the beta diversity to a larger extent, while host‐associated and environmental factors, especially diet and initial fish weight, had a higher impact on the alpha diversity. Salmon had a higher alpha diversity and higher abundance of Enterococcus and Staphylococcus than trout, which had higher abundance of Weissella and Mycoplasma. The results of this meta‐analysis fill in a critical knowledge gap that demonstrate technical methodologies must be standardized and factors associated with host and environment need to be accounted for in the future design of salmonid gut microbiota experiments.

First Report of Colletotrichum cordylinicola Causing Anthracnose on Cordyline fruticosa in China.

Cordyline fruticosa is a shrub plant, commonly used in landscape, and distributed in the tropical regions of southern China. In September 2022, anthracnose symptoms were found on this species in Nanning, Guangxi, China. The disease incidence was between 30% to 80% and disease severity was 10% to 30% in five surveyed planting areas. The symptoms initially appeared as small, round, brown spots on leaves. As the disease developed, the lesions turned gray-white with brown borders and yellow halos. Some spots coalesced into larger irregular shapes and even leading to leaf blight. Small segments of the diseased tissues (3×3 mm) were cut from the leaves, surface-sterilized by dipping in a 1% sodium hypochlorite solution for 1 min, rinsed three times with sterile distilled water, and plated on potato dextrose agar (PDA). These plates were incubated at 28°C in the dark for 5 days. Ten fungal isolates with similar morphology were consistently isolated from these diseased tissues. The colonies on PDA were initially white with sparse aerial mycelia and turned pale orange with abundant orange conidial masses on the center after 8 days of culture. The reverse color was pale orange. No sclerotia or setae were found in culture. Conidia were single-celled, hyaline, straight, cylindrical with round ends, and 12.2 to 17.8 µm long (mean 14.9 µm) and 3.9 to 7.3 µm wide (mean 4.8 µm, n=50). The morphological characteristics of these isolates were similar to the Colletotrichum cordylinicola (Sharma et al., 2014). Genomic DNA of two isolates Z3 and Z4 generated from monospore culture was extracted using a fungal DNA extraction kit (Solarbio, Beijing, China). Partial sequences of internal transcribed spacer (ITS), partial actin (ACT), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and beta-tubulin (TUB2) were amplified using the primer pairs ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-345R, GDF1/GDR1, and BT2A/BT2B (Lin et al., 2022), respectively. All the sequences (GenBank accession nos. OQ509909, OQ509910, OQ658690, OQ658691, and OK649310 to OK649314) showed 99% to 100% identity with those of C. cordylinicola in GenBank database. A phylogenetic tree based on concatenated sequences of ITS, ACT, CHS-1, TUB, and GAPDH using maximum likelihood analysis by MEGA X software revealed that Z3 and Z4 clade with reference strains of C. cordylinicola (OJX010226 and MK935473). Based on morphological observation and multi-gene sequence analysis, the isolates were identified as C. cordylinicola (Phoulivong et al., 2010). To assess their pathogenicity, conidial suspensions (106 conidia/ml) of C. cordylinicola were inoculated onto 10 healthy living leaves wounded by slight puncturing (10 μl/wounded spot). Control leaves were treated with sterile water. All inoculated and control plants were maintained under high relative humidity (~90%) and 28℃ in a climate chamber. After 8 days, all the inoculated leaves showed brown lesions resembling natural symptoms, whereas the control group remained symptom-free. The same fungus was re-isolated from the symptomatic leaves, thus completing Koch's postulates. C. cordylinicola is a species of the C. gloeosporioides complex (Weir et al., 2012). It has been reported to cause anthracnose on C. fruticosa in USA and Thailand (Phoulivong et al., 2010; Sharma et al., 2014). To our knowledge, this is the first report of C. cordylinicola causing anthracnose on C. fruticosa in China. Knowing the causal agent is essential to control the serious disease effectively.