Phylogenetic Identification Research Articles

Genetic differentiation of Oncomelania hupensis robertsoni in hilly regions of China: Using the complete mitochondrial genome.

Oncomelania hupensis robertsoni is the only intermediate host of Schistosoma japonicum in hilly regions of south-west China, which plays a key role during the transmission of Schistosomiasis. This study aimed to sequence the complete mitochondrial genome of Oncomelania hupensis robertsoni and analyze genetic differentiation of Oncomelania hupensis robertsoni. Samples were from 13 villages in Yunnan Province of China, with 30 Oncomelania hupensis snails per village, and the complete mitochondrial genome was sequenced. A comprehensive analysis of the genetic differentiation of Oncomelania hupensis robertsoni was conducted by constructing phylogenetic trees, calculating genetic distances, and analyzing identity. A total of 26 complete mitochondrial sequences were determined. The length of genome ranged from 15,181 to 15,187 bp, and the base composition of the genome was A+T (67.5%) and G+C content (32.5%). This genome encoded 37 genes, including 13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes. The phylogenetic trees and identity analysis confirmed that Oncomelania hupensis robertsoni was subdivided into Oncomelania hupensis robertsoni Yunnan strain and Sichuan strain, with a genetic distance of 0.0834. Oncomelania hupensis robertsoni Yunnan strain was further subdivided into two sub-branches, corresponding to "Yunnan North" and "Yunnan South", with a genetic distance of 0.0216, and the samples exhibited over 97% identity. Oncomelania hupensis robertsoni is subdivided into Oncomelania hupensis robertsoni Yunnan strain and Sichuan strain. Oncomelania hupensis robertsoni Yunnan strain exhibits a higher level of genetic identity and clear north-south differentiation. This work reported the first mitochondrial genome of Oncomelania hupensis robertsoni Yunnan strain, which could be used as an important reference genome for Oncomelania hupensis, and also provide the important information for explaining the distribution pattern of Oncomelania hupensis robertsoni and control of Schistosoma japonicum.

Complete Chloroplast Genomes and Phylogenetic Analysis of Woody Climbing Genus Phanera (Leguminosae).

Phanera Lour., a genus in the subfamily Cercidoideae of the family Leguminosae, is characterized by woody liana habit, tendrils, and distinctive bilobate or bifoliolate leaves. The genus holds important medicinal value and constitutes a complex group characterized by morphological diversity and unstable taxonomic boundaries. However, limited information on the chloroplast genomes of this genus currently available constrains our understanding of its species diversity. Hence, it is necessary to obtain more chloroplast genome information to uncover the genetic characteristics of this genus. We collected and assembled the complete chloroplast genomes of nine representative Phanera plants, including Phanera erythropoda, Phanera vahlii, Phanera aureifolia, Phanera bidentata, Phanera japonica, Phanera saigonensis, Phanera championii, Phanera yunnanensis, and Phanera apertilobata. We then conducted a comparative analysis of these genomes and constructed phylogenetic trees. These species are each characterized by a typical quadripartite structure. A total of 130-135 genes were annotated, and the GC content ranged from 39.25-42.58%. Codon usage analysis indicated that codons encoding alanine were dominant. We found 82-126 simple sequence repeats, along with 5448 dispersed repeats, mostly in the form of forward repeats. Phylogenetic analysis revealed that 16 Phanera species form a well-supported monophyletic group, suggesting a possible monophyletic genus. Furthermore, 10 hypervariable regions were detected for identification and evolutionary studies. We focused on comparing chloroplast genome characteristics among nine Phanera species and conducted phylogenetic analyses, laying the foundation for further phylogenetic research and species identification of Phanera.

Identification and Characterization of Four Novel Viruses in Balclutha incisa.

Balclutha incisa (Cicadellidae: Deltocephalinae), a leafhopper prevalent in tropical and temperate regions, is notably abundant in grasses and rice. The virome of B. incisa was investigated using deep transcriptome sequencing, leading to the first identification of four viruses belonging to the families Aliusviridae, Iflaviridae, and Totiviridae in B. incisa. These viruses have been provisionally named B. incisa ollusvirus 1 (BiOV1), B. incisa ollusvirus 2 (BiOV2), B. incisa iflavirus 1 (BiIV1), and B. incisa totivirus 1 (BiTV1). The complete genome sequences of these viruses were obtained through rapid amplification of cDNA ends (RACE). BiOV1 has a linear genome of 15,125 nucleotides (nt), while BiOV2 possesses a circular genome of 14,853 nt. The BiIV1 genome, excluding the poly(A) tail, is 10,903 nt in length and encodes a single open reading frame (ORF) for a polyprotein consisting of 3194 amino acids (aa). The BiTV1 genome is 4357 nt long and contains two overlapping ORFs, with the viral RNA-dependent RNA polymerase (RdRp) translated via a -1 ribosomal frameshift. Phylogenetic and sequence identity analyses suggest that all these viruses are novel members of their respective families. This study significantly expands our understanding of the virome associated with B. incisa by reporting and characterizing these novel viruses.

Xanthomonas sontii, and Not X. sacchari, Is the Predominant Vertically Transmitted Core Rice Seed Endophyte.

Seed endophytes, particularly the abundant, core, and vertically transmitted species, are major areas of focus in host microbiome studies. Apart from being the first members to colonize, they accompany the plant throughout its development stages and to the next generation. Recently published studies have reported the keystone species to be Xanthomonas sacchari, a core endophyte that is vertically transmitted in rice with probiotic properties. Furthermore, the Xanthomonas species was reported to be involved in the assembly of beneficial bacteria after early inoculation in rice seeds. However, the strains discussed in these studies were misclassified as X. sacchari, a well-known pathogen of sugarcane. By including nonpathogenic Xanthomonas species with plant-protective functions reported from rice seeds, we have correctly established the phylogenetic and taxonomic identity of the keystone species as X. sontii. This will enable researchers to use the correct reference or lab strain of X. sontii for further systematic and in-depth studies as a model endophyte in plant-microbe interactions apart from its exploitation in seed health.

Molecular cluster, transmission characteristics, origin and dynamics analysis of HIV-1 CRF59_01B in China: A molecular epidemiology study

PurposeThis study investigated for the HIV-1 CRF59_01B epidemic's spatiotemporal dynamics and its transmission networks in China. MethodsBetween 2007 and 2020, a total of 250 partial pol gene sequences of HIV-1 CRF59_01B were collected from four regions (10 Chinese provinces). Phylogenetic tree construction and cluster identification were then performed. The Bayesian skyline and birth–death susceptible–infected–removed models were employed for the phylodynamic analyses of subtypes and large clusters, respectively. Phylogenetic analyses and trait diffusion of these sequences were performed using Bayesian phylogenetic methods (beast-classic package). Distance-based molecular network analyses were performed to identify putative relationships. ResultsUsing a genetic distance threshold of 1.3 %, We identified 45 clusters that included 62.40 % (156/250) of the sequences. Three clusters (6.67 %, 3/45) had 10 or more sequences, and were considered "large clusters". Six clusters (13.33 %) included sequences from different regions (Southeast, Northeast, Southeast, and Central China). Thirteen clusters (28.89 %) included sequences of men who had sex with men only, three clusters (6.67 %) included sequences of heterosexuals only, and 12 clusters (26.67 %) included sequences of both groups. The substitution rate of CRF59_01B was 1.91 × 10–3 substitutions per site per year [95 % highest posterior density (HPD) interval: 1.39 × 10−3–2.49 × 10−3)], the time to the most recent common ancestor of CRF59_01B was to be 1992.83 (95 % HPD: 1977.97–2002.81). A Bayesian skyline plot revealed that the effective population size of CRF59_01B increased from 2000 to 2015 and remained stable after 2015. The large clusters showed continuous growth from 2013 to 2020. Phylogeographic analysis showed that CRF59_01B B most likely originated in Southeast China, with a posterior probability of 97.44 %, and then spread to other regions. ConclusionsOur study revealed the temporal and geographical origins of HIV-1 CRF59_01B as well as the process of transmission among various regions and risk groups in China, which can help develop targeted HIV prevention strategies.

Pangenomic and biochemical analyses of Helcococcus ovis reveal widespread tetracycline resistance and a novel bacterial species, Helcococcus bovis.

Helcococcus ovis (H. ovis) is an opportunistic bacterial pathogen of a wide range of animal hosts including domestic ruminants, swine, avians, and humans. In this study, we sequenced the genomes of 35 Helcococcus sp. clinical isolates from the uterus of dairy cows and explored their antimicrobial resistance and biochemical phenotypes in vitro. Phylogenetic and average nucleotide identity analyses classified four Helcococcus isolates within a cryptic clade representing an undescribed species, for which we propose the name Helcococcus bovis sp. nov. By establishing this new species clade, we also resolve the longstanding question of the classification of the Tongji strain responsible for a confirmed human conjunctival infection. This strain did not neatly fit into H. ovis and is instead a member of H. bovis. We applied whole genome comparative analyses to explore the pangenome, resistome, virulome, and taxonomic diversity of the remaining 31 H. ovis isolates. An overwhelming 97% of H. ovis strains (30 out of 31) harbor mobile tetracycline resistance genes and displayed significantly increased minimum inhibitory concentrations of tetracyclines in vitro. The high prevalence of mobile tetracycline resistance genes makes H. ovis a significant antimicrobial resistance gene reservoir in our food chain. Finally, the phylogenetic distribution of co-occurring high-virulence determinant genes of H. ovis across unlinked and distant loci highlights an instance of convergent gene loss in the species. In summary, this study showed that mobile genetic element-mediated tetracycline resistance is widespread in H. ovis, and that there is evidence of co-occurring virulence factors across clades suggesting convergent gene loss in the species. Finally, we introduced a novel Helcococcus species closely related to H. ovis, called H. bovis sp. nov., which has been reported to cause infection in humans.

First Molecular and Phylogenetic Identification of Toxoplasma Gondii in Sheep Liver Intended for Human Consumption in Northern Tunisia.

The aim of this study was to estimate the molecular infection prevalence of Toxoplasma gondii in sheep liver tissues destined for human consumption. A total number of 224 liver tissues were collected from slaughtered sheep in Sejnane slaughterhouse (Northwest Tunisia). PCR was used to detect T. gondii DNA in liver tissues followed by phylogenetic analysis of amplicons. The phylogenetic tree was then constructed to compare the partial sequences of the ITS1 gene with GenBank sequences.The overall molecular prevalence of T. gondii in sheep livers was 25% (56/224). The highest molecular prevalence of T. gondii was recorded in sheep aged of less than one year old (27.3%; 52/190). Infection prevalence was significantly higher in Noire de Thibar breed (33%; 17/51) compared to other breeds (p = 0.023). There were no differences depicted according to sheep's gender. The T. gondii sequences obtained in the present study (GenBank accession numbers: OR509829 and OR509830) were 98.40-100% homologous to T. gondii sequences published in the GenBank. These results highlight a high level of T. gondii contamination of tissues destined for human consumption. Further studies are needed to improve our knowledge on different genotypes of T. gondii that infect Tunisian sheep population.

Discovery and Genomic Analysis of Three Novel Viruses in the Order Mononegavirales in Leafhoppers.

Leafhoppers are economically important pests and may serve as vectors for pathogenic viruses that cause substantial crop damage. In this study, using deep transcriptome sequencing, we identified three novel viruses within the order Mononegavirales, including two viruses belonging to the family Rhabdoviridae and one to the family Lispiviridae. The complete genome sequences were obtained via the rapid amplification of cDNA ends and tentatively named Recilia dorsalis rhabdovirus 1 (RdRV1, 14,251 nucleotides, nt), Nephotettix virescens rhabdovirus 1 (NvRV1, 13,726 nt), and Nephotettix virescens lispivirus 1 (NvLV1, 14,055 nt). The results of a phylogenetic analysis and sequence identity comparison suggest that RdRV1 and NvRV1 represent novel species within the family Rhabdoviridae, while NvLV1 is a new virus belonging to the family Lispiviridae. As negative-sense single-strand RNA viruses, RdRV1 and NvRV1 contain the conserved transcription termination signal and intergenic trinucleotides in the non-transcribed region. Intergenomic sequence and transcriptome profile analyses suggested that all these genes were co-transcriptionally expressed in these viral genomes, facilitated by specific intergenic trinucleotides and putative transcription initiation sequences.

A First Approach for the In Vitro Cultivation, Storage, and DNA Barcoding of the Endangered Endemic Species Euonymus koopmannii.

Euonymus koopmannii is a rare and protected species in Kazakhstan, valued for its ecological role in soil stabilization and its ornamental properties. This study presents the first use of micropropagation and phylogenetic analysis for the endemic plant E. koopmannii. Seedlings of E. koopmannii proved to be more effective than internodes as primary explants for plant micropropagation of in vitro culture, with a multiplication coefficient of 28.5 from seedlings and 6.1 from internodes. On MSR I medium supplemented with 0.5 mg/L IBA and 0.05 mg/L IAA, a higher success rate of 67% was achieved for root formation of test tube-grown E. koopmannii plants. Using mannitol as an osmotic agent at a concentration of 8 mg/L prolonged the storage time of E. koopmannii under slow growth conditions when compared to CCC and abscisic acid. Phylogenetic relationships and species identification were analyzed using four DNA-barcoding markers, comparing E. koopmannii with species from NCBI. All candidate barcoding markers showed sufficient levels of interspecific genetic variation among Euonymus species. In addition, ITS region and rbcL gene sequences effectively distinguished E. koopmannii from other species. These results provide fundamental information that will be valuable for future biotechnological and molecular studies.

Antibacterial activity evaluation of a novel K3-specific phage against Acinetobacter baumannii and evidence for receptor-binding domain transfer across morphologies

Acinetobacter baumannii (A. baumannii) poses a serious public health challenge due to its notorious antimicrobial resistance, particularly carbapenem-resistant A. baumannii (CRAB). In this study, we isolated a virulent phage, named P1068, from medical wastewater capable of lysing CRAB, primarily targeting the K3 capsule type. Basic characterization showed that P1068 infected the A. baumannii ZWAb014 with an optimal MOI of 1, experienced a latent period of 10 ​min and maintained stability over a temperature range of 4–37 ​°C and pH range of 3–10. Phylogenetic and average nucleotide identity analyses indicate that P1068 can be classified as a novel species within the genus Obolenskvirus of the Caudoviricetes class as per the most recent virus classification released by the International Committee on Taxonomy of Viruses (ICTV). Additionally, according to classical morphological classification, P1068 is identified as a T4-like phage (Myoviridae). Interestingly, we found that the tail fiber protein (TFP) of P1068 shares 74% coverage and 88.99% identity with the TFP of a T7-like phage (Podoviridae), AbKT21phiIII (NC_048142.1). This finding suggests that the TFP gene of phages may undergo horizontal transfer across different genera and morphologies. In vitro antimicrobial assays showed that P1068 exhibited antimicrobial activity against A. baumannii in both biofilm and planktonic states. In mouse models of intraperitoneal infection, P1068 phage protected mice from A. baumannii infection and significantly reduced bacterial loads in various tissues such as the brain, blood, lung, spleen, and liver compared to controls. In conclusion, this study demonstrates that phage P1068 might be a potential candidate for the treatment of carbapenem-resistant and biofilm-forming A. baumannii infections, and expands the understanding of horizontal transfer of phage TFP genes.

Exploring Canine Picornavirus Diversity in the USA Using Wastewater Surveillance: From High-Throughput Genomic Sequencing to Immuno-Informatics and Capsid Structure Modeling.

The SARS-CoV-2 pandemic resulted in a scale-up of viral genomic surveillance globally. However, the wet lab constraints (economic, infrastructural, and personnel) of translating novel virus variant sequence information to meaningful immunological and structural insights that are valuable for the development of broadly acting countermeasures (especially for emerging and re-emerging viruses) remain a challenge in many resource-limited settings. Here, we describe a workflow that couples wastewater surveillance, high-throughput sequencing, phylogenetics, immuno-informatics, and virus capsid structure modeling for the genotype-to-serotype characterization of uncultivated picornavirus sequences identified in wastewater. Specifically, we analyzed canine picornaviruses (CanPVs), which are uncultivated and yet-to-be-assigned members of the family Picornaviridae that cause systemic infections in canines. We analyzed 118 archived (stored at -20 °C) wastewater (WW) samples representing a population of ~700,000 persons in southwest USA between October 2019 to March 2020 and October 2020 to March 2021. Samples were pooled into 12 two-liter volumes by month, partitioned (into filter-trapped solids [FTSs] and filtrates) using 450 nm membrane filters, and subsequently concentrated to 2 mL (1000×) using 10,000 Da MW cutoff centrifugal filters. The 24 concentrates were subjected to RNA extraction, CanPV complete capsid single-contig RT-PCR, Illumina sequencing, phylogenetics, immuno-informatics, and structure prediction. We detected CanPVs in 58.3% (14/24) of the samples generated 13,824,046 trimmed Illumina reads and 27 CanPV contigs. Phylogenetic and pairwise identity analyses showed eight CanPV genotypes (intragenotype divergence <14%) belonging to four clusters, with intracluster divergence of <20%. Similarity analysis, immuno-informatics, and virus protomer and capsid structure prediction suggested that the four clusters were likely distinct serological types, with predicted cluster-distinguishing B-cell epitopes clustered in the northern and southern rims of the canyon surrounding the 5-fold axis of symmetry. Our approach allows forgenotype-to-serotype characterization of uncultivated picornavirus sequences by coupling phylogenetics, immuno-informatics, and virus capsid structure prediction. This consequently bypasses a major wet lab-associated bottleneck, thereby allowing resource-limited settings to leapfrog from wastewater-sourced genomic data to valuable immunological insights necessary for the development of prophylaxis and other mitigation measures.

Phylogenetic and Pathogenic Evidence Reveals Novel Host-Pathogen Interactions between Species of Lasiodiplodia and Citrus latifolia Dieback Disease in Southern Mexico.

Mexico ranks second in the world for Persian lime (Citrus latifolia) exports, making it the principal citrus exporter within the national citrus industry, exporting over 600,000 tons per year. However, diseases are the main factor reducing production, resulting in significant economic losses. Among these diseases, fungal diseases like dieback, caused by species of Lasiodiplodia, are an emerging issue in Persian lime. Symptoms include gummosis, twig and branch dieback, cankers, the necrosis of bark and wood, fruit mummification, and tree decline. The aim of this study was to investigate the occurrence and pathogenicity of the fungal species associated with twig and branch dieback, cankers, and decline of Persian lime trees in southern Mexico, and to elucidate the current status of the Lasiodiplodia species causing the disease in Mexico. During June, July, and August of 2023, a total of the 9229 Persian lime trees were inspected across 230 hectares of Persian lime orchards in southern Mexico, and symptoms of the disease were detected in 48.78% of the trees. Branches from 30 of these Persian lime trees were collected. Fungal isolates were obtained, resulting in a collection of 40 strains. The isolates were characterized molecularly and phylogenetically through the partial regions of four loci: the internal transcribed spacer region (ITS), the β-tubulin gene (tub2), the translation elongation factor 1-alpha gene (tef1-α), and the DNA-directed RNA polymerase II second largest subunit (rpb2). Additionally, pathogenicity was assessed, successfully completing Koch's postulates on both detached Persian lime branches and certified 18-month-old Persian lime plants. Through multilocus molecular phylogenetic identification, pathogenicity, and virulence tests, five species were identified as causal agents: L. iraniensis, L. lignicola, L. mexicanensis, L. pseudotheobromae, and L. theobromae. This study demonstrates that in southern Mexico, at least five species of the genus Lasiodiplodia are responsible for dieback in Persian lime. Additionally, this is the first report of L. lignicola and L. mexicanensis as causal agents of the disease in citrus, indicating novel host interactions between species of Lasiodiplodia and C. latifolia.

Plastome evolution of Engelhardia facilitates phylogeny of Juglandaceae

BackgroundEngelhardia (Juglandaceae) is a genus of significant ecological and economic importance, prevalent in the tropics and subtropics of East Asia. Although previous efforts based on multiple molecular markers providing profound insights into species delimitation and phylogeography of Engelhardia, the maternal genome evolution and phylogeny of Engelhardia in Juglandaceae still need to be comprehensively evaluated. In this study, we sequenced plastomes from 14 samples of eight Engelhardia species and the outgroup Rhoiptelea chiliantha, and incorporated published data from 36 Juglandaceae and six outgroup species to test phylogenetic resolution. Moreover, comparative analyses of the plastomes were conducted to investigate the plastomes evolution of Engelhardia and the whole Juglandaceae family.ResultsThe 13 Engelhardia plastomes were highly similar in genome size, gene content, and order. They exhibited a typical quadripartite structure, with lengths from 161,069 bp to 162,336 bp. Three mutation hotspot regions (TrnK-rps16, ndhF-rpl32, and ycf1) could be used as effective molecular markers for further phylogenetic analyses and species identification. Insertion and deletion (InDels) may be an important driving factor for the evolution of plastomes in Juglandoideae and Engelhardioideae. A total of ten codons were identified as the optimal codons in Juglandaceae. The mutation pressure mostly contributed to shaping codon usage. Seventy-eight protein-coding genes in Juglandaceae experienced relaxed purifying selection, only rpl22 and psaI genes showed positive selection (Ka/Ks > 1). Phylogenetic results fully supported Engelhardia as a monophyletic group including two sects and the division of Juglandaceae into three subfamilies. The Engelhardia originated in the Late Cretaceous and diversified in the Late Eocene, and Juglandaceae originated in the Early Cretaceous and differentiated in Middle Cretaceous. The phylogeny and divergence times didn’t support rapid radiation occurred in the evolution history of Engelhardia.ConclusionOur study fully supported the taxonomic treatment of at the section for Engelhardia species and three subfamilies for Juglandaceae and confirmed the power of phylogenetic resolution using plastome sequences. Moreover, our results also laid the foundation for further studying the course, tempo and mode of plastome evolution of Engelhardia and the whole Juglandaceae family.

Molecular Identification of Kocuria Species Isolated from Pediatric Teaching Hospital in Sulaymaniyah, Iraq

Abstract Background: Kocuria species is now increasingly being recognized as an emerging human pathogen, suggesting that this genus has mostly been neglected or misidentified. Objective: This study attempts to use molecular techniques to correctly identify Kocuria species isolated from inpatient children, and to compare between the identification results obtained from two methods: VITEK 2 and 16S rRNA gene sequencing. Materials and Methods: VITEK 2, an automated system, was used to identify clinical isolates. Isolates identified as Kocuria spp. were submitted to DNA extraction and polymerase chain reaction testing. A partial sequence of the 16S rRNA gene was constructed and compared to GenBank sequences of the 16S rRNA gene. To confirm the strains’ identity, a phylogenetic tree including all Kocuria species was constructed. Results: Three isolates of Kocuria spp. were identified; the first two isolates were confirmed as K. kristinae by VITEK 2, pairwise 16S rRNA sequencing, and phylogenetic tree. However, the third isolate was identified as K. rosea, contradicting the pairwise 16S rRNA sequencing and phylogenetic tree identification as K. turfanensis. Conclusion: It is crucial to utilize molecular techniques for correctly identifying Kocuria species. In this study, it is found that K. turfanensis emerges as a pathogenic species and the first known cause of meningitis in humans and K. kristinae is identified as the first known cause of pediatric urinary tract infections.

Isolation, characterization and identification of root nodule bacteria from Macrotyloma uniflorum (Lam.) Verdc

The present study was conducted to characterize the native plant growth promoting rhizobacteria (PGPRs) from the nodules of Macrotyloma uniflorum (Lam.) Verdc. The seeds of Macrotyloma uniflorum was grown in two different soils i.e., sterile and non-sterile soil and four bacterial isolates were isolated. The bacterial isolates were characterised on the basis of morphological and different biochemical analysis. Phylogenetic identification was done on the basis of 16S rRNA analysis. Four isolates obtained belongs to Bacillus velezensis and Enterobacter cloacae.

Leaf Spot Disease of Red Clover Caused by Leptosphaeria weimeri (=Longiseptatispora meliloti) in China

Leaf Spot Disease of Red Clover Caused by Leptosphaeria weimeri (=Longiseptatispora meliloti) in China

Phylogenetic Identification of the Predator Orius tristicolor1 Associated with Ornamental Sunflower (Helianthus annuus L.) in Mexico State, Mexico

Phylogenetic Identification of the Predator Orius tristicolor1 Associated with Ornamental Sunflower (Helianthus annuus L.) in Mexico State, Mexico

Phylogenetic identification of microbes from fermented botanicals used in gluten-free composite flour mixes

Phylogenetic information on microbial communities involved in fermenting botanicals has important implications for the food industry since it can provide a valuable perspective on the diversity, composition, and techno-functional properties and characteristics of the final product. Microbial phylogenetic analysis illustrates the evolutionary history of microbes through visual representational graphs (phylogenetic trees) showing the beginning and advancement of their assemblage.&#x0D; In this study, we used molecular methods to determine the phylogenetic identities of microbes occurring in spontaneously fermented sweet potato, maize, and pigeon pea samples after a 72-hourly evaluation every 12 h. The sequences obtained were edited using the bioinformatics algorithm against similar sequences downloaded from the National Center for Biotechnology Information (NCBI) database using BLASTN and aligned using ClustalX. The neighbor-joining technique was applied to extrapolate the chronicle of the isolates evolution.&#x0D; Molecular identification from the BLASTN results showed the following bacterial isolates: Lysinibacillus macrolides, Klebsiella pneumoniae, Lactococcus lactis, Providencia stuartii, Enterobacter cloacae, Limosilactobacillus fermentum, Lactobacillus fermentum, Staphylococcus edaphicus, and Bacillus flexus, as well as the following fungal isolates: Trichosporon asahii, Mucor irregularis, Cladosporium tenuissimum, and Aspergillus niger. The sequences obtained from the isolates produced an exact match with the NCBI non-redundant nucleotide (nr/nt) database. L. lactis had the highest percentage occurrence for bacteria (38.46%), while T. asahii and A. niger showed the highest occurrence for fungi (37.50%).&#x0D; Identifying and characterizing the microorganisms involved in the fermentation process would allow optimizing fermentation conditions to enhance the quality and nutritional value of the final products.

Life after isolation: Reconstructing the phylogenetic identity and a centennial chronology of historical Cryphonectria isolates

AbstractFungi of the genus Cryphonectria are of special interest to tree pathologists because they cause bark disease in numerous species of the Fagaceae and Betulaceae. Despite this special attention, several Cryphonectria species were not recognized as such for a long time. This also affected the identity of eight isolates with outdated names that have been deposited in our laboratory since 1954. The present study reconstructs the history of the Cryphonectria cultures M282–M289 using primary bibliographic sources. To verify the species identity, all isolates were DNA barcoded and taxonomic affiliation was assessed using a phylogenetic approach. The taxonomic identity combined with the history of the isolates confirms that the chestnut blight epidemic (Cryphonectria parasitica) in Spain was caused by the planting of Asian chestnuts and that the first documented case of bark canker in 1940 was caused by Cryphonectria radicalis on Japanese chestnuts imported from France. The history of isolate M289 could be traced back to CBS 165.32, which proved to be the oldest preserved isolate of the rare Cryphonectria naterciae, confirming the presence of this fungus in Europe for at least 100 years. Based on the ITS barcoding of the 32‐year‐old type specimen, Cryphonectria decipiens must be reclassified as a later synonym of Cryphonectria radicalis. The reconstruction of the history of isolates M282–M289 takes us on a scientific journey from Italy to France, Spain and Portugal in the early twentieth century and offers new insights into the historical context of the invasion of Cryphonectria parasitica in Europe.

Tick-borne Rickettsia, Anaplasma, Theileria, and enzootic nasal tumor virus in ruminant, PET, and poultry animals in Pakistan.

Pakistan is an agricultural country; most of its income is based on livestock rearing. The increasing prevalence of tick-borne pathogens among animals may affect the animal production and livelihood of owners, which eventually derange the economy of a country. To further comprehend TBPs, 213 ticks were collected from different animals, including ruminants, pets, and poultry. After molecular and phylogenetic analysis identification, ticks were managed into different pools based on their species level (Hyalomma anatolicum = 80, Rhipicephalus microplus = 35, Hyalomma scupense = 23, Rhipicephalus turanicus = 70, and Rhipicephalus sanguineus = 5). After tick species identification, further molecular PCR amplification was carried out to screen out the pathogens for the presence of Theileria, Rickettsia, Anaplasma, and enzootic nasal tumor virus (ENTV). The following pathogens were detected: 11 (5.16%) for Anaplasma, 1 (0.47%) for Rickettsia, and 9 (4.23%) for Theileria. Nevertheless, other TBPs that had not been reported so far in Pakistan 3 (1.41%), were positive for enzootic nasal tumor virus (ENTV). Besides, phylogenetic analysis of the enzootic nasal tumor virus (ENTV) strain confirmed its resemblance to the Chinese strain, while Anaplasma has comparability with Pakistan and China, Rickettsia with Pakistan, China, and Iran, and Theileria with India, South Africa, United States, Japan, and Spain. This study reveals that there is a considerably wider range of TBPs held in Pakistan that take in various contagious zoonotic pathogens than was previously thought. This information advances TBP epidemiology and will contribute to upgrade future control measure.