16S Genes Research Articles

Investigation of Babesia spp. and Theileria spp. in ticks from Western China and identification of a novel genotype of Babesia caballi

Babesia spp. and Theileria spp. are tick-borne protozoan parasites with veterinary importance. In China, epidemiological and genetic investigations on many Babesia and Theileria species were still absent in many areas and many tick species. From Aug 2021 to May 2023, 645 ticks were collected from the body surface of domestic animals (camels, goats, sheep, and cattle) using tweezers in seven counties in three provinces including Xinjiang (Qitai, Mulei, Hutubi, and Shihezi counties), Chongqing (Youyang and Yunyang counties), and Qinghai (Huangzhong county). Three tick species were morphologically and molecularly identified (334 Hyalomma asiaticum from Xinjiang, 245 Rhipicephalus microplus from Chongqing, and 66 Haemaphysalis qinghaiensis from Qinghai). A total of three Babesia species and two Theileria species were detected targeting the 18S gene. The COI and cytb sequences were also recovered from Babesia strains for further identification. In R. microplus from Chongqing, Babesia bigemina, the agent of bovine babesiosis, was detected. Notably, in H. asiaticum ticks from Xinjiang, a putative novel genotype of Babesia caballi was identified (0.90%, 3/334), whose COI and cytb genes have as low as 85.82% and 90.64–90.91% nucleotide identities to currently available sequences. It is noteworthy whether the sequence differences of its cytb contribute to the drug resistance of this variant due to the involvement of cytb in the drug resistance of Babesia. In addition, Theileria orientalis and Theileria annulata were detected in R. microplus from Chongqing (12.20%, 31/245) and H. asiaticum from Xinjiang (1.50%, 5/334), respectively. These results suggest that these protozoan parasites may be circulating in domestic animals in these areas. The pathogenicity of the novel genotype of B. caballi also warrants further investigation.

16S rDNA Sequencing for Bacterial Identification in Preterm Infants with Suspected Early-Onset Neonatal Sepsis.

The high prevalence of suspected early-onset neonatal sepsis among preterm infants leads to immediate antibiotic administration upon admission. Notably, most blood cultures for suspected early-onset neonatal sepsis do not yield a causative pathogen. This study aimed to assess polymerase chain reaction (PCR) targeting the variable region V4 of the 16S ribosomal gene (16S rDNA) and Sanger sequencing for bacterial identification in preterm infants with suspected early-onset neonatal sepsis. Therefore, this prospective study was conducted. Preterm infants with suspected early-onset neonatal sepsis were included in this study. The three groups were formed based on the risk of infection and clinical sepsis. Blood samples were collected upon admission to the neonatal unit for culture and molecular analysis. PCR amplification and subsequent Sanger sequencing of the V4 region of the 16S rDNA were performed. Twenty-eight patients were included in this study. Blood cultures were negative in 100% of the patients. Amplification and sequencing of the V4 region identified bacterial genera in 19 patients across distinct groups. The predominant taxonomically identified genus was Pseudomonas. Amplifying the 16S rDNA variable region through PCR and subsequent Sanger sequencing in preterm neonates with suspected early-onset neonatal sepsis can enhance the identification of microbial species that cause infection, especially in negative cultures.

16s gene metagenomic characterization in healthy stallion semen

Studies on the bacterial composition of seminal samples have primarily focused on species isolated from semen and their effects on fertility and reproductive health. Culture-independent techniques, such as 16S rRNA gene sequencing and shotgun metagenomics, have revolutionized our ability to identify unculturable bacteria, which comprise >90% of the microbiome. These techniques allow for comprehensive analysis of microbial communities in seminal samples, shedding light on their interactions and roles. In this study, we characterized the taxonomic diversity of seminal microbial communities in healthy stallions using 16S rRNA gene sequencing. Semen samples were collected from four stallions during the reproductive season, and DNA was extracted for sequencing. The results revealed a diverse array of bacterial taxa, with Firmicutes, Bacteroidota, and Proteobacteria being predominant phyla. At the family and genus levels, significant variations were observed among individuals, with individual variability in microbial richness and diversity standing out. Moreover, each stallion showed a distinct microbial fingerprint, indicating the presence of a characteristic microbial core for each stallion. These results underscore the importance of considering individual microbial profiles in understanding reproductive health and fertility outcomes.

Changes in the gut microbiome of patients with esophageal cancer: A systematic review and meta-analysis based on 16S gene sequencing technology

BackgroundEsophageal cancer (EC) possesses a high degree of malignancy and exhibits poor therapeutic outcomes and prognosis. However, its pathogenesis remains unclear. With the development of macrogene sequencing technology, changes in the intestinal flora have been found to be highly related to the development of EC, although discrepancies and controversies remain in this research area. Materials and methodsWe comprehensively searched the PubMed, EMBASE, and Cochrane's Central Controlled Trials Register and the Scientific Network's database search projects based on systematically reviewed preferred reporting projects and meta-analyses. We used Engauge Digitizer for data extraction and Stata 15.1 for data analysis. In addition, we used the Newcastle–Ottawa Scale for grade grading and forest and funnel plots, sensitivity, and Egger and Beggar tests to evaluate the risk of bias. ResultsThis study included 10 studies that assessed stool, tumor, and nontumor esophageal mucosa (gastroscopy and surgical resection) samples from 527 individuals, including 273 patients with EC and 254 healthy control group. We observed remarkable differences in microbial diversity in EC patients compared to healthy controls. The Chao1 index (46.01 vs. 42.67) was significantly increased in EC patients, whereas the Shannon index (14.90 vs. 19.05), ACE (39.24 vs. 58.47), and OTUs(28.93 vs. 70.10) were significantly lower. At the phylum level, the abundance of Bacteroidetes (37.89 vs. 32.77) increased significantly, whereas that of Firmicutes (37.63 vs. 38.72) decreased significantly; the abundance of Clostridium and Verruciformis increased, while that of Actinobacteria and Proteobacteria decreased to varying degrees. The abundance of Bacteroides (8.60 vs. 15.10) and Streptococcaceae (15.08 vs. 27.05) significantly reduced in EC. ConclusionsAccording to our meta-analysis, in patients with EC, the Chao1 index increased, whereas the Shannon and the OTUs decreased. At the phylum level, the abundance of Firmicutes decreased significantly, whereas that of Bacteroidetes and Proteobacteria increased significantly. At the genus/family level, the abundance of Bacteroidaceae, Prevotellaceae and Streptococcaceae decreased significantly, whereas that of Veillonellaceae increased. This meta-analysis identified changes in gut microbiota in patients with EC; however, its conclusions were inconsistent.

Comparative genomics analysis of Mycoplasma capricolum subsp. capricolum MOR20

Mycoplasma capricolum subsp. capricolum (Mcc) is an etiological agent of Contagious Agalactia (CA) in small ruminants and belongs to Mycoplasma mycoides cluster. Until today, six strains genomes and candidates of Mcc were sequenced in Germany, USA, France and China. The aim of the present study is to analyze the genomic sequence of Mcc MOR20 strain and to compare it with the other sequenced strains. Indeed, the genome sequence of Mcc MOR20 strain was obtained using Illumina NovaSeq 6000 platform. NGS sequencing reads were assembled de novo using CLC genomics v12 (Qiagen). The variant calling on MOR20 strain was conducted using SNIPPY tools. The phylogenomic analysis of Mcc MOR20 strain was performed using BuscoPhylo pipeline. Thus, the genome of Mcc MOR20 characterized by 960,401 bp and encoded for 820 protein-coding genes, 15 rRNA genes (5S, 16S, and 23S), 34 tRNA genes, and three ncRNA genes. In addition, Mcc MOR20 is genetically closer to Mcc isolate human case with percentage of 98.58 %. In addition, the Mcc MOR20 show a genomic relationship with Mccp clade, and they share the same must of potential virulence genes that were identified, including variable surface lipoproteins and metabolic enzymes. The results of this study could be of interest for contagious agalactia vaccine development and the conserved gene might be taken as candidate for new markers for Mcc diagnosis.

Environmental DNA reveals fine‐scale spatial and temporal variation of marine mammals and their prey species in a Scottish marine protected area

AbstractMarine mammal foraging grounds are popular focal points for marine protected area (MPA) implementation, despite being temporally dynamic, requiring continuous monitoring to infer prey availability and abundance. Marine mammal distributions are assumed to be driven by their prey in foraging areas, but limited understanding of prey distributions often prevents us from exploring how shifting prey availability impacts both seasonal and long‐term marine mammal distributions. Environmental DNA (eDNA) metabarcoding could enhance the understanding of marine mammal habitat use in relation to their prey through simultaneous monitoring of both. However, eDNA applications focused on marine mammals or predator–prey dynamics have been limited to date. In this study, we assess spatiotemporal changes in the distribution and abundance of cetaceans, minke whales (Balaenoptera acutorostrata), bottlenose dolphins (Tursiops truncatus) and harbor porpoises (Phocoena phocoena) in relation to key prey species in a newly established MPA, employing eDNA metabarcoding. We recovered 105 molecular operational taxonomic units (OTUs) from marine vertebrates using two primer sets targeting 12S and 16S genes, along with 112 OTUs from a broader eukaryotic primer set targeting 18S rRNA. Overall, key forage fish prey species, sandeels and clupeids, were the most abundant teleost fishes detected, although their availability varied temporally and with distance from shore. We also found clear spatial partitioning between coastal bottlenose dolphins and the more pelagic minke whales and harbor porpoises, paralleling availability of their main prey species. Other species of conservation interest were also detected including the critically endangered European eel (Anguilla anguilla), Atlantic bluefin tuna (Thunnus thynnus), and the invasive pink salmon (Oncorhynchus gorbuscha). This study demonstrates the application of eDNA to detect spatiotemporal trends in the occurrence and abundance of cetacean predators and their prey, furthering our understanding of fine‐scale habitat use within MPAs. Future long‐term monitoring of predator–prey dynamics with eDNA could improve our ability to predict climate‐induced shifts in foraging grounds and enhance rapid responses with appropriate management actions.

Base-substitution rates of nuclear and mitochondrial genes for polyclad flatworms

The increase in the use of molecular methodologies in systematics has driven the necessity for a comprehensive understanding of the limitations of different genetic markers. Not every marker is optimal for all species, which has led to multiple approaches in the study of the taxonomy and phylogeny of polyclad flatworms. The present study evaluates base-substitution rates of nuclear ribosomal (18S rDNA and 28S rDNA), mitochondrial ribosomal (16S rDNA), and protein-codifying (cytb, cox1) markers for this taxonomic group, with the main objective of assessing the robustness of these different markers for phylogenetic studies. Mutation rates and Ti/Tv ratios of the other markers were assessed for the first time. We estimated substitution rates and found cytb to be the most variable, while 18S rDNA was the least variable among them. On the other hand, the transition to transversion (Ti/Tv) ratio of the different genes revealed differences between the markers, with a higher number of transitions in the nuclear gene 28S and a higher number of transversions in the mitochondrial genes. Lastly, we identified that the third codon position of the studied protein-codifying genes was highly variable and that this position was saturated in the cox1 marker but not in cytb. We conclude that it is important to assess the markers employed for different phylogenetic levels for future studies, particularly in the order Polycladida. We encourage the use of mitochondrial genes cytb and 16S for phylogenetic studies at suborder, superfamily, and family levels and species delimitation in polyclads, in addition to the well-known 28S and cox1.

First Report of Powdery Mildew Caused by Podosphaera xanthii on Sphagneticola trilobata in Hainan Province, China.

Sphagneticola trilobata (L.) Pruski is a perennial creeping herb of the Asteraceae family, which is native to South America. It was introduced into Southern China as a groundcover in the 1970s (Zhang et al. 2023). Now it is mainly used for folk medicine to treat various kinds of inflammatory, incuding joint pain, rheumatic diseases, arthritis, in addition to treating persistent wounds, ulcers, and edemas (Gonçalves et al. 2022). In February and November 2023, powdery mildew symptoms were observed on 60% of S. trilobata plants on the Hainan Medical University campus (19° 58' 53″ N; 110° 19' 47″ E) in Haikou, Hainan Province, China. Powdery mildew colonies covered the leaf surfaces and stems of affected plants, causing discoloration and defoliation. Mycelia were superficial and hyphal appressoria were nipple-shaped. Conidiophores (n =30) were unbranched, cylindrical, 74 to 161 × 10 to 14 µm, and produced three to five immature conidia in chains with a crenate outline. Foot cells (n =30) were cylindrical, straight or sometimes curved at the base, and 27 to 56 µm long. Conidia (n =100) were ellipsoid-ovoid to doliiform, 17 to 30 ×14 to 28 µm (length/width ratio = 1.1 to 1.9), with well-developed fibrosin bodies, and produced germ tubes from the lateral position. Based on these morphological characteristics, the pathogen was provisionally identified as Podosphaera xanthii (Braun and Cook 2012). The teleomorph was not observed. A specimen was deposited in the Hainan Medical University Plant Pathology Herbarium as HMST-23. To confirm the genus identification and ascertain a putative species, genomic DNA was extracted from mycelium, conidiophores, and conidia using a fungal DNA kit (Omega Bio-Tek, USA). The rDNA internal transcribed spacer (ITS) region was amplified with primers ITS1/ITS4 (White et al. 1990) and sequenced directly. The resulting 577-bp sequence was deposited in GenBank (accession no. OR784549). A BLASTn search in GenBank of this sequence showed 100% similarity with the ITS sequences of P. xanthii isolates from China (MT260063, MN203658, OP765400, and MT739423), Thailand (LC270780), and Vietnam (KM260731, KM260730, and KR779870). Additionally, the 28S rDNA region was amplified using the primer pairs NL1 and NL4 (O´Donnell 1993; accession no. OR784550). This region shared 100% similarity with P. xanthii isolates (LC371334, LC270782, AB936277, and OP765401) as well. Powdery mildew from Hainan sample belonged to the P. xanthii group with strong bootstrap values support 99% in maximum likelihood phylogenetic tree based on ITS and 28S gene sequences. To confirm pathogenicity, five healthy potted plants of S. trilobata were inoculated by gently pressing a powdery mildew-infected leaf onto 15 young leaves. Five non-inoculated plants served as controls. All plants were maintained in a greenhouse at 24 to 30°C, 70% relative humidity, with a 16-h photoperiod. After 7 days, inoculated leaves showed powdery mildew symptoms whereas no symptoms were observed on control plants. The fungal colonies observed on inoculated plants were morphologically identical to those found on the originally infected leaves collected from Hainan Province. Based on the morphological characteristics and molecular identification, the fungus was identified as P. xanthii. In different countries and regions, P. xanthii has been previously reported on S. trilobata in Taiwan (Yeh et al. 2021). To our knowledge, this is the first record of P. xanthii infecting S. trilobata in Hainan Province, China. S. trilobata is often planted as an ornamental plant on both sides of the road, and we are concerned that it may serve as a new host, spreading this pathogen to other economic crops.

Island hitchhikers: pathogen agents of Madeira and Azores ticks

Ticks are blood-sucking arthropods that can transmit pathogens to their host. As insular ecosystems can enhance tick-host interactions, this study aimed to understand tick diversity, pathogen presence, and their respective associations in the Azores and Madeira archipelagos. Unfed or partially engorged ticks (n = 120) were collected from 58 cats and dogs in the Azores (n = 41 specimens) and Madeira (n = 79 specimens) from November 2018 to March 2019. Vector identification was based on morphology and molecular criteria. For pathogen sequencing, 18S gene fragment for Babesia/Hepatozoon and gltA for Rickettsia were performed. Sequence data was explored using BLAST and BLAST and phylogenetic inference tools. In the Azores, Ixodes hexagonus, I. ventalloi, and Rhipicephalus sanguineus (n = 6; 14.6%, n = 6; 14.6%, and n = 29; 70.7% respectively) were found and in Madeira I. ricinus and R. sanguineus (n = 78, 98.7%; and n = 1, 1.3%; respectively) were identified. Tick COI markers confirmed species highlighting confirmation of R. sanguineus s.s. and genotype A of I. ventalloi. In the Azores Islands, the detected Rickettsia massiliae was linked to R. sanguineus (dogs and cats) and I. hexagonus (dogs), and in Madeira Island, R. monacensis (dogs) and Hepatozoon silvestris (cats) were found associated with I. ricinus. Further, I. ventalloi presence in the Azores expands west its known range, and Hepatozoon silvestris in Madeira may suggest that I. ricinus could have a role as a potential vector. Finally, as R. massiliae and R. monacensis presence underlines public health risks, surveillance by health authorities is crucial as pathogen-tick interactions may drive disease spread, therefore monitoring remains pivotal for disease prevention.

Brain invasion of bovine coronavirus: molecular analysis of bovine coronavirus infection in calves with severe pneumonia and neurological signs.

Although the role of bovine coronavirus (BCoV) in calf diarrhea and respiratory disorders is well documented, its contribution to neurological diseases is unclear. This study conducted virological investigations of calves showing diarrhea and respiratory and neurological signs. An outbreak of diarrhea, respiratory, and neurological disorders occurred among the 12 calves in July 2022 in Istanbul, Türkiye. Two of these calves exhibited neurological signs and died a few days after the appearance of symptoms. One of these calves was necropsied and analyzed using molecular and histopathological tests. BCoV RNA was detected in the brain, lung, spleen, liver, and intestine of the calf that had neurological signs by real-time reverse transcription polymerase chain reaction. Immunostaining was also observed in the intestine and brain. A 622 bp S1 gene product was noted on gel electrophoresis only in the brain. Phylogenetic analysis indicated that the BCoV detected in this study had a high proximity to the BCoV strain GIb with 99.19% nucleotide sequence homology to the strains detected in Poland, Israel, Türkiye, and France. No distinct genetic lineages were observed when the brain isolate was compared with the respiratory and enteric strains reported to GenBank. In addition, the highest identity (98,72%) was obtained with the HECV 4408 and L07748 strains of human coronaviruses. The strain detected in a calf brain belongs to the GIb-European lineage and shares high sequence homology with BCoV strains detected in Europe and Israel. In addition, the similarity between the human coronaviruses (4408 and L07748) raises questions about the zoonotic potential of the strains detected in this study.

Testing multiple environmental DNA substrates for detection of the cryptic and Critically Endangered burrowing freshwater crayfish Engaewa pseudoreducta

AbstractEffective conservation of endangered species depends on knowledge of their distributions, but species detection can often be challenging. An example of this is provided by the Critically Endangered Margaret River burrowing crayfish (Engaewa pseudoreducta), which is highly cryptic. Due to the burrowing habit of this crayfish, detection of this species currently requires a great deal of effort, the results are often non‐conclusive, and, as it involves manual excavation of their burrows, the habitat of this and other species is destroyed in the detection process. In response to these challenges, this study developed and optimized a species‐specific probe‐based qPCR assay targeting the 16S gene region to detect the target species from environmental samples. Three test substrates—chimney pellets (soil expelled by a crayfish as it digs its burrow), burrow scrapes (soil lining the inside of a burrow), and burrow water (water that is filling the burrow space)—were tested from 11 crayfish burrows thought to have been constructed by the target species to see if eDNA could be detected. DNA from the target species was successfully amplified from both chimney pellets (6/11 samples) and burrow scrapes (3/11 samples); however, E. pseudoreducta was not detected in any water samples. As previously stated, sampling techniques to confirm the presence of this species have relied on burrow excavation (resulting in habitat destruction) and were often not definitive; therefore, replacing the traditional survey method with a noninvasive eDNA‐based technique could be of enormous benefit to the management and conservation of this (and similar) species.

Genetic Variability and Antibacterial Sensitivity of Dichelobacter nodosus and Fusobacterium necrophorum Infection in Sheep Sulaimani Province, Kurdistan Region, Iraq

Footrot is a significant health problem in ruminants, especially sheep, caused by the bacteria Dichelobacter nodosus and Fusobacterium necrophorum. Infected animals may become lame and lose body condition. The aim of this study was to investigate whether sheep had mixed infections with D. nodosus and F. necrophorum, the global genetic relationships of both target genes (lktA and 16S rRNA genes) with different reference genes, and the degree of genetic heterogeneity between the standard serogroups of D. nodosus were studied. In addition, to detect the antibacterial sensitivity against these two pathogens. One hundred and seventy-five sheep interdigital spaces were investigated (2016-2022). The D. nodosus field isolate (Dn Sul/016-KY399851) showed 96% compatibility with other global isolates (NR_104942/USA, JN008724/India, and DQ016290/Sweden) when its 16S rRNA sequence was compared to some reference genes. The local isolate (Fn Sul/016- KY399852) and other IktA gene strains from other nations (FJ230831/New Zealand, JX678872/Australia, JX648295/India) were compared, and the results showed 99% and 96% identity. Additionally, within the multiple sequence alignment, a single nucleotide variation at position 389 (CA) was seen in the partial lktA sequence when compared to the field isolate (Fn Sul/16-KY399852). This modification corresponds to a switch at residue 130 from alanine (Ala) to glutamic acid (Glu). This study showed the initial molecular identification of D. nodosus and F. necrophorum from sheep with footrot in the Iraqi province of Sulaimani‎‎‎‎‎.

Chitosan Potential as Delivery Agent for S1 Gene from SARS-CoV-2 with pEGFP-N1 as its Vectors

Coronavirus Disease-19 (COVID-19) merupakan penyakit yang disebabkan oleh severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), penyakit ini mulai muncul pada akhir tahun 2019 dan menjadi pandemi hingga saat ini. Vaksinasi dapat berperan penting dalam prevensi penyebaran penyakit, sehingga banyak dilakukan pengembangan vaksin salah satunya ialah vaksin DNA. Sistem penghantaran seperti kitosan dapat meningkatkan efektifitas dari vaksin. Tujuan dari penelitian ini adalah untuk mengoptimasi dan mengevaluasi formula pEGFP-N1-S1 dengan sistem penghantaran kitosan sebagai kandidat vaksin DNA. Plasmid dilakukan kloning pada E.coli DH5α dan dilakukan ekstraksi untuk evaluasi selanjutnya dengan enzim restriksi dan sekuensing. Plasmid EGFP-N1-S1 kemudian diformulasikan dengan nanopartikel kitosan dengan rasio massa Plasmid DNA:kitosan sebesar 1:0,1; 1:0,2; 1:0,3; 1:0,4; 1:0,5; 1:0,6 dan 1:0,7. Hasil plasmid yang dilakukan isolasi memiliki konsentrasi 2182,32 ng/L dan kemurnian sebesar 1,911. Hasil restriksi menunjukkan bahwa gen S1 (684 bp) berhasil diinsersi pada plasmid pEGFP-N1 (4813 bp). Rasio massa DNA:kitosan sebesar 1:0,6 merupakan formulasi yang optimal untuk DNA berikatan dengan kitosan secara sempurna.

A New Dual Fluorescence Method for Rapid Detection of Infectious Bronchitis Virus at Constant Temperature.

Infectious bronchitis virus (IBV) causes infectious bronchitis in chicken, an acute, highly contagious respiratory infection. Because of genetic mutations and recombination, IBV forms many subtypes, which makes it difficult to treat the disease and apply commercial vaccines. Therefore, to detect IBV in time and stop the virus from spreading, a novel and convenient IBV detection technology based on reverse transcription recombinase-aided amplification (RT-RAA) was established in this study. According to the S1 gene of IBV CH I-V and Mass genotypes and S1 gene of IBV CH VI genotype, a set of optimal primers were designed and selected to establish a real-time dual fluorescence RT-RAA method. The lowest detection line was 10 copies/μL of RNA molecules and the method exhibited no cross-reactivity with avian reticuloendotheliosis virus (REV), infectious bursal disease virus (IBDV), avian leukosis virus (ALV), Newcastle disease virus (NDV), chicken infectious anemia virus (CIAV), infectious laryngotracheitis virus (ILTV), Marek's disease virus (MDV), and H9N2 avian influenza virus (H9N2), demonstrating high specificity. When compared to qPCR detection results, our method achieved a sensitivity of 96.67%, a specificity of 90%, and a Kappa value of 0.87 for the IBV CH I-V and Mass genotypes, and achieved a sensitivity of 100%, a specificity of 97.73%, and a Kappa value of 0.91 for the IBV CH VI genotype.

Accumulation of 5-methyltetrahydrofolate and other bioactive compounds, in the course of fermentation of green tea (Camellia sinensis) kombucha

Kombucha is a potential probiotic tea-based drink with increasing worldwide consumption. Studies on this probiotic beverage are growing rapidly, especially about micronutrients and microbial population. As such, the present study performed the molecular identification of the microorganism and evaluated 5-methyltetrahydrofolate content by HPLC-DAD, phenolic compounds, flavonoids, carotenoids, antioxidant activity by spectrophotometric methods, and physicochemical composition of green tea kombucha on fermentation days 1, 3, 7, 14, and 21. DNA sequencing identified the Microbacterium genus as predominant. However, was unable to safely determine the species level because of the rRNA 16S gene sequence similarity between four species M. ureisolvens, M. yannicii, M. chocolatum e M. atlanticum. The concentration of 5-methyltetrahydrofolate found on the third day was 39.12 ± 1.32 μg/mL (liquid) and 45.78 ± 8.42 μg/mL (polymeric biofilm); On the twenty-first day it was 50.87 ± 3.56 μg/mL (liquid) and 54.88 ± 3.89 μg/mL (polymeric biofilm). Total phenolic compounds increased with fermentation; however, flavonoids and carotenoids were degraded by the process. The information on 5-methyltetrahydrofolate is unprecedented and highly relevant for food guidelines, since related deficiencies can lead to fetal malformation in the first three months of pregnancy. Lastly, the best fermentation time to obtain 5-methyltetrahydrofolate and others bioactive compounds is between days 7–14. Further analyses are also encouraged to understand the bioavailability of the vitamin.

The impact of space, host dissimilitude, and environment on prokaryotic communities of golf ball sponges

AbstractGolf ball sponges are small, sometimes inconspicuous, sponges. They can be found across a range of habitats varying from perturbed and pristine coral reefs to harbours and marine lakes and from the deep sea to shallow waters. They can be difficult to distinguish in the field and have presented some problems with taxonomists lumping and splitting species due to the difficulty in defining clear species boundaries. In the present study, we sampled golf ball sponges from Indo‐Pacific and Caribbean locations and used 16S gene amplicon sequencing to study their prokaryotic communities. We show that golf ball sponges harbour a wide variety of prokaryotic communities. Among the most prevalent operational taxonomic units (OTUs), several belonged to a range of taxa, including the bacterial AqS1 and EC94 groups, which have been associated with genes known to facilitate interactions between hosts and microbes. Certain host taxa were enriched with OTUs classified to the SAR202 clade of Chloroflexi. Our findings show that prokaryotic dissimilarity varied as a function of space (geographical distance) and host dissimilitude. The importance of space and host dissimilitude, however, varied depending on the data transformation with host dissimilitude a more important predictor of untransformed data whereas space was a more important predictor of log‐transformed data. Given that log‐transformation downscales the influence of abundant taxa, we interpret these results by the tendency of closely related host organisms to host similar sets of abundant symbiotic microorganisms; distantly sampled specimens, in contrast, tend to harbour less abundant prokaryotic microorganisms found in the surrounding environment (e.g., seawater or sediment).

Deep learning for predicting 16S rRNA gene copy number

Culture-independent 16S rRNA gene metabarcoding is a commonly used method for microbiome profiling. To achieve more quantitative cell fraction estimates, it is important to account for the 16S rRNA gene copy number (hereafter 16S GCN) of different community members. Currently, there are several bioinformatic tools available to estimate the 16S GCN values, either based on taxonomy assignment or phylogeny. Here we present a novel approach ANNA16, Artificial Neural Network Approximator for 16S rRNA gene copy number, a deep learning-based method that estimates the 16S GCN values directly from the 16S gene sequence strings. Based on 27,579 16S rRNA gene sequences and gene copy number data from the rrnDB database, we show that ANNA16 outperforms the commonly used 16S GCN prediction algorithms. Interestingly, Shapley Additive exPlanations (SHAP) shows that ANNA16 can identify unexpected informative positions in 16S rRNA gene sequences without any prior phylogenetic knowledge, which suggests potential applications beyond 16S GCN prediction.

Tobacco crop rotation enhances the stability and complexity of microbial networks.

The effects of continuous cropping and rotation cropping, two important tobacco cultivation practices, on soil microbial communities at different stages remain unclear. Different planting patterns have been shown to influence soil physical and chemical properties, which in turn can affect the composition and diversity of soil microbial communities. In order to investigate the impact of different planting methods on soil microbial community structure, we selected two representative planting methods: continuous cropping (tobacco) and rotational cropping (tobacco-maize). These methods were chosen as the focal points of our research to explore the potential effects on soil microbial communities. High-throughput sequencing technology was employed to investigate the structure of soil microbial communities, as well as their relationships with soil environmental factors, by utilizing the 16S rRNA, ITS, and 18S genes. Furthermore, the interaction among microorganisms was explored through the application of the Random Matrix Theory (RMT) molecular ecological network approach. There was no significant difference in α diversity, but significant difference in β diversity based on Jaccard distance test. Compared to continuous cropping, crop rotation significantly increased the abundance of beneficial prokaryotes Verrucomicrobia and Rhodanobacter. These findings indicate that crop rotation promotes the enrichment of Verrucomicrobia and Rhodanobacter in the soil microbial community. AP and NH4-N had a greater effect on the community structure of prokaryotes and fungi in tobacco soil, while only AP had a greater effect on the community structure of protist. Molecular ecological network analysis showed that the network robustness and Cohesion of rotation were significantly higher than that of continuous cropping, indicating that the complexity and stability of molecular ecological networks were higher in the rotational, and the microbial communities cooperated more effectively, and the community structure was more stable. From this point of view, rotational cropping is more conducive to changing the composition of soil microbial community, enhancing the stability of microbial network structure, and enhancing the potential ecological functions in soil.

First Report of Powdery Mildew on Cardamine violifolia in China.

Cardamine violifolia, also called Cardamine hupingshanensis, is an economically important medicinal plant renowned for accumulating selenium (Guo et al., 2022). Selenium is an essential trace element with anti-oxidant, anti-inflammatory, anti-cancer, and immune regulatory functions. In July 2023, an outbreak of powdery mildew was detected, infecting the leaves of numerous C. violifolia plants in Enshi (30°11'5.27''N; 109°48'48.45''E), Hubei Province, China. This disease caused severe damage to plant leaves and stems, starting as individual spots and merging into a large mold that covers the entire leaf. It affected nearly 25% all C. violifolia plants, resulting in significant yield loss, disruption of normal metabolism, and premature aging. The lower leaf blades and underside of the leaves were particularly vulnerable. The affected leaves were collected and subjected to morphological diagnostic analysis (Mori et al., 2000) (Fig. S1). The powdery mildew species aggressively spread throughout the leaves, pedicels, and pods, persisting until present and often covering the entire surface. The conidiophores were upright, cylindrical, composed of 3 to 4 cells, and measured 92.3 ± 12.9 × 9.2 ± 0.6 μm (n = 30). Conidial pedicels had 21.6 ± 3.4 μm (n = 50) long cylindrical podocytes. The monoconidia were columnar or barrel-columnar, 30.60-55.59 × 9.11-20.00 μm in size. Conidia lacked an obvious cellulose body. The bud tubes formed from the end of conidia, and papillary appressoria developed on the epiphytic mycelia. ITS region sequences were amplified using the specific powdery mildew universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3'), PM6 (5'-GYCRCYCTGTCGCGAG-3') for partial sequences of 18S and 28S ribosomal DNA genes (Takamatsu et al., 2001). The sequence was deposited in the GenBank under the accession number OR506156 and aligned with available sequences on NCBI, which were 99.2%(528/532) identical to the E. cruciferarum (MT309701, MF192845, and KY660929) sequences (Fig. S2). The ITS sequence from GenBank was used to conduct maximum likelihood phylogenetic analysis using MEGA 11.0. The analysis results showed both the strain and E. cruciferarum clustered on the same branch. To confirm Koch's postulates, pathogenicity testing was carried out using an illuminating incubator. Infected leaves were attached to healthy leaves of C. violifolia seedlings (n=8). All the plants were incubated under 25℃ and >80% relative humidity. After one month, all inoculated plants presented the same symptoms as those initially observed in the field. Morphological and molecular analysis confirmed the isolated fungi's identity as the same pathogen. Therefore, C. violifolia is a suitable host for E. cruciferarum in China. The growers must be informed of these findings to prevent serious economic losses caused by this pathogenic white powder and to prepare for proper management practices. To our knowledge, this is the first report of E. cruciferarum infecting C. violifolia in China.

Novel Theileria sp. as an Etiology of Cutaneous Theileriosis among the Vulnerable Arabian Oryx.

The Arabian Peninsula's endemic ungulate, Oryx leucoryx, was on the verge of extinction at the end of the 1970s. Despite the different reintroduction programs, the International Union for Conservation of Nature is still classifying it as Vulnerable. Among other factors, their vulnerability lies in their susceptibility to specific etiological agents that affect livestock, necessitating health monitoring and strict preventive/biosecurity measures. Within this frame, the current work investigated the determination of the etiological agent potentially involved with cutaneous lesions observed in eight males of Arabian oryx within one of the several national governance conservation programs. Microscopic examination from one animal specimen suggested theileriosis association, which was confirmed by molecular tools using 18S gene sequencing and the report of a novel Theileria sp. not clustering with previously reported antelope sequences. This finding prompts further explorations into the disease dynamics within the Arabian oryx population, especially with the scarcity of data in Qatar about tick-borne pathogens and their transmission.