Gene Regulatory Sequences Research Articles

Oral microbiome and preterm birth.

The etiology of preterm birth (PTB) is heterogeneous and not yet well known. Maternal periodontal disease has been investigated for decades and is a known risk factor for adverse pregnancy outcomes. However, no particular bacterial species or higher taxonomic order has been found as causative of PTB, leading to studies of the whole oral microbiome. In order to determine if and how the composition of the oral microbiome is associated with PTB, we performed a large case-control study including women with term (TB) and PTB. We compared oral microbiomes in PTB to TB, to examine differences in the microbial richness, diversity, and differential abundance of specific taxa. We obtained oral swab samples from 152 Caucasian pregnant women who were classified as either PTB (≤36 6/7 weeks, n = 61) or TB (≥38 0/7 weeks, n = 91) in exclusion of any other major medical or obstetric conditions. The oral microbiomes of these women were characterized by 16S ribosomal RNA (rRNA) gene sequencing of the V3-V4 region on the MiSeq platform. The dominant microorganisms at the phylum level in all pregnant women regardless of birth week outcomes as belonging to Firmicutes, Proteobacteria, Bacteroidetes, Fusobacteria, and Actinobacteria. The phyla Firmicutes and Bacteroidetes were relatively more abundant in women with a PTB than in women with a TB, while Proteobacteria was less prevalent in women with a PTB. At the genus level, Veillonella, Prevotella, and Capnocytophaga were enriched in the PTB, and while many of the members of these genera could not be resolved to the species level, Veillonella massillensis was shown to be increased in the PTB group. We identified the genera Veillonella, Prevotella, and Capnocytophaga in the maternal oral microbiome as being associated with PTB independently of clinically apparent infection, uterine anomalies, and other pregnancy complications, including placenta previa, and placental abruption. The clarification of the role of those taxa in the etiology of PTB merits further research.

Evaluation of the Zybio EXS3000 mass spectrometry in routine identification of Clinical isolates

The matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been widely applied in routine clinical microbiology laboratories as an efficient and reliable technique for diagnostic purpose. In this work, we evaluated the performance of the newly developed Zybio EXS3000 (Zybio Inc., China) in microbial identification and compared it with VITEK MS (bioMérieux, France). For this study, a total of 1340 isolates from various clinical specimens were collected. These isolates were analyzed simultaneously on both EXS3000 and VITEK MS. The inconsistent or unidentifiable data were further identified using the help of either 16S rRNA gene or ITS region sequencing. During the study, we observed that EXS3000 and VITEK MS provided positive confirmatory diagnostics for 95.0% and 96.5% of the isolates, respectively, which were consistent with the sequencing results. However, it is worth noting that the EXS3000 system needs to improve the identification performance of Candida albicans in the follow-up. There are no significant differences between the two devices in terms of microbial identification performance. The advantage of EXS3000 over VITEK MS is in its ability to perform in significantly lesser time period. In conclusion, the results of this investigation showed that EXS3000 can be used to identify microorganisms in clinical microbiology laboratories.

Identification and Characterization of Pseudomonas aeruginosa 16S rRNA Gene Isolated from Contaminated Soil With Oil Residues

Pseudomonas aeruginosa was isolated from fifty soil samples were collected from different sites of contaminated soil with oil residues in Ministry of science& technology. Morphological, biochemical tests and 16S rRNA genes sequencing was performed for bacterial isolates identification. Thirty (30) isolates of P. aeruginosa bacteria were confirmed according to morphological, biochemical tests. For molecular analysis, all isolates that tested positive for P. aeruginosa underwent amplification of the 16S rRNA gene using previously described primers that amplified a particular DNA fragment of 956 bp. PCR product was delivered to Macrogen Corporation - Korea for Sanger sequencing utilizing an automated DNA sequencer called the ABI3730XL. The results were emailed to us, and we used geneious software to analyze them. The investigation of phylogenetic relationships between different strains of Pseudomonas aeruginosa has frequently been conducted on the sequencing of the 16S rRNA gene region, which is a viable technique for species identification. To determine the degree of genetic similarity between the organisms, a distance tree was built. Consequently, gene sequencing of the 16S rRNA region was an appropriate method for isolating isolates at the molecular level. The a novel local strain of Ps. aeruginosa which isolated from contaminated soil with oil residues samples (MSZ. IRQ20) and we will registration of isolate in GenBank under accession number of MT832126.1. Distance Tree Using Blast Tool in the Geneious software.

P-323 Associations between endometrial microbiome and the local immune cell composition during the mid-secretory phase

Abstract Study question Is there an association between the endometrial microbiome and the local immune cell composition during the mid-secretory phase? Summary answer The percentage of endometrial neutrophils was negatively related to the highest number of microorganisms, while B-cells and T-helpers were positively related to certain bacterial taxa. What is known already Endometrial receptivity is strongly influenced by the local immune cell composition and its inflammatory status. Meanwhile, many studies on the endometrial microbiome and its impact on receptivity are arising. Knowing that bacterial composition has an effect on the immune system, it is worth studying the potential associations between the microbiome and immune cell populations in the endometrium during the mid-secretory phase. This relationship could adjust the magnitude of inflammation which has a strong effect on embryo implantation process. Investigations on the endometrial bacterial communities’ effect on the local immune cell composition could be applied in improving assisted reproductive technologies. Study design, size, duration Prospective observational study performed between April 2021 and January 2023 in a private in vitro hospital. Endometrial biopsies were collected from 30 women aged between 34 and 45 years during the mid-secretory phase, on day 7 after LH surge. Patients with history of recent inflammatory disease, chronic endometritis, recent antibiotic treatment, endocrinological disorders, autoimmune diseases, oncological diseases, moderate or severe endometriosis, adenomyosis, uterine hyperplasia, and endometrial polyps were not included. Participants/materials, setting, methods B cells, T cells, T helpers, NK cells, macrophages and neutrophils in the endometrial stroma were identified by immunohistochemical staining with antibodies against CD79α (IS62, Dako), CD3 (BRB063, Zytomed), CD4 (IS649, Dako), CD56 (А00121-0007, ScyTek), CD14 (Е-AB-71017, Elabscience) and neutrophil elastase (950334, NOVUSBIO), respectively. The immune cell percentages were calculated by HALO image analysis software (version 2.3, IndicaLabs). Endometrial microbiota (EM) composition was analyzed after DNA isolation using 16S rRNA (v4-v5 region) gene sequencing. Main results and the role of chance Totally 271 bacterial species and 668 genera were identified in the endometrial samples. The median (range) immune cell percentages found were: 0.083% (0.49) B-cells, 0.974% (4.93) T-cells, 0.066% (0.44) T-helpers, 0.931% (5.36) NK-cells, 1.129% (3.11) macrophages and 0.207% (3.86) neutrophils. Spearman analysis revealed significant correlations between T-helpers percentage and the relative abundance of Cutibacterium acnes (R = 0.475, p = 0.012) and its genus Cutibacterium (R = 0.477, p = 0.012) as well as between B-cells and the relative abundance of Bacteroides vulgatus (R = 0.532, p = 0.003) and its genus Bacteroides (R = 0.457,p = 0.013). NK-cells presence was not related to any of the found bacterial species or genus (p > 0.05). Neutrophils demonstrated significant correlations with the relative abundance of the highest number of species: Prevotella melaninogenica (R = −0.404, p = 0.03), Butyrivibrio crossotus (R = 0.433,p = 0.02), Moraxella osloensis (R = −0.433, p = 0.019), Blautia sp. (R = 0.427, p = 0.021), Alicyclobacillus acidocaldarius (R = −401,p = 0.031) also related with the T-cells percentage (R = −0.380, p = 0.042) and Acinetobacter lwoffii (R = −0.458, p = 0.008) also related with the macrophages (R = −0.454, p = 0.013). Among the studied species Alloprevotella tannerae was showing significant correlations with neutrophils (R = −0.0602, p = 0.001), macrophages (R = −0.416, p = 0.025) and T-cells (R = −0.464, p = 0.011). At genus level, most of the bacteria were related to the neutrophils – Pseudoalteromonas (R = −0.540, p = 0.002), Aliterella (R = −0.617, p<0.001), Endhydrobacter (R = −0.433,p = 0.019) and AKIW781 (R = −0.493, p = 0.011). Aliterella and AKIW781 were also related to the macrophages (R = −0.473, p = 0.01 and R = −0.423, p = 0.031, respectively) and T-cells (R = −0.387, p = 0.038 and R = −0.519, p = 0.007, respectively). Limitations, reasons for caution The study was limited in sample size. Wider implications of the findings The results of this study indicated that there is a quantitative relation between the main endometrial immune cells and the endometrial core microbiome. The association between these two determinants of endometrial receptivity could help in understanding the fine tuning of the endometrium for accepting the embryo. Trial registration number The current research was funded by National Science Fund, Ministry of Education, Bulgaria, Contract № KP-06-N53/14/16.11.2021.

P-317 Associations between endometrial microbiome composition and cellular senescence

Abstract Study question Is there an association between the percentage of senescent cells and microbiome composition in human endometrium? Summary answer We found that cellular senescence is negatively associated with the relative abundance of certain bacterial taxa. What is known already Recent studies indicated that endometrial microbiome characteristics and certain levels of cellular senescence are essential for the establishment of normal endometrial receptivity and successful embryo implantation. The secretion of senescence-associated secretory phenotype (SASP) components is responsible for immune cell activation and might cause endometrial dysbiosis. On the other hand, some studies suggest that the presence of specific metabolic groups of bacteria and their metabolites could influence cellular stress response that might suppress the development of senescent cells. However, the direct relationship between the quantity of senescent cells and microbiome content in the endometrium has never been studied. Study design, size, duration This was a prospective observational cohort study performed between April 2021 and January 2023. Endometrial biopsies were collected from 30 women aged between 34 and 45 during the mid-luteal phase (LH + 7) in a natural cycle. Each biopsy was divided for genetic and immunohistochemical analyses. The exclusion criteria were history of recent inflammatory disease, chronic endometritis, recent antibiotic treatment, endocrinological disorders, autoimmune diseases, oncological diseases, moderate or severe endometriosis, adenomyosis, uterine hyperplasia, and endometrial polyps. Participants/materials, setting, methods We used immunohistochemical biomarker p16ink4a (MAD-000690QD–7, Master Diagnostica) to identify senescent cells. The percentages of positively stained cells in the endometrial stroma and in the luminal epithelium were calculated by HALO image analysis software (version 2.3, IndicaLabs). Endometrial microbiota composition was analyzed after DNA isolation from the endometrial samples using 16S rRNA (v4-v5 region) gene sequencing. Statistical analysis was performed by Spearman’s correlation test using SPSS v.21 (IBM Corp., Armonk, NY, USA). Main results and the role of chance A total of 271 distinct bacterial species and 668 genera were identified across the studied 30 endometrial samples. The percentage of p16+ senescent endometrial stromal cells ranged from 0 to 1.7%, with a mean value of 0.37%, while the percentage of p16+ luminal epithelial cells ranged between 2.5% and 74.0%, with a mean value of 21.5% Spearman analysis revealed significant negative correlation between the percentage of p16+ stromal cells and the relative abundance of Porphyromonas sp. (R = −0.41, p = 0.03) and Sphingobacterium sp. (R = −0.37, p = 0.05). The percentage of p16+ luminal senescent cells correlated negatively with the abundance of Micrococcus luteus (R = −0.44, p = 0.02), Moraxella osloensis (R = −0.37, p = 0.05), Rubellimicrobium sp. (R = −0.46, p = 0.02), Stenotrophomonas sp. (R = − 0.45, p = 0.03), Chryseobacterium sp. (R = −0.41, p = 0.04), Carnobacterium sp. (R = −0.40, p = 0.04), Deinococcus sp. (R = −0.38, p = 0.05), and Enhydrobacter sp. (R = − 0.37, p = 0.05). Limitations, reasons for caution The study was limited in sample size. The assessment includes only endometrial samples from the mid-luteal phase of the cycle. Wider implications of the findings We found that the level of senescence is negatively associated with the relative abundance of certain bacteria taxa in human endometrium, including some related to impaired reproductive function and early embryonic development arrest. These findings suggest a possible specific interaction between senescent cells and microbiome that might influence endometrial receptivity. Trial registration number The current research was funded by National Science Fund, Ministry of Education, Bulgaria, Contract № KP-06-N53/14/16.11.2021

Morphology and molecular phylogeny of Erysiphe japonica var. japonica found on Quercus aliena in Korea

AbstractIn October 2022, Erysiphe powdery mildew from the section Typhulochaeta was found on Quercus aliena for the first time in Korea. Based on morphological characteristics and molecular‐phylogenetic analysis of the internal transcribed spacer region (ITS) and large subunit (LSU) gene sequences of the rDNA, the fungus was identified as Erysiphe japonica var. japonica. This is the first report of E. japonica var. japonica in Korea and the third report of this mildew on Q. aliena, with this current fungus‐host association spreading to China and Japan. Based on Korean samples, this study provides detailed morphology and molecular phylogeny of E. japonica var. japonica.

Children and adolescents with attention deficit hyperactivity disorder and autism spectrum disorder share distinct microbiota compositions

ABSTRACT An association has been suggested between altered gut microbiota, and attention deficit hyperactivity disorder (ADHD), and autism spectrum disorder (ASD), respectively. Thus, we analyzed the gut microbiota composition in children and adolescents with or without these disorders and evaluated the systemic effects of these bacteria. We recruited study participants diagnosed with ADHD, ASD, and comorbid ADHD/ASD, while the control groups consisted both of siblings and non-related children. The gut microbiota was analyzed by 16S rRNA gene sequencing of the V4 region, while the concentration of lipopolysaccharide-binding protein (LBP), cytokines, and other signaling molecules were measured in plasma. Importantly the gut microbiota compositions of cases with ADHD and ASD were highly similar for both alpha- and beta-diversity while differing from that of non-related controls. Furthermore, a subset of ADHD and ASD cases had an increased LBP concentration compared to non-affected children, which was positively correlated with interleukin (IL)-8, 12, and 13. These observations indicate disruption of the intestinal barrier and immune dysregulation among the subset of children with ADHD or ASD.

Molecular identification and sequencing of Pseudomonas aeruginosa 16S rRNA gene isolated from a variety of raw cow milk samplesin Iraq

Pseudomonas aeruginosa is Gram-negative bacterium which is notably known as a pathogen in humans, animals, and plants.Suspect raw milk samples were analyzed to amplify the 16S rRNA gene and confirm Pseudomonas aeruginosa strains. This study was conducted at different farmland in diwaniya and al-Najaf., a total of 70 samples were collected (28 from mastitic cows and 42 from apparently healthy cows ) the detection of the 16S rRNA gene in this study revealed 11 ( positive isolates of Pseudomonas were identified from samples after culture on cetrimid agar. New sets of primer pairs were designed using the NCBI database search tool. The phylogenetic relationship between different strains of Pseudomonas aeruginosa has been studied through using 16S rRNA gene region sequencing. A distance tree was constructed to determine the genetic similarity between species. as result of the 9 isolate gene sequencing, it was determined, and submitted in GenBank accession numbers (MZ799357, MZ817077, MZ820788, MZ820878 , MZ821016, MZ823355, MZ823356 MZ823387. MZ823389) , in conclusion the PCR is a reliable technique that identifies Pseudomonas aeruginosa at the molecular level

Ocular conjunctival microbiome profiling in dry eye disease: A case control pilot study.

Keratoconjunctivitis sicca (KCS) or dry eye disease (DED) is a multifactorial disease that results in discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface. A pilot study was undertaken to determine if there were any major substantial differences in the ocular microbiome in DED patients versus healthy controls. The bacterial communities residing in the conjunctiva of patients with DED (n = 4) and healthy controls (n = 4) were assessed by 16S ribosomal RNA (rRNA) gene sequencing of the V4-V5 region. The phyla Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes were most dominant and accounted for 97% and 94.5% of all bacterial sequences in patients and controls, respectively. At the genus level, 27 bacterial genera were found with more than two-fold difference between patients and controls. Four of these - Acinetobacter, Corynebacterium, Lactobacillus, and Pseudomonas spp. - dominated the ocular microbiome of all subjects, but were proportionately lower in DED (16.5%) compared to controls (37.7%). Several bacterial genera were found to be unique in DED (34) and controls (24). This pilot study is an attempt to profile the ocular microbiome in patients with DED that demonstrated a higher concentration of microbial DNA compared to controls, with Firmicutes phyla dominating the bacterial population in patients with DED.

High-throughput time series expression profiling of Plasmopara halstedii infecting Helianthus annuus reveals conserved sequence motifs upstream of co-expressed genes

Downy mildew disease of sunflower, caused by the obligate biotrophic oomycete Plasmopara halstedii, can have significant economic impact on sunflower cultivation. Using high-throughput whole transcriptome sequencing, four developmental phases in 16 time-points of Pl. halstedii infecting Helianthus annuus were investigated. With the aim of identifying potential functional and regulatory motifs upstream of co-expressed genes, time-series derived gene expression profiles were clustered based on their time-course similarity, and their upstream regulatory gene sequences were analyzed here. Several conserved motifs were found upstream of co-expressed genes, which might be involved in binding specific transcription factors. Such motifs were also found associated with virulence related genes, and could be studied on a genetically tractable model to clarify, if these are involved in regulating different stages of pathogenesis.

The Oral Microbiome as Mediator between Oral Hygiene and Its Impact on Nasopharyngeal Carcinoma

Oral hygiene and the alteration of the oral microbiome have been linked to nasopharyngeal carcinoma (NPC). This study aimed to investigate whether the oral microbiome plays a mediating role in the relationship between oral hygiene and NPC, and identify differential microbial taxonomies that potentially mediated this association. We conducted a case-control study that involved 218 NPC patients and 192 healthy controls. The 16S rRNA gene sequencing of the V4 region was performed to evaluate the composition of the oral microbiome. Mediation analysis was applied to explore the relationship among oral hygiene, the oral microbiome and NPC. We found that dental fillings and poor oral hygiene score were associated with increased risks of NPC (OR = 2.51 (1.52-4.25) and OR = 1.54 (1.02-2.33)). Mediation analysis indicated that dental fillings increased the risk of NPC by altering the abundance of Erysipelotrichales, Erysipelotrichaceae, Solobacterium and Leptotrichia wadei. In addition, Leptotrichia wadei also mediated the association between oral hygiene score and the risk of NPC. Our study confirmed that poor oral hygiene increased the risk of NPC, which was partly mediated by the oral microbiome. These findings might help us to understand the potential mechanism of oral hygiene influencing the risk of NPC via the microbiome.

Phylogenetic analysis of Prunus genus using nuclear and chloroplast gene markers as a bioorganic structure profiling

Prunus species are known as model species for the family Rosaceae and other woody plants with their small genome size and relatively small life cycle as woody plants. Also Prunus species are economically important plants in the subfamily Amygdaloideae of the Rosaceae have a controversial taxonomic history due to the lack of a phylogenetic framework. Therefore, mapping, classification, determination the genetic structure and characterization of genes as a spesific chemical and fuctional units of nucleic acids and making phylogenetic analysis in these species have important implications. The aim of this study to find any gene markers as a bioorganic – chemical structure profiling to determine genetic diversity of different Prunus species from Iraq by using nuclear and chloroplast DNA markers to evaluate the phylogeny of Prunus. A total of 12 species Prunus subgenus amygdalus were collected from Iraq. Ten gene region markers on both nuclear DNA and chloroplast DNA have been tested to evaluate the genetic variation at their molecular levels by using multiplex PCR with 10 gene region primers and sequence analysis for matK. The name of the gene regions using in this study are ITS (ITS1+ITS2+5.8S coding region), rbcL, matK, rpl16, trnL-trnF, atpB-rbcL, rp136-infA-rps8, trnK-rps16, pbsM-trnD, psbA-trnH. As a result, five different phylogenetic trees were obtained from the gene sequences of the samples and the base sequences were close to each other. The estimated value of the shape parameter for the discrete Gamma Distribution is 0.05. The nucleotide frequencies are A = 29.41%, T/U = 37.37%, C = 17.52%, and G = 15.67%. The nucleotide diversity of the sequence was low with the 0.004113 value. There were 14 different informative base site between the samples. The polymorphism observed was less in agreement with higher level of conservation of the gene regions. The Prunus species from Iraq region were grouped on two closed clusters on the phylogenetic trees.

Erysiphe lonicerigena sp. nov., a Powdery Mildew Species Found on Lonicera harae

A powdery mildew (Erysiphaceae) has been continuously collected on the leaves of Lonicera harae in the southern part of the Korean Peninsula, where this shrub is indigenous. Microscopic examination of the asexual morphs revealed that the current collections are differentiated from the all known Erysiphe species on Lonicera spp. by its longer conidiophores and longer conidia. Although the morphology of the chasmothecia is reminiscent of Erysiphe ehrenbergii and E. lonicerae, the specimens on L. harae differ from them in having smaller ascospores. A phylogenetic tree generated from a combined dataset of the internal transcribed spacer region and 28S rDNA gene sequences demonstrates that sequences obtained from three powdery mildew collections on L. harae clustered together as an independent species clade with high bootstrap values distant from other Erysiphe species on Lonicera, representing a species of its own. Based on morphological differences and molecular-phylogenetic results, the powdery mildew on L. harae is proposed as a new species, Erysiphe lonicerigena, and the holomorph of the fungus is described and illustrated in this study.

First Report of Neopestalotiopsis Disease in Ohio caused by an emerging and novel species of Neopestalotiopsis on Strawberry.

In October 2021, strawberry (Fragaria x ananassa) plants (cv. Ruby June) that had dark brown lesions with a diffuse black halo and light brown center and / or dark brown V-shaped necrotic areas often starting from the edge of the leaves were observed in a commercial planting in Washington County. The grower reported 50% incidence in the field when the sample was first submitted and two weeks later reported 80% incidence. The morphology of conidia present on symptomatic leaf tissue was consistent with species of Neopestalotiopsis (Maharachchikumbura et al. 2014). The conidia were ellipsoid to fusiform, five-celled, with three light brown colored median cells and one hyaline apical and basal cell. The apical cells had two to four flexuous appendages and the basal cell had one non-flexuous appendage. Average (N=30) conidia length, not including the appendages, and width was 24.1 ± 2.7 and 6.5 ± 1.4 µm respectively. Two isolates (MLI267-21 and MLI268-21) were purified on potato dextrose agar, producing a dense white mycelial mat with undulate margins. The underside color of the mycelial mat was pinkish-orange. Conidiomata formed randomly in the colonies and extruded black gelatinous spores. To confirm the identity of these isolates the genome of MLI267-21 was sequenced using the NextSeq 2000 Illumina platform and Nextera DNA CD indexes (OSU Applied Microbiology Service Laboratory, Columbus, OH). Partial internal transcribed spacer (ITS) region, β-tubulin (TUB), and translation elongation factor 1-alpha (TEF-1α) gene sequences (Accession numbers: OM649904, OM649905, and OM649906 respectively) were extracted from the MLI267-21 genome, concatenated, and aligned to published reference sequences. These same genes were amplified and sequenced from MLI268-21. Maximum likelihood phylogenetic analysis performed in IQ-TREE (Minh et al. 2020, Kalyaanamoorthy et al. 2017, Chernomor et al. 2016) with the aligned sequences revealed the clustering of MLI267-21 and MLI268-21 with seven other Neopestalotiopsis isolates, from strawberry (17-43L; Baggio et al. 2021) and pomegranate (GEV3426 to GEV3431; Xavier et al. 2021) leaves in Florida, which form a unique and emerging species group. The ITS, TUB, and TEF-1α sequences from both Ohio isolates were 100% similar to the same sequences from 17-43L and GEV3426 - GEV3431. Pathogenicity tests were performed using MLI267-21 by spray inoculating (~104 spore/ml) four-week-old 'Cabrillo' strawberry plants (n=4) and placing three drops (10µl each) of spore suspension (~104 spore/ml) on the calix area of detached fruit (n=4). Non-inoculated plants and fruit (n= 4 each) served as negative controls. The plants were covered with transparent plastic bags and maintained at 25 °C for 72 hours before the bags were removed (Baggio et al. 2021). Five days post-inoculation, dark brown circular spots on the leaves and petioles were observed on all four inoculated plants and acervuli were observed within the necrotic spots after an additional 72 hours in a moist chamber. Fruits were incubated in a moist chamber at 25 °C and after 72 hours orange-brown lesions formed on the fruit. After five days, fruit were mushy and covered with white mycelia, acervuli, and conidiomata. Neopestalotiopsis disease has been reported on strawberry in Florida (Baggio et al. 2021) and in several South American (Obregon et al. 2018, Hidrobo et al. 2021) and European (Chamorro et al. 2016, Gilardi et al. 2019) countries. The disease can cause rapid plant death when conditions are warm and wet. Research to investigate host susceptibility and to identify effective chemical and biological control has been initiated in Ohio to establish preventative management programs for commercial field operations.

First Report of Fusarium ipomoeae causing Fusarium wilt on Glycine max in South Korea.

Soybean (Glycine max L.) is one of the most important crops worldwide. In South Korea, three species of Fusarium have been reported as causal pathogens of Fusarium wilt of soybean (KSPP, 2021). From 2017 to 2018, wilted soybeans were observed in two soybean fields in Daegu (36.62°126.91°) and Yesan (35.89°128.44°), South Korea. The incidence rate was about 2 to 5% of the total 0.1ha, respectively. The diseased soybeans were yellowed from the lower leaves or dried up, and the inside of the root and stem were turned brown. Fragments (each 5 mm × 5 mm) of the symptomatic vascular tissue were surface-sterilized with 1% NaOCl for 1 min, and then rinsed twice in sterilized distilled water. The seven pieces each from two diseased plants were placed on water agar and incubated at 25°C for 5 days. Two single spore isolates were cultured on carnation leaf agar at 25°C for 14 days under near ultra violet/dark conditions for 12 hours. Macroconidia of two isolates were mostly 3- to 5-septate, dorsiventral curvature, hyaline, apical cell hooked to tapering, basal cell foot-shaped, and measured 51.3 - 62.2 × 3.7 - 4.7 μm (DG43821) and 63.8-74.8×3.1-4.4 μm (YS37232). Microconidia were not observed. Chlamydospores were produced in chains or pairs, subglobose and thick walled. The color of the aerial mycelium was pinkish white and the reverse of the colony was brownish orange on potato dextrose agar. Based on morphological and cultural characteristics, the two isolates were identified as belonging to Fusarium incarnatum-equiseti species complex (Leslie and Summerell 2006). To confirm the accurate species identification of the two isolates, DNA sequencing of the internal transcribed spacers and intervening 5.8S (ITS), partial translation elongation factor 1-alpha (TEF) and RNA polymerase II largest subunit (RPB2) genes was carried out using primer sets of ITS1/ITS4, EF1 / EF2 and 7cf / 11ar, respectively (O'Donnell et al. 2010). The nucleotide sequences obtained of two isolates were deposited in GenBank with accession numbers of MW375694, MW375695, MW382963, MW382964, MZ364324 and MZ364325. Identities of the ITS region, TEF and RPB2 gene sequences of the two isolates were 490/492, 482/483, 632/633, 631/632, 870/870 and 931/931 with those of ex-type strain F. ipomoeae LC12165 (MK280832, MK289599 and MK289752) in GenBank, respectively. Thus, based on molecular characteristics, the two isolates were confirmed as F. ipomoeae. A pathogenicity test of the two isolates was conducted using root-dip inoculation on seedlings of one soybean cultivars, Pyeongwon. A spore suspension was prepared by flooding 10-day-old cultures on PDA with sterilized distilled water. Fifteen soybean seedlings at the VC stage per each isolate were inoculated by dipping the roots in the spore suspension (1 × 106 conidia/mL) for 2 hours. Inoculated plants were transplanted into pots containing sterilized soil and maintained in the greenhouse at 28±3°C with 14 h/10 h light/dark. An equal number of plants inoculated with sterilized distilled water served as controls. Five days after inoculation, withered symptoms were observed on two or four of the inoculated seedlings, and by 10 days after inoculation, all inoculated plants had withered and died. No symptoms were observed in the non-inoculated control soybeans. The pathogen was consistently re-isolated from only inoculated plants, thus fulfilling Koch's postulates. To our knowledge, this is the first report of F. ipomoeae causing Fusarium wilt on soybean in South Korea, as well as worldwide. This pathogen has been reported on peanut in China as a causal agent of leaf spot (Xu et al., 2021). Understanding the host range of this pathogen and the distribution of F. ipomoeae affecting legume crops in South Korea is important, to ensure an effective management of Fusarium wilt on soybeans.

C. elegans Gene Regulatory Alleles and Reporter Bashing Studies.

Gene regulation has been studied in C. elegans for over 30 years. In this analysis of 102 publications, we find that most transcriptional cis-regulatory elements are located within 5,000 bp of the transcription start site. Over 75% of studies conclude that transcriptional elements and 5'UTRs activate-, while 3'UTRs repress gene expression. While gene regulatory mutations make up less than 0.8% of alleles in forward genetics screens, recent CRISPR-Cas approaches are increasing the number of tested mutations. This work provides a resource of known gene regulatory sequences in C.elegans .

First Report of Powdery Mildew Caused by Podosphaera fuliginea on Veronica spicata in China.

Veronica spicata L. (syn. Pseudolysimachion spicatum Opiz), Plantaginaceae, is a perennial herb and frequently cultivated in gardens as an ornamental plant in China. In June 2017, powdery mildew infections were observed on V. spicata in Jingyuetan National Forest Park (43.80°N, 125.46°E), Changchun, China. A voucher specimen was deposited in the Herbarium of Mycology of Jilin Agricultural University under the accession no. HMJAU-PM91763. The incidence of the disease on leaves and stems was about 30 to 50%. The disease initially appeared as thin white colonies, which subsequently developed into dense effuse white colonies on the plant. Hyphae were flexuous to straight, septate, 3.0 to 8.0 μm wide. Hyphal appressoria were indistinct or nipple-shaped, solitary. Conidiophores (n=30) arise from the upper surface of hyphal mother cells, erect to straight, 133.4 to 176.2 × 7.8 to 10.7 μm. Foot-cells (n=40) were cylindrical, straight or slightly flexuous, 39.1 to 78.5 × 7.4 to 9.7 μm, and followed by 1 to 3 short cells. Conidia (n=45) were catenescent, ellipsoid, oval, or doliiform, with fibrosin bodies, 17.8 to 27.8 × 12.2 to 17.4 μm, length/width ratio 1.3 to 2.1. Germ tubes were produced at the subterminal to lateral part of conidia, straight or sinuous, without a distinct terminal appressorium. The sexual morph was not observed in the collected samples. The morphological characteristics of the asexual morph were consistent with Podosphaera fuliginea (Schltdl.) U. Braun & S. Takam. (Braun and Cook 2012). To confirm the identification, the complete internal transcribed spacer (ITS) region and partial 28S rRNA gene sequences of the pathogen were amplified by semi-nested PCR with the primers ITS5/P3 followed by ITS5/ITS4, and LSU1/TW14 followed by LSU1/LSU2, respectively. The sequences of 566 bp ITS (MF543026) and 609 bp 28S rDNA (MF543027) were obtained and showed 100% identity with P. fuliginea (AB046986, ON073893) on V. spicata from USA (Hirata et al. 2000). Based on the morphological and molecular characteristics, the fungus was identified as P. fuliginea. To perform pathogenicity assays, three healthy annual plants of P. spicatum were inoculated by gently pressing a diseased leaf onto the leaves, with three non-inoculated plants as controls. All plants were placed in a greenhouse at 21 to 29 °C, 60% relative humidity, with 16 h/8 h light/dark cycle. Nine days after inoculation, typical powdery mildew colonies started to appear on the inoculated plants, while the control plants remained symptomless. The morphology of the fungus on the inoculated leaves was identical to that observed on the originally diseased leaves. Powdery mildews on V. spicata (P. spicatum) were previously referred to as Erysiphe (Golovinomyces) orontii in Italy (Garibaldi et al. 2006) and Sphaerotheca (Podosphaera) fuliginea in many countries, such as Poland, Russia, Switzerland, Wisconsin, Ukraine, etc. (Amano 1986; Braun and Cook 2012; Farr and Rossman 2022; Heluta et al. 2011). To our knowledge, this is the first record of P. fuliginea on V. spicata from China and the first report of this species at all. The results of this study provide important information for horticultural management and plant protection in China. Acknowledgements This work was supported by the National Natural Science Foundation of China (31970019, 31670022).

Genetic Diversity Relationship in Azakheli Buffalo Inferred from mtDNA and MC1R Sequences Comparison.

Azakheli is relatively smaller riverine breed with a very peculiar characteristics kept under unique traditional husbandry practices in comparison with rest of the Pakistani buffalo breeds; however, milk production is comparable. The present study was conducted to evaluate the genetic diversity of Azakheli breed. A total of sixty-six blood samples were collected for the amplification of mtDNA D-loop region and MC1R gene sequencing analysis. Median-joining network analysis of 191 mtDNA D-loop sequences of Azakheli and eight Indian riverine buffalo breeds clustered into three clades. Ancient Azakheli Region 1 clade was the oldest with the highest mutation steps and was present close to the root of UPGMA phylogenetic tree. There was 5 mutated lines distance between Pakistan buffalo and Indian riverine buffaloes. The populations of neighboring countries did not share any haplotypes with Azakheli buffalo of Pakistan. Possibly, residing for so long in the cold atmosphere and high elevation regions caused the mutation in mtDNA D-loop, though these conditions did not affect the overall performance of Azakheli as milch buffalo breed of Pakistan. MC1R analyses showed high mutations in Azakheli of Albino phenotype and all the black phenotype individuals of Azakheli buffalo share haplotypes with dominant Chinese and Indian black phenotypes buffaloes in MC1R median-joining network, indicating the reason of black coat color is due to MC1R gene. The haplotype diversity and nucleotide diversity was (H. 0.923, Pi: 0.00895) in Azakheli. Current results illustrated Asian ancestry for Azakheli buffalo, and mtDNA and MC1R analyses provided further evidence. Additional genetic analyses and archeological studies may provide further insight into the domestication period and history of Azakheli buffalo breed. The further studies are required on different coat colors with different genes on Azakheli buffalo to understand the phenotype variation.

Varieties of Lettuce Forming Distinct Microbial Communities Inhabiting Roots and Rhizospheres with Various Responses to Osmotic Stress

A plant microbiome is an important factor in plant growth, stress resistance, health status, and consumer quality and safety. The rhizosphere microbiome evolves in a negotiation between microbial communities that inhabit soil and plant root tissue. In this study, the rhizosphere and root internal tissue microbiome of six varieties of lettuce were analyzed in normal conditions and under salinity stress. The metabarcoding analysis used 16S rRNA gene and ITS2 region sequencing. The microbiomes of root samples were significantly less diverse with different members of the community compared to those of the rhizosphere. A significant effect of lettuce variety was found on the diversity index for bacteria and fungi. Varieties formed very different communities of bacteria in roots. Pseudomonas, Herbaspirillum, Mycobacterium, potentially pathogenic Enterobacter, and other genera were more prevalent in certain varieties. Salinity stress had a significant negative impact on bacterial diversity and community composition, whereas the diversity of fungi has not changed significantly, and the fungal community has changed less than the bacterial one. Changes were more evident in varieties that were more resistant to salinity stress than in sensitive varieties.

Clinical and Microbiological Characteristics of Aspergillosis at a Chinese Tertiary Teaching Hospital.

Aspergillus spp. infection in immunocompromised patients results in increasing morbidity and mortality. This study investigated clinical and microbiological characteristics of aspergillosis in a Chinese tertiary teaching hospital. A total of 114 patients with aspergillosis were included over a 5-year period at Ruijin Hospital. In sum, 114 Aspergillus strains were isolated and identified at species level using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, confirmed by ITS gene region and β-tubulin (BenA) gene sequencing. Sensititre YeastOne was used in vitro to test susceptibility to antifungal drugs: amphotericin B, itraconazole, voriconazole, posaconazole, isavuconazole, micafungin, anidulafungin, and caspofungin. The median age of the patients was 61 (19) years, men accounted for 53.5% (n=61) of the sample, about 64% were immunocompromised, and 36% had underlying diseases. Pulmonary diseases accounted for 27.2%. Aspergillus isolates were mainly isolated from sputum (n=42, 36.8%). Antifungal therapy was administered to 106 (93.0%) patients and voriconazole (n=76, 66.7%) was the most frequently used as empirical therapy. Aspergillus fumigatus (n=69, 60.5%) was the most common species. There was a 73.7% concordance between MALDI-TOF MS and molecular identification. All Aspergillus isolates showed good susceptibility to anidulafungin and caspofungin. Immunocompromised patients are an at-risk population for aspergillosis, and voriconazole was used as empirical therapy in Ruijin Hospital, China. A. fumigatus was the predominant Aspergillus species causing aspergillosis, and A. flavus - as non-A. fumigatus species are increasing - the second-leading cause of aspergillosis. Anidulafungin and caspofungin were the most active in vitro against the Aspergillus isolates tested. The MALDI-TOF MS method showed good accuracy for identification of common Aspergillus spp. In vitro antifungal-susceptibility testing is crucially important for decisions on effective therapy with aspergillosis.