Partial 16S rRNA Gene Sequences Research Articles

Integrating short- and full-length 16S rRNA gene sequencing to elucidate microbiome profiles in Pacific white shrimp (Litopenaeus vannamei) ponds.

Despite their immense economic value as a key aquaculture species, the production of Pacific white shrimp (Litopenaeus vannamei) faces significant challenges from intensive farming practices and disease outbreaks. Routine microbial profiling for disease surveillance could be a promising approach to anticipate and control disease outbreaks. To achieve this, accuracy in microbial profiling in shrimp ponds is crucial for enabling targeted action and prevention. Extensive documentation emphasizes that, beyond biological factors (related to the host, diet, or health status during the rearing period), technical elements, including sequencing techniques significantly influence bacterial community profiling. This study investigated the influence of short- and long-read sequencing of 16S rRNA genes on the microbial profiles in shrimp intestines, water, and sediments. The origin of the samples (intestine or environmental) in shrimp culture ponds primarily drove the observed differences in core microbial species. The ecological niches accounted for 56% of bacterial community variations in culture ponds. Both sequencing approaches showed consistent results in identifying higher-rank taxa and assessing alpha and beta diversity. However, at the species level, full-length 16S rRNA gene sequences provided better resolution than V3-V4 sequences. For routine microbial profiling in shrimp culture ponds, our study suggests that short-read sequences were sufficient for determining overall bacterial community.IMPORTANCEThis interdisciplinary study investigated the influence of sequencing techniques on bacterial communities profiling within Pacific white shrimp (Litopenaeus vannamei) ponds. By integrating aquaculture, microbiology, and environmental science, we revealed the role of ecological niches and factors like salinity and pH on microbiota diversity and composition in shrimp intestines, pond water, and sediment. Additionally, we compared the taxonomic resolution using partial versus full-length 16S rRNA gene sequences, highlighting the value of longer amplicons for precise identification of key taxa. These findings provide novel insights into microbial dynamics underlying environmental effects in shrimp aquaculture. Comprehensive characterization of the pond microbiome could lead to management strategies that promote shrimp health and productivity. Furthermore, the potential of a multi-omics approach for integrating complementary data streams to elucidate environment-microbiome-host interactions was highlighted.

Halorubrum miltondacostae sp. nov., a potential polyhydroxyalkanoate producer isolated from an inland solar saltern in Rio Maior, Portugal

One hundred and sixty-three extreme halophiles were recovered from a single sample collected from an inland solar saltern in Rio Maior. Based on random amplified polymorphic DNA (RAPD) profiles and partial 16S rRNA gene sequencing 125 isolates were identified as members of the Archaea domain within the genus Halorubrum. Two strains, RMP-11T and RMP-47, showed 99.1 % sequence similarity with the species Halorubrum californiense based on phylogenetic analysis of the 16S rRNA gene sequence. However, phylogenetic analysis based on five housekeeping genes, atpB, EF-2, glnA, ppsA and rpoB’, showed Halorubrum coriense as the closest related species with 96.7 % similarity. The average nucleotide identity (ANI) of strains RMP-11T, RMP-47 and species Hrr. coriense were within the range of 90.0–90.5 %, supporting that strains RMP-11T and RMP-47 represent a novel species of the genus Halorubrum. These strains formed red-pigmented colonies that were able to grow in a temperature range of 25–50 °C. Polyhydroxyalkanoate (PHA) granules were detected in both strains. The polar lipid profile was identical to the neutrophilic species of the genus Halorubrum. The Rio Maior sample from which both strains were isolated was metagenome sequenced. We identified five metagenome-assembled genomes representing novel Halorubrum species but distinct from the species represented by strains RMP-11T and RMP-47. Based on phylogenetic, phylogenomic, comparative genomics, physiological and chemotaxonomic parameters, we describe a new species of the genus Halorubrum represented by strains RMP-11T (=CECT 30760T = DSM 115521T) and RMP-47 (=CECT 30761 = DSM 115541) for which we propose the name Halorubrum miltondacostae sp. nov.

Changes in microbial communities during high-rate microbial selenate reduction in an up-flow anaerobic fluidized bed reactor

Biological fluidized bed reactor (FBR) is a promising treatment option for removing selenium oxyanions from wastewater by converting them into elemental selenium. The process can achieve high rates and be efficiently operated at low hydraulic retention times (HRT). However, the effects of HRT on the changes in microbial community in the FBR process have not been previously explored. In this study, dynamic changes of microbial communities both on biofilm carrier and in suspension of a selenate-reducing FBR were explored at various HRTs (0.3–120 h). Based on partial 16S rRNA gene sequencing of the microbial communities, alpha diversity of microbial communities in suspension rather than in the biofilm were impacted by low HRTs (0.3 h–3 h). Members from genera Geobacter, Geoalkalibacter, and Geovibrio were the main selenate-reducing bacteria on carrier throughout the FBR process. Genus Geobacter was dominant in FBR carrier at HRT of 24 h–120 h, whereas Geoalkalibacter and Geovibrio dominated at low HRT of 0.3 h–6 h. Suspended microbial communities detected in the FBR effluent were more sensitive to HRT changes than that in biofilm. “Shock loading” at HRT of 0.3 h had a great impact on microbial community compositions both in the biofilm and effluent. Reactor operation in batch mode and long HRT of 24 h helped recover the community from “shock loading” and improved selenite reduction and ethanol oxidation. Redundancy analysis revealed that HRT, influent pH and selenate loading were key operational parameters impacting both the FBR performance and the composition of microbial communities associated with both the FBR carrier and effluent. Overall, the microbial communities in FBR biofilm flexibly responded to the changes of HRT and showed resilience to the temporary shock loading, enabling efficient selenate removal.

Exploring Listeria monocytogenes in Ewe Milk: ssrA Gene-based Real-time PCR Identification, Phylogenetic Analysis, and Antibacterial Assessment of Magnesium Oxide Nanoparticles Synthesized with Myrtus communis Leaf Extract

Background: Listeria monocytogenes, a zoonotic pathogen affecting humans and animals, exhibits a global distribution, including Iraq. This study focused on the rapid identification and phylogenetic analysis of L. monocytogenes in freshly collected ewe milk samples from various farms in Al-Qadisiyah province, Iraq.Methods: The study was conducted with care and precision, involving 150 milk samples. These samples were subjected to traditional bacterial isolation and identification using the enrichment culture method and biochemical tests, with the PCR technique confirming the results. The antibacterial activity of MgONPs was then assessed using the disc diffusion method, ensuring a comprehensive and reliable approach to the study.Result: The results show that 150 ewe milk samples underwent real-time PCR (RT-PCR) targeting the ssrA gene, followed by partial 16S rRNA gene sequencing (PSGS) of purified conventional PCR products. Furthermore, the study entails the biosynthesis of magnesium oxide nanoparticles using Myrtus communis leaf extract, followed by a comprehensive characterization utilizing UV-spectra, FTIR, SEM, and TEM techniques. The Agar well diffusion method assessed the antibacterial efficacy of these Biosynthesized nanoparticles against L. monocytogenes. The RT-PCR results revealed the presence of L. monocytogenes in 36 out of 150 samples (24%). Subsequent PCR analysis confirmed the presence of the pathogen in 30 out of these 36 positive samples (83.33%). Sequencing of two purified PCR products demonstrated 100% nucleotide identity with global isolates from Iraq and Turkey. Furthermore, the study demonstrated that L. monocytogenes exhibited substantial sensitivity (24.66 ± 0.3) to the biosynthesized magnesium oxide nanoparticles. These findings underscore the speed and precision of the RT-PCR method for detecting L. monocytogenes in fresh ewe milk samples.Conclusion: This comprehensive investigation enhances our understanding of L. monocytogenes prevalence in ewe milk and highlights the potential of Myrtus communis -derived nanoparticles for combating this pathogen.Keywords: Antibacterial nanoparticles; Listeria monocytogenes; Magnesium oxide; Myrtus communis sheep milk; ssrA gene

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Three species of nematodes within Acrobeloides (Cobb, 1924); Thorne, 1937 (Rhabditida, Cephalobidae) were collected and recorded for the first time in Iraq, based on morphometric and molecular data, these species, A. saeedi Siddiqi, Ley & Khan, 1992, A. apiculatus (Thorne, 1925), and A. bodenheimeri (Steiner, 1936), were molecularly characterized using the partial 28S rRNA gene sequences. The phylogenetic tree has been constructed to separate Acrobeloides species from closely related species.

First Record of Alkalinema sp. (Cyanobacteria) Thermophilic Strains in Negros Oriental, Philippines

Negros Oriental in the Philippines provides a rich diversity of geothermal habitats. Despite the abundance, there are only a few published thermophile studies in the area. This study aimed to isolate thermophilic cyanobacteria from a hot spring in Negros Oriental and use morphological and molecular features for characterization and identification. Six filamentous, homocystous cyanobacteria designated as CV1, CV2, CV3, CV4, CV5, and CV6 were successfully isolated and cultured. Morphological characterization showed light green to blue-green sheathed trichomes that form an interwoven pattern similar to the recently identified cyanobacterium, Alkalinema. Partial 16S rRNA gene sequences revealed 98.58% identity of the strains with Alkalinema pantanalense DCHY213, and phylogenetic analysis placed them under the family Leptolyngbyaceae – hence forming a separate cluster from other A. pantanalense strains. The differences in ecotype and morphotype, as well as the distinct clustering in the phylogenetic tree of the Negros Oriental strains, imply the possibility of belonging to a different species. Amylase activity was detected in all strains except CV4, hence implying their potential for bioactive metabolite studies. These results highlight the importance of untapped extreme environments in the country for the discovery of novel or recently identified microorganisms that have taxonomical, ecological, and biotechnological applications.

Characterization of Erwinia amylovora isolates from the northern region of Saudi Arabia, including CRISPR genotyping

AbstractFire blight, caused by the bacterium Erwinia amylovora, poses a global threat to the pome fruit industry, leading to reduced yields and significant economic losses. A comprehensive countrywide survey was carried out to determine the possible E. amylovora distribution in pome fruit‐growing regions. During the period 2020–2023, 85 isolates characteristic of E. amylovora were obtained from pear across 23 locations in three main pear‐producing regions (Al‐Jouf, Hail and Tabuk) in northern Saudi Arabia (SA). Isolates were identified using biochemical, molecular and pathogenicity assays. Molecular characterization of Saudi strains was achieved using partial 16S rRNA and rpoB gene sequencing, rep‐PCR (specifically, BOX‐PCR) and CRISPR array analysis. The CRISPR genotyping in particular, using an existing scheme of three CRISPR arrays, CR1, CR2 and CR3, provided consistent patterns of the Saudi isolates with high similarity to those of E. amylovora strains from the UK and France, hinting at a possible origin of the fire blight infections from these countries. Some variation was observed in patterns between local strains, highlighting the importance of continued surveillance and genetic studies for effective disease management strategies. Identification of fire blight in three regions over several years should serve as a warning to the Ministry of Environment, Water and Agriculture in Saudi Arabia, prompting the initiation of a more comprehensive survey and an active disease management programme. This programme should include early detection and advice to growers on prevention and control measures. This is the first scientific study on the occurrence and genetic variability of E. amylovora in Saudi Arabia.

Study of Multiple Metal Resistant Citrobacter sp. Isolated From Industrial Effluent

The pollution of the environment with toxic heavy metals is spreading throughout the world along with industrial progress. Microbe related technologies may provide an alternative or addition to conventional method of metal removal or metal recovery. The present study deals with isolation, identification and characterization of heavy metal resistant bacteria from industrial effluent. a total58 isolates were screened for heavy metal resistant from industrial effluent. The GOZPRN isolate show multiple metal resistant and antibiotic resistances. On the basis of morphological, biochemical, partial 16S rRNA gene sequencing revealed that this isolates belongs to Citrobacter sp.. The isolate showed optimum growth at 30°C and pH 7.0. This isolate was found to be resistant to nickel (Ni), lead (Pb), zinc (Zn), Chromium (Cr). The minimal tolerance concentration (MTC) of this isolate against Cr, Ni, Pb, and Zn was 2.8 mM, 80 mM, 800 mM, and 500 mM respectively. It also exhibited resistance against tetracycline. This Citrobacter Sp. has potential to be useful for the bioremediation of heavy metals.

Characterization of Bacillus velezensis DCJ 2 isolated from the rhizosphere soil of aglaonema ‘Maria’ and investigation of its plant growth-promoting effects

Aglaonema ‘Maria’ is one of the key foliage plants that has gained high demand in foreign destinations in the last few years. This study was conducted to characterize Bacillus velezensis DCJ 2, assess its plant growth promotion effect and investigate the effectiveness of different delivery methods of the bacterial inoculum for the growth promotion of Aglaonema ‘Maria’. In this context, DCJ 2, isolated from the rhizosphere soil of A. Maria was screened to investigate its potential as a plant growth-promoting bacterium. The pure culture of DCJ 2 maintained on nutrient agar was utilized for morphological, biochemical and physiological examinations. The molecular characterization of the strain was performed with different primer pairs targeting 16SrRNA, gyrA, pgsB and sugar kinase genes of DCJ 2. A pot experiment was carried out to investigate the plant growth promotion effect. It was conducted in the greenhouse as per the complete randomized block design with eight treatments and five replicates. . Healthy plants of A. ‘Maria’ established in field soil and solarized soil were treated with 100 ml of the suspension of the DCJ 2 at the rate of 10 8 CFU/ml either as a soil drench or foliar spray. The partial 16SrRNA, gyrA, pgsB and sugar kinase gene sequences of DCJ 2, BLAST searched in the NCBI database revealed that it has a high similarity to Bacillus velezensis. The pot experiment showed that the application of strain DCJ 2 as a soil drench to solarized soil (Treatment 6) yielded a significant increase in growth parameters at p=0.05. Based on molecular analysis, morphological, biochemical, and physiological reactions, DCJ 2 was identified as Bacillus velezensis. This study revealed that B. velezensis DCJ 2 has the potential to boost the growth of A. Maria and hence serve as a base for developing a biofertilizer.

Lactobacillus paracasei Jlus66 relieves DSS-induced ulcerative colitis in a murine model by maintaining intestinal barrier integrity, inhibiting inflammation, and improving intestinal microbiota structure.

Ulcerative colitis (UC) is a serious health problem with increasing morbidity and prevalence worldwide. The pathogenesis of UC is complex, currently believed to be influenced by genetic factors, dysregulation of the host immune system, imbalance in the intestinal microbiota, and environmental factors. Currently, UC is typically managed using aminosalicylates, immunosuppressants, and biologics as adjunctive therapies, with the risk of relapse and development of drug resistance upon discontinuation. Therefore, further research into the pathogenesis of UC and exploration of potential treatment strategies are necessary to improve the quality of life for affected patients. According to previous studies, Lactobacillus paracasei Jlus66 (Jlus66) reduced inflammation and may help prevent or treat UC. We used dextran sulfate sodium (DSS) to induce a mouse model of UC to assess the effect of Jlus66 on the progression of colitis. During the experiment, we monitored mouse body weight, food and water consumption, as well as rectal bleeding. Hematoxylin-eosin staining was performed to assess intestinal pathological damage. Protein imprinting and immunohistochemical methods were used to evaluate the protein levels of nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and tight junction (TJ) proteins in intestinal tissues. Fecal microbiota was analyzed based on partial 16S rRNA gene sequencing. Jlus66 supplementation reduced the degree of colon tissue damage, such as colon shortening, fecal occult blood, colon epithelial damage, and weight loss. Supplementation with Jlus66 reduced DSS-induced upregulation of cytokine levels such as TNF-α, IL-1β, and IL-6 (p < 0.05). The NF-κB pathway and MAPK pathway were inhibited, and the expression of TJ proteins (ZO-1, Occludin, and Claudin-3) was upregulated. 16S rRNA sequencing of mouse cecal contents showed that Jlus66 effectively regulated the structure of the intestinal biota. In conclusion, these data indicate that Jlus66 can alter the intestinal biota and slow the progression of UC, providing new insights into potential therapeutic strategies for UC.

Metagenomic evaluation of peanut rhizosphere microbiome from the farms of Saurashtra regions of Gujarat, India

The narrow zone of soil around the plant roots with maximum microbial activity termed as rhizosphere. Rhizospheric bacteria promote the plant growth directly or indirectly by providing the nutrients and producing antimicrobial compounds. In this study, the rhizospheric microbiota of peanut plants was characterized from different farms using an Illumina-based partial 16S rRNA gene sequencing to evaluate microbial diversity and identify the core microbiome through culture-independent (CI) approach. Further, all rhizospheric bacteria that could grow on various nutrient media were identified, and the diversity of those microbes through culture-dependent method (CD) was then directly compared with their CI counterparts. The microbial population profiles showed a significant correlation with organic carbon and concentration of phosphate, manganese, and potassium in the rhizospheric soil. Genera like Sphingomicrobium, Actinoplanes, Aureimonas _A, Chryseobacterium, members from Sphingomonadaceae, Burkholderiaceae, Pseudomonadaceae, Enterobacteriaceae family, and Bacilli class were found in the core microbiome of peanut plants. As expected, the current study demonstrated more bacterial diversity in the CI method. However, a higher number of sequence variants were exclusively present in the CD approach compared to the number of sequence variants shared between both approaches. These CD-exclusive variants belonged to organisms that are more typically found in soil. Overall, this study portrayed the changes in the rhizospheric microbiota of peanuts in different rhizospheric soil and environmental conditions and gave an idea about core microbiome of peanut plant and comparative bacterial diversity identified through both approaches.

Bacterial Lipopeptides Are Effective against Pear Fire Blight.

Fire blight, a devastating disease caused by Erwinia amylovora, poses a significant threat to pear and apple trees in Xinjiang province, China. In an effort to combat this pathogen, we isolated 10 bacteria from various components of apple and crabapple trees and conducted screenings to assess their ability to inhibit E. amylovora in vitro. Through biochemical tests and partial 16S rRNA gene sequencing, we identified two promising strains, Priestia megaterium strain H1 and Bacillus subtilis strain I2. These strains were then evaluated for their efficacy in biocontrol under controlled laboratory conditions, focusing on immature fruits and leaves. Remarkably, all selected antagonists exhibited the capability to reduce the severity of the disease on both fruit and leaves. P. megaterium strain H1 and B. subtilis strain I2 exhibited significant reductions in disease incidence on both immature fruits and leaves compared to the control. Specifically, on immature fruits, they achieved reductions of 53.39% and 44.76%, respectively, while on leaves, they demonstrated reductions of 59.55% and 55.53%, respectively. Furthermore, during the study, we detected the presence of lipopeptides, including surfactin, iturins, bacillomycin D, and fengycins, in the methanol extract obtained from these two antagonistic bacteria using thin-layer chromatography (TLC). Based on the results obtained, B. subtilis strain I2 and P. megaterium strain H1 exhibit considerable potential for controlling fire blight. However, further evaluation of their efficacy under natural field conditions is essential to validate their practicality as a biocontrol method.

Isolation and characterization of pure cultures for metabolizing 1,4-dioxane in oligotrophic environments.

1,4-Dioxane concentration in most contaminated water is much less than 1 mg/L, which cannot sustain the growth of most reported 1,4-dioxane-metabolizing pure cultures. These pure cultures were isolated following enrichment of mixed cultures at high concentrations (20 to 1,000 mg/L). This study is based on a different strategy: 1,4-dioxane-metabolizing mixed cultures were enriched by periodically spiking 1,4-dioxane at low concentrations (≤1 mg/L). Five 1,4-dioxane-metabolizing pure strains LCD6B, LCD6D, WC10G, WCD6H, and WD4H were isolated and characterized. The partial 16S rRNA gene sequencing showed that the five bacterial strains were related to Dokdonella sp. (98.3%), Acinetobacter sp. (99.0%), Afipia sp. (99.2%), Nitrobacter sp. (97.9%), and Pseudonocardia sp. (99.4%), respectively. Nitrobacter sp. WCD6H is the first reported 1,4-dioxane-metabolizing bacterium in the genus of Nitrobacter. The net specific growth rates of these five cultures are consistently higher than those reported in the literature at 1,4-dioxane concentrations <0.5 mg/L. Compared to the literature, our newly discovered strains have lower half-maximum-rate concentrations (1.8 to 8.2 mg-dioxane/L), lower maximum specific 1,4-dioxane utilization rates (0.24 to 0.47 mg-dioxane/(mg-protein ⋅ d)), higher biomass yields (0.29 to 0.38 mg-protein/mg-dioxane), and lower decay coefficients (0.01 to 0.02 d-1). These are characteristics of microorganisms living in oligotrophic environments.

A new sympagic nematode species Daptonema gelida sp. n. (Monhysterida, Xyalidae) from the White Sea landfast ice

A new ice-associated species of Xyalidae family, Daptonema gelida sp. n., is described from the first-year fast sea ice of the Ermolinskaya Inlet, White Sea, Russia. The new species of Daptonema is a morphologically distinct species from other species of the genus. Daptonema gelida sp. n. is characterized by (1) elongated, slender body, (2) short, evenly distributed throughout the body somatic setae, (3) circular amphideal fovea, (4) thin and strongly curved, L-shaped spicules with round-shaped manubrium, (5) gubernaculum with short dorsal-caudal apophysis by triangular-shaped outline, (6) post-vulval uterine sac present, and (7) two short terminal setae. We barcoded partial 18S rRNA gene sequences from the new sympagic species. Phylogenetic reconstructions showed that the specimen of Daptonema gelida sp. n. formed a distinct lineage with high support among other Daptonema species and was genetically related to D. carnulentum, D. setosum, D. hirsutum, Metadesmolaimus sp. and Daptonema spp. We provided a list of valid Daptonema species, including key morphological characteristics of males, and additionally reviewed 30 species inquirenda.

Bacillus amyloliquefaciens: a versatile antimicrobial Firmicute for the suppression of wilt caused by Ralstonia pseudosolanacearum in tomato

ABSTRACT Rhizosphere bacteria based biological control strategy is an eco-friendly approach to protect crops from various plant diseases. Among these, spore-forming Bacillus, abundant in the rhizosphere, provide unique advantages for plant health. This study focused on characterising two tomato rhizosphere-associated Bacillus amyloliquefaciens strains (Trb7 and Trb11), highly effective against the bacterial wilt pathogen Ralstonia pseudosolanacearum in vitro. Species identification was based on partial 16S rRNA gene sequencing, supported by phylogenetic analysis. Five antimicrobial peptide (AMP) genes (surfactin, fengycin, bacilysin, subtilin, and spore protein) were detected in both the isolates of B. amyloliquefqciens using specific molecular markers. Greenhouse trials showed that prophylactic application of these isolates led to an 83.4% reduction in tomato bacterial wilt, with only 16.6% wilt incidence compared to the 100% in the R. pseudosolanacearum-inoculated control. Moreover, these B. amyloliquefaciens isolates exhibited plant-growth-promoting traits in vitro. Interestingly, they also showed significant inhibition of mycelial growth against Sclerotium rolfsii and Alternaria alternata, major fungal pathogens in tomatoes. Overall, our findings highlight the potent bactericidal role of B. amyloliquefaciens isolates in suppressing R. pseudosolanacearum in tomatoes. Key highlights Two among the 100 Bacillus displayed antibacterial activity against Ralstonia pseudosolanacearum. Antibacterial firmicutes was identified as Bacillus amyloliquefaciens through the 16S rRNA gene sequence analysis. Five AMP genes (srfAA, fenD, bacA, spas, and spoVG) were identified in the isolates. Delivery of B. amyloliquefaciens drenching on rhizosphere significantly reduced wilt. Additionally, B. amyloliquefaciens showed the antifungal activity on Sclerotium rolfsii and Alternaria alternata.

Ipomoea obscura Represents a New Host of Phytoplasma Belonging to 16SrII Group Associated with Witches'-Broom Symptoms in China.

Phytoplasmas are phloem-limited plant pathogenic prokaryotes which can not be cultured in vitro. The pathogens could cause various plant symptoms such as witches'-broom, virescence, and leaf yellows. Ipomoea obscura is a valuable plant species belonging to the family Convolvulaceae, mainly used as a traditional Chinese medicine used to treat diseases such as dehydration and diuresis. In western countries it is commonly referred to as 'obscure morning glory'. During 2020 to 2021, plants showing abnormal symptoms including witches'-broom, internode shortening, and small leaves were found in Hainan Province, a tropical island of China. Approximately 30 % of I. obscura plants in the sampling regions which spanned 400 acres, showed symptoms. In order to identify the associated pathogen, six symptomatic samples and three asymptomatic samples were collected and total DNA were extracted from 0.10 g fresh plant leaf tissues using CTAB DNA extraction method. 16S rRNA and secA gene fragments, specific to phytoplasmas, were PCR amplified using primers R16mF2/R16mR1 and secAfor1/secArev3. The target PCR bands were obtained from the DNA of six symptomatic samples, whereas not from the DNA of the asymptomatic samples. The PCR products of phytoplasma 16S rRNA and secA gene obtained from the diseased samples were cloned and sequenced by Biotechnology (Shanghai) Co., Ltd. (Guangzhou, China). The 16S rRNA and secA gene sequences identified in the study were all identical with the length of 1330 bp (GenBank accession: OR625212) and 720 bp (OR635662) respectively. According to methods and protocols of phytoplasma identification and classification (Wei and Zhao, 2022), the phytoplasma strain identified in the study was described as Ipomoea obscura witches'-broom (IoWB) phytoplasma, IoWB-hnld strain. The partial 16S rRNA gene sequence of IoWB showed 100 % sequence identity over the full 1330 bp sequence to phytoplasmas belonging to 16SrII group like cassava witches'-broom phytoplasma (KM280679). The BLAST search of the 720 bp partial secA gene fragment of IoWB showed 100% sequence identity for the full sequence to phytoplasmas belonging to 16SrII group like 'Sesamum indicum' phyllody phytoplasma (OQ420657). RFLP analysis based on the 16S rRNA gene using iPhyClassifier demonstrated that the IoWB strain was a member of 16SrII-A subgroup with the similarity coefficient 1.00 to the reference phytoplasma strain (L33765). Phylogenetic analysis based on 16S rRNA and secA genes by MEGA 7.0 employing neighbor-joining (NJ) method with 1000 bootstrap value indicated that IoWB-hnld was clustered into one clade with the phytoplasmas belonging to 16SrII group, with 98% and 100% bootstrap value separately. To our knowledge, this is the first report that Ipomoea obscura can be infected by phytoplasmas belonging to 16SrII-A subgroup in China. This report adds to the host range of 'Ca. Phytoplasma aurantifolia', documenting the symptoms on I. obscura which will assist in monitoring and control of the associated pathogen.

Fluctibacter corallii gen. nov., sp. nov., isolated from the coral Montipora capitata on a reef in Kāne'ohe Bay, O'ahu, Hawai'i, reclassification of Aestuariibacter halophilus as Fluctibacter halophilus comb. nov., and Paraglaciecola oceanifecundans as a later heterotypic synonym of Paraglaciecola agarilytica.

Strain AA17T was isolated from an apparently healthy fragment of Montipora capitata coral from the reef surrounding Moku o Lo'e in Kāne'ohe Bay, O'ahu, Hawai'i, USA, and was taxonomically evaluated using a polyphasic approach. Comparison of a partial 16S rRNA gene sequence found that strain AA17T shared the greatest similarity with Aestuariibacter halophilus JC2043T (96.6%), and phylogenies based on 16S rRNA gene sequences grouped strain AA17T with members of the Aliiglaciecola, Aestuariibacter, Lacimicrobium, Marisediminitalea, Planctobacterium, and Saliniradius genera. To more precisely infer the taxonomy of strain AA17T, a phylogenomic analysis was conducted and indicated that strain AA17T formed a monophyletic clade with A. halophilus JC2043T, divergent from Aestuariibacter salexigens JC2042T and other related genera. As a result of monophyly and multiple genomic metrics of genus demarcation, strain AA17T and A. halophilus JC2043T comprise a distinct genus for which the name Fluctibacter gen. nov. is proposed. Based on a polyphasic characterisation and identifying differences in genomic and taxonomic data, strain AA17T represents a novel species, for which the name Fluctibacter corallii sp. nov. is proposed. The type strain is AA17T (= LMG 32603T = NCTC 14664T). This work also supports the reclassification of A. halophilus as Fluctibacter halophilus comb. nov., which is the type species of the Fluctibacter genus. Genomic analyses also support the reclassification of Paraglaciecola oceanifecundans as a later heterotypic synonym of Paraglaciecola agarilytica.

Ampelopsis grossedentata Represents a New Host of the 16SrI Group of Phytoplasma Associated with Yellow Leaf Symptoms in China.

Ampelopsis grossedentata, commonly known as "Vine Tea" and well-recognized for its rich flavonoid content, is mainly distributed in the southern regions of the Yangtze River basin in China. These regions include Hunan, Hubei, Jiangxi, and Guizhou provinces. Vine Tea is mainly consumed as an herbal tea and has garnered attention for its reported health benefits, including antioxidant, anti-inflammatory, anti-tumor, anti-diabetic, and neuroprotective properties. It has been used to alleviate coughs and sore throats (Zhang et al., 2021; Wang et al., 2017; Gao et al., 2009). In the Zhangjiajie region of Hunan province alone, the Vine Tea planting area reached 7,670.5 hectares and produced commercial goods worth 1.417 billion RMB in 2022. In May 2021, leaf margins and veins fading to yellowing mottling, and crumpling of leaf blades in the shape of a boat symptoms were found in ~16% of Vine Tea plants in the Sanjiakuan Township, Yongding District, Zhangjiajie region (29°15'E, 110°30' N) (Figure 1a, b, c). (Figure 1a, b, c). Phytoplasma-like microbial cells (small oval shaped bacterial cells, around 1000 nm in size) were observed in sieve tube cells in the phloem of diseased leaves using transmission electron microscopy. No such cell was observed in the phloem of healthy leaves (Figure 2a, b). To investigate the potential association between phytoplasma and the observed symptoms of the diseased plants, total DNA was isolated from ten diseasedeaves and compared with ten healthy leaves from the same field using SteadyPure Plant Genomic DNA Extraction Kit. The isolated DNAs were analyzed first in a direct PCR using universal phytoplasma primer pair R16mF2/R16mR1 targeting the 16S rRNA gene (Gundersen and Lee 1996) and specific pair rpF1/rpR1 (Lee et al. 1998) targeting the DNA fragment encoding partial ribosomal proteins (rp) L22 (complete) and S3 and S19 (partial). The initial amplified products were used as templates and further amplified by nested PCR respectively with primer pair R16F2n/R16R2 for the 16S rRNA gene (Lee et al. 1998) and the rpF2/rpR2 primer pair for the rp gene (Martini et al. 2007). No amplification was obtained with DNA from healthy leaf samples using any of the four primer pairs. The amplified fragments from diseased leaves by nested PCR were cloned and sequenced (Qingke Biotech, China). The obtained sequences have been deposited in GenBank with accession numbers OR282806 for the 16S rRNA gene and GenBank OR353012 for the rp gene. BLASTn analysis revealed that the partial 16S rRNA gene sequence in our sample shared 99.4% nucleotide sequence identity with 'Candidatus Phytoplasma sp.' (MW364378) and 'Peony yellows phytoplasma' (KY814723) of the 16SrI group. Similarly, our rp gene sequence shared 99.6% nucleotide identity with the rpI group of phytoplasma such as the 'Balsamine virescence phytoplasma' (JN572890) and 'Paulownia witches'-broom phytoplasma' (HM146079). Phylogenetic analysis of the 16S rRNA and rp sequences using MEGA version 7.0 revealed that the phytoplasma strain associated with A. grossedentata yellow leaf syndrome in our study site belonged to the 16SrI (Candidatus Phytoplasma asteris) group of phytoplasma (Figure 3a, b). Using the interactive online phytoplasma classification tool iPhyClassifier (Zhao et al., 2009), virtual restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene sequences showed our strain having a distinct RFLP map but was closest to that of the onion yellow phytoplasma 16SrI-B subgroup (GenBank accession number: AP006628), with a similarity coefficient of 0.94 (Figure 4a, b). To confirm phytoplasma transmission, healthy plants were inoculated with three scions of infected plants of A. grossedentata. After 16 days, the new leaves of the inoculated A. grossedentata showed yellow leaf symptoms (Figure 5a, b, c), akin to the symptoms originally observed in the field, and the outer contour of the leaf margin appeared chlorotic. After 26 days, primer pairs R16mF2/R16R1 and R16F2n/R16R2 were used for nested PCR detection of phytoplasma in symptomatic A. grossedentata leaves. Phytoplasma was detected in the first and second leaves of symptomatic branches and leaves while negative control showed no amplification. Sequencing of the amplified fragments showed 100% nucleotide identity to the strain from the grafting source. Our results indicated that the pathogen and the disease can be transmitted by tissue grafting, consistent with the biological characteristics of phytoplasma, and further confirmed that the phytoplasma was the pathogen of yellow leaf syndrome of A. grossedentata. Toour knowledge, this is the first report of phytoplasma of group 16SrI affecting A. grossedentata.

First Report of Pantoea ananatis and Pantoea eucalypti causing onion leaf blight and bulb decay in Central Chile.

Nearly 5,400 hectares of long-day onions (Allium cepa) are cultivated in the Central Zone of Chile (UTM 33°3´S to 37°24´S). During summer 2021-22 and 2022-23 (December to February), followed by high temperatures (around 35°C) and high humidity conditions, symptoms showing yellowing, soft rot, blight on old leaves, necrotic tips, and soft bulbs were observed. The affected plants were observed in "spots" into the fields, and the incidence reached 5-10%. The severity was high, and 70-80% of the affected plants died. Symptomatic plants from different fields from the Coquimbo, Metropolitan, O´Higgins and Maule regions were sampled. Isolations were made using casamino-acid peptone glucose (CPG) agar medium (Schaad, 2001). Yellow-pigmented, circular to irregular shaped colonies were observed. Molecular identification was carried out by partial 16S rRNA gene sequencing, resulting in the identification of 18 isolates of Pantoea spp. from twelve different fields. Selected strains were biochemically analyzed using the GEN III BIOLOG microtest system (Hayward, CA) and were identified as Pantoea spp. BLAST analyses of the 16S rRNA sequences (602 nt) of selected strains (GenBank Accession No. OR527817 to OR527819) against the NCBI Database resulted in the identification of Pantoea species with 100% coverage and 100% identity. To determine the Pantoea species of each strain, housekeeping gene gyrB (Delétoile et al. 2009) was amplified and sequenced (GenBank Accessions No. OR544061 to OR544063). BLAST analysis (802 nt) of selected strains resulted in 100% coverage and 100% identity, identifying three different species: P. ananatis, P. eucalypti, and P. agglomerans. Pantoea species were isolated from both leaves and bulbs and no more than one species was observed per field. Pathogenicity assays in onion plants and bulbs were performed based on the methodology described by Asselin et al. (2018). Five onion plants cv. Cimarron of thirteen-week-old were inoculated by wounding an external leave with a sterile toothpick previously immersed in a bacterial suspension at ~ 108 CFU/mL and maintained at 26-28°C for 30 days in high humidity conditions. Control plants were inoculated with sterile water. Plants inoculated with Pantoea spp. showed chlorosis, soft rot, and necrosis mainly in older leaves, as observed in the field, while negative control plants remained healthy. Pantoea spp. were re-isolated from the inoculated onion plants. Toothpicks dipped in the inoculum were stuck 4 cm into the shoulders of onion bulbs and incubated at 26°C for 20 days. Water was inoculated as a negative control. At the end of the incubation period, the bulbs were opened longitudinally across their inoculation sites and shrunken, brownish, watery scales were observed. Pantoea species have been previously described as causing leaf blight of onions in Georgia and Michigan in the USA and South Africa, Brazil, and Uruguay (Hattingh and Walters 1981; Gitaitis and Gay 1997; Edens et al. 2006; Tho et al. 2015; De Armas et al. 2022; Rosende et al. 2022). P. agglomerans was recently reported in Chile (Sepúlveda et al. 2023), but this is the first report of P. ananatis and P. eucalypti affecting onions in central Chile. This detection is an alert call for the onion's growers and exporters in Chile for upcoming seasons, where conditions predisposing to disease may continue to occur. It is crucial to continue analyzing the factors that caused the appearance of this new disease in onions.

Phylogenetic analysis and haplotype networking of Hepatozoon felis infecting wild animals in Gir National Park, Gujarat, India.

The present study was aimed to determine the phylogenetic relationship, haplotype network, and demographic dynamics of H. felis infecting the endangered Asiatic lions in Gir National Park, Gujarat, India, on the basis of partial 18S rRNA gene. The phylogenetic analysis based on the partial 18S rRNA gene sequences of H. felis exhibited the presence of two distinct genotypes of H. felis (HfG1 and HfG2) infecting the Indian wild felids, viz., the Asiatic lion, Royal Bengal tiger, and Indian leopard. The HfG1 and HfG2 genotypes exhibited 97.6-100% and 99.7-100%, and 96.9-98.7% nucleotide identity within and between themselves, respectively. The HfG1 genotype exhibited a higher genetic diversity as compared to HfG2. A total of 22 molecular signatures were identified in the 18S rRNA gene between these genotypes. Further, analysis of a total 67 sequences of H. felis (13 different host species from 13 countries of Africa, South America, Asia, and Europe) that were downloaded from GenBankTM, generated 30 haplotypes. Among all the haplotypes, Hap_17 (h=12) was the most frequent followed by Hap_12 (h=09) and Hap_4 (h=05). Out of 13 location-wise populations, India (h=12) shared the highest number of haplotypes followed by Japan (h=08), and the least number of haplotypes were found in Hungary (h=02). Population dynamics study involving neutrality tests and mismatch distribution, and genetic differentiation indices, revealed the presence of phylogeographic population structure and a constant population size indicating a uniform gene flow among the populations worldwide. In conclusion, a high genetic diversity along with the presence of two distinct genotypes of H. felis were observed on the basis of 18S rRNA gene sequence analysis.