Amplified 16S rDNA Research Articles

Identifying the hidden parasitic intruders: Molecular insights into the health of common Myna

Birds are known to act as the parasite reservoir and can transmit them to other organisms through food chains. This study aims to report the molecular prevalence and phylogenetic evaluation of various blood borne pathogens (Toxoplasma gondii, Isospora spp., Plasmodium spp., Haemoproteus spp., Leucocytozoan spp. and Neospora caninum) in blood samples of common Myna (Acridotheres tristis: N = 80) collected from four region (Jhang, Khanewal, Multan and Muzaffargarh) in Punjab Pakistan. Effect of pathogens on the complete blood count of the host was also determined. Results revealed by 2/80 Myna (2.5 %) amplified ITS-1 gene of Toxoplasma (T.) gondii (confirmed by DNA sequencing) while 2/80 (2.5 %) birds amplified 18S rDNA gene and Isospora spp. Phylogenetic analysis of both pathogens showed that Pakistani isolates were clustered together and were closely related to isolates that were reported from worldwide countries.Risk factor analysis revealed that prevalence of both pathogens was not restricted to a particular sampling site or a particular bird sex (P > 0.05). T. gondii infected birds had elevated red cell distribution width while Isospora sp. infected birds had elevated % monocytes and platelet distribution width while decreased mean cell hemoglobin, mean corpuscular hemoglobin concentration and platelets hematocrit than their respective uninfected birds. In conclusion, we are reporting the presence of T. gondii and Isospora sp. among Pakistani common Myna that had disturbed the complete blood count parameters that may have affected their normal physiology.

Study of bacterium associated with Spinacia oleracea and nematode, Pseudomonas fragi using 16S rDNA from Limpopo Province, South Africa

Microorganisms known as Plant-associated Pseudomonas can thrive as parasites or saprophytes on plant surfaces and within plant tissues. They are crucial in enhancing plant growth by suppressing pathogenic microorganisms, synthesizing growth-stimulating plant hormones, and boosting plant disease resistance. At the University of Limpopo in South Africa, a molecular study was conducted in 2023 to identify a Pseudomonas bacterium in a spinach field associated with a free-living nematode, Acrobeles complexus, in Limpopo Province. The study utilized the Chelex method to extract DNA and the 16S rDNA marker to identify the bacterium as Pseudomonas fragi. Specific primers were then used to amplify 16S rDNA, which identified the bacterium, and Nblast analysis revealed a 96% similarity to a strain from India. Phylogenetic analysis placed this species with those identified as P. fragi in the same clade with highly supported (100) bootstrap values. This marker (16S rDNA) is recommended to use. But for a better understanding of P. fragi's phylogeny, the other DNA markers are recommended to identify this species.

Onion (Allium cepa L.) as a new host for 'Candidatus Arsenophonus phytopathogenicus' in Germany.

Onion (Allium cepa L.) is the most produced vegetable after tomato worldwide and is grown on about 15,000 ha in Germany. In Lampertheim, Hesse in southwest Germany (49°40'02.3"N, 8°26'00.0"E) bulbs of the cultivar 'Red Baron F1' were harvested in September 2023 in an apparently healthy state. Four months later some of the onions showed rotting symptoms, which could not be assigned to a known storage disease. At first, the bulbs became glassy, later they showed soft rot. They originated from a field located in a growing region severely affected by "Syndrome Basses Richesses" (SBR). 'Candidatus Arsenophonus phytopathogenicus' as well as 'Candidatus Phytoplasma solani' are associated with this disease in sugar beet (Gatineau et al. 2002). Moreover, 'Ca. A. phytopathogenicus' was recently reported in association of bacterial wilt and yellowing in potato (Behrmann et al. 2023). Both phloem-restricted bacteria are vectored by the polyphagous planthopper Pentastiridius leporinus (Therhaag et al. 2024), which is highly abundant in this region. To examine, if the unknown symptoms in onion might be related to the presence of these pathogens, DNA of 69 bulbs showing a different degree of softening were analyzed. The samples were tested for the presence of 'Ca. Phytoplasma solani' in a TaqMan assay (Behrmann et al. 2022). All showed negative results. To demonstrate the presence of 'Ca. A. phytopathogenicus', universal and genus-specific primers for the amplification of 16S rDNA and a real-time qPCR assay amplifying an hsp20 fragment were employed (Christensen et al. 2004, Zübert and Kube 2021). Two bulbs of the five positive samples were in an apparently healthy state, the other three showed light to moderate softening symptoms. The 16S rDNA fragments from two samples were sequenced on both strands and aligned. Both fragments were homologous. One fragment of 1474 bp fragment showing 100% homology to the 16S rDNA from SBR (accession no. AY057392) was submitted to GenBank (accession no. PP400342). Other taxa of 'Ca. Arsenophonus' showed 16S rDNA homologies of less than 99.3 %. To corroborate the finding onion samples were subjected to PCR reactions employing genus-specific primers for the conserved tufB, secY and manA gene, which had been derived from multiple alignments of 'Ca. A. spp' sequence submissions (Sela et al. 1989, Lee et al. 2010). The tufB, secY and manA primers amplified fragments of about 980 bp, 640 bp and 930 bp, respectively, from all previously positive samples. Samples which had been tested negative for 'Ca. P. phytopathogenicus' remained negative. Fragments from two accessions were sequenced and the sequences from both isolates were 100 % identical. A BLAST search of the partial tufB gene (acc. no. PP950434) showed 98.57 % sequence identity to a yet unnamed Arsenophonus endosymbiont (acc. no. OZ026540) and 91.85 to 91.83 % to 'Ca. A. nasoniae' and 'Ca. A. apicola', respectively. A similar result was obtained for the partial secY sequence (acc. no. PP950433). The manA sequence (acc. no. PP942231) was identical to a partial sequence of 'Ca. A. phytopathogenicus' strain HN (acc. no. OK335757) and 97.42 % to 'Ca. A. nasoniae and about 87 % to related Arsenophonus species. The finding of 'Ca. A. phytopathogenicus' in onion is novel and might indicate an expanding host range of vector and pathogen in the regional crop rotation. As a correlation between the pathogen and the soft rot symptom is unclear at present, further investigations are needed.

First Report of Bacterial Leaf Streak of Rice Caused by Pantoea ananatis in Guangdong Province, China.

Rice (Oryza sativa L.) is a crucial staple crop worldwide, and bacterial diseases are among the primary factors affecting rice yield. In late October 2022, bacterial leaf streak disease was observed on the leaves of the rice variety Meixiangzhan 2 across multiple fields (approximately 130 hm2) in Leizhou City, Guangdong Province, China. The incidence rate was up to 30% in each field. Infected rice leaves exhibited distinctive symptoms at the boundary between diseased and healthy tissue, featuring dark green to yellow-brown streaks, while most of the leaf margin exhibited symptoms of either leaf edge or sheath rot. Disease progression from the leaf tip inwards revealed gray-white or dehydrated lesions with a bluish-gray color. Some leaves exhibited wrinkling at the edges, and severe symptoms at the leaf tip resembled those of bacterial leaf blight in rice. Ten leaves were collected from 10 infected rice plants in three distinct fields, and leaf pieces at the border of diseased and healthy areas were surface disinfected with 75% anhydrous ethanol for 60 seconds, rinsed three times with sterile water, and then soaked in sterile water for 8 hours. The obtained bacterial suspension was diluted at a ratio of 1: 106, and 100 μL of the diluted samples were plated on Potato Dextrose Agar (PDA) plates. After incubation at 28°C for 48 hours, the yellow bacterial colonies that appeared, were purified on PDA plates. To confirm the bacterial species, the amplification of genes gyrB, leuS, rpoB, and 16S rDNA was performed on six randomly selected isolates from the three different fields using the primers 27F/1492R, gyrB-F/R, leuS-F/R and rpoB-F/R, as reported by Yu et al (2022), respectively. PCR products were sequenced. All six isolates had identical sequences for all genes sequenced.The gene sequences of 16S rDNA (960 bp), gyrB (953 bp), leuS (733 bp), and rpoB (877 bp) for LZ1, were deposited in the NCBI database under accession numbers PP048830 , PP068625 , PP068626, and PP068627, respectively. These sequences were subsequently compared using BLASTn tool against the NCBI nr/nt database. The 16S rDNA, gyrB, leuS, and rpoB of LZ1 showed similarities of 99.90%, 99.16%, 99.73%, and 99.89%, with the corresponding sequences of P. ananatis TZ39 (GenBank accession numbers MZ800600.1 for 16S rDNA, and CP081342.1 for gyrB, leuS and rpoB ). MLSA analysis using concatenated sequences of gyrB, leuS, and rpoB genes indicated that the isolated strain LZ1 belongs to P. ananatis. In the tillering stage of rice varieties Meixiangzhan 2 and Huahangyuzhan, P. ananatis LZ1 was inoculated at a concentration of 108 CFU/mL using the leaf-cutting method, with sterile water used as a control (Toh et al., 2019). After 14 days of bacterial inoculation, the inoculated leaves gradually became necrotic, changing from light green to brown showing identical symptoms as those in the field, while the control plants remained symptom-free. Subsequent 16S rDNA, gyrB, leuS and rpoB gene sequencing results further confirmed the identity of the pathogen as P. ananatis, thereby fulfilling Koch's postulates. Previous reports have already identified P. ananatis as the pathogen causing rice bacterial leaf streak (Kini et al., 2017; Arayaskul et al., 2019; Yu et al., 2022; Lu et al., 2022; Luna et al., 2023; Yuan et al., 2023). This is the first report of rice bacterial leaf streak caused by P. ananatis in Guangdong Province, China, laying the foundation for future research to establish strategies for the prevention and control of this disease.

Effects of Palm Oil Deodorizer Distillate on the Ruminal Environment of Sheep.

This study aimed to assess the impact of palm oil deodorizer distillate (POD) on the ruminal environment, including (i) microbial community, (ii) ruminal degradability, and (iii) apparent digestibility in sheep. The data used were derived from twenty rumen-cannulated sheep fed five isoproteic and isofiber diets based on elephant grass (Pennisetum purpureum Schum. cv. Roxo) silage supplemented with 0, 25, 50, 75, or 100 g kg-1 POD on a dry matter (DM) basis. Rumen fluid samples were collected three hours after feeding directly from the ventral sac of the rumen via a cannula and then subjected to DNA extraction, which was subsequently used for 16S rDNA amplification, followed by sequencing and diversity analysis. In this study, the microbial diversity was dominated by Bacteroidetes and Firmicutes, followed by Euryarchaetoa, Actinobacteria, and Tenericutes, in the ruminal environment, and was slightly modified when supplemented with the POD up to 100 g/kg (10%), leading to only a slight decrease in the diversity index. The ruminal degradability, ruminal fermentation parameters, and apparent digestibility were slightly compromised by the inclusion of up to 25 g of POD per kg of DM, and larger inclusions interfered with the ruminal degradability of fibrous fractions and the apparent digestibility of dry matter. This lipid supplement showed good results for feeding sheep and is an inexpensive and abundant alternative in the regional market.

Potential biomarkers of acute myocardial infarction based on the composition of the blood microbiome

BackgroundIt is difficult to distinguish between acute myocardial infarction (AMI) and unstable angina (UA) due to their similar clinical features. In recent years, studies have shown that microbiomes have great potential in distinguishing diseases. The purpose of this study is to describe the composition of serum microbiome in the AMI and UA by 16S rDNA sequencing. MethodsBased on the high-throughput detection platform and 16S rDNA amplification sequencing technology, this study detected the blood microbial composition of 55 patients with AMI and 62 patients with UA. Alpha diversity and Beta diversity analysis were used to compare the differences in microbial composition and bacterial colony structure between AMI and UA groups. We perform PCoA (Principal Co-ordinates Analysis) based on Unweighted Unifrac distance. In addition, various statistical methods were employed to examine the significance of differences in microbial composition and genus between the two groups. PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was employed to predict KEGG (Kyoto Encyclopedia of Genes and Genomes) function from 16S sequencing data. Random forest was applied to identify biomarkers and construct the diagnostic model. Subsequently, the stability of the model was verified by 10-fold cross and the diagnostic effectiveness was evaluated through ROC (Receiver Operating Characteristic). ResultsIn this study, we found that alpha diversity index of serum microbiome in AMI group was significantly higher than in UA group. T-test analysis demonstrated that the UA group presented a higher abundance of Ralstonia, Faecalibaculum and Gammaproteobacteria, while Bacteroides, Sphingomonas, Faecalibaculum, Haemophilus, Serratia, Bifidobacterium and Chloroplast were more abundant in the AMI group. The LefSe (LDA Effect Size) analysis showed that the Gammaproteobacteria, Proteobacteria, Ralstonia pickettli, Ralstonia, Burkholderiaceae and Burkholderiales were enriched in UA group, and Bacteroidales, Bacteroidia, Bacteroidota, Clostridia and Firmicutes were more abundant in the AMI group. Ten bacterial diagnostic models were constructed in the random forest. The area under the curve (AUC) in the training set was 88.01%, and the AUC value in the test set was 95.04%. ConclusionIn this study, the composition of blood microorganisms in the groups of patients with AMI and UA has been analyzed, providing novel insights for understanding the pathogenesis of AMI; Blood microbiome may serve as novel diagnostic biomarkers of AMI.

First Report of Pantoea anthophila Causing Shoot Dieback Disease of Ceylon Ironwood (Mesua ferrea) in Malaysia

Ceylon ironwood (Mesua ferrea Linn.) or Penaga lilin is one of Asia's most popular tropical herbal plants, including Malaysia (Sharma et al., 2017). The trees are cultivated for their aesthetic value and pharmacological properties, especially as traditional remedies for asthma, dermatopathy, inflammation, and rheumatic conditions (Adib et al., 2019). In August 2022, a disease survey was conducted on Ceylon ironwood trees ranging from 5 to 12 years old in Botanical Park, Putrajaya, Malaysia, with 80% exhibiting shoot dieback disease of the 15 trees exhibiting shoot dieback disease. Symptoms include irregular, water-soaked with brown lesions on young leaves and shoots, where the small lesion coalesced and formed broad necrotic regions, subsequently causing dieback and gradual defoliation. Three infected shoots were collected from each tree, excised into small pieces (10 to 20 mm), immersed with 75% ethanol for 3 min, washed with 2% NaOCl solution for 1 min, and rinsed twice for 1 min in sterilized distilled water. A 10 µl aliquot of the sample suspension was streaked onto nutrient agar (NA) and incubated for 24 h to 48 h at 35 °C. A total of 15 isolates with similar morphology were obtained, and each isolate was re-streaked three times to obtain pure colonies that were round, smooth, with irregular edges, and produced yellow pigment in culture. All isolates were Gram-negative, negative for indole production, and utilized glucose, maltose, trehalose, sucrose, D-lactose, and pectin. Three representative isolates (C001, C002, and C003) with similar morphology were selected for further characterization. The total genomic DNA of all isolates was extracted from overnight cultures using Geneaid™ DNA Isolation Kit (Geneaid Biotech Ltd., Taiwan). PCR amplification of 16S rDNA (Zhou et al., 2015) and species-specific infB (Brady et al., 2008) genes was performed, and each of the ~1500 bp and ~900 bp amplicons were sequenced. BLASTn and phylogenetic analyses revealed all isolates were 100% identical to Pantoea anthophila (P. anthophila) LGM 2558 strains (Accession Nos. NR_116749 and NR_116113) for the 16S rDNA gene. They were 99% identical to P. anthophila CL1 strain (Accession Number CP110473) for infB gene. These sequences were later deposited in the GenBank (Accession Nos. OQ772233, OQ772234, and OQ772235 for 16S rDNA gene, and OQ803527, OQ803528, and OQ803529 for infB gene). For the pathogenicity test, healthy Ceylon ironwood seedlings' shoots were inoculated with 10 mL of each isolate suspension (1 x 108 CFU/ml) by spraying the inoculum on the young shoots using a sterilized spray bottle. Control seedlings were inoculated with sterile water. The inoculated shoots were covered with a sealed plastic bag to maintain the moisture and were kept in the greenhouse with temperatures ranging from 26 to 35 °C. The experiments were repeated twice, with three replicates for each treatment. Inoculated shoots showed dieback symptoms like natural infection, including irregular, water-soaked, and brown lesions on leaves and young shoots at 10 days post-inoculation. Control seedlings remained asymptomatic. The pathogen was re-isolated and identified via sequencing of the 16S rDNA and infB genes, thus fulfilling Koch’s postulates. Previously, P. anthophila has been reported to cause soft rot in wampee plants in China (Zhou et al., 2015) and leaf blight of cotton in Pakistan (Tufail et al., 2020). To our knowledge, this is the first report of P. anthophila causing shoot dieback disease of Ceylon ironwood trees in Malaysia. Plant disease management strategies need to be established to reduce losses due to P. anthophila infection since the pathogen could limit Ceylon ironwood tree production in Malaysia.

First report of Pectobacterium aroidearum causing soft rot on leaf mustard in China.

Leaf mustard (Brassica juncea [L.] Czern. et Coss.) is widely planted in China as an important leaf vegetable. In March 2022, water-soaked and mushy rot symptoms were observed on leaf mustard plants in the field of Zhaotong (27.85°N; 105.05°E), Yunnan, China. The incidence of symptomatic leaf mustard was approximately 10%. The isolation of the causal agent followed the method of Peng et al. (2023). Briefly, infected tissues from four diseased plants were mixed and teased apart, and homogenized by vortex shaking. The bacterial suspension was diluted and spread on nutrient agar (NA). About 10 single colonies exhibiting different colony morphologies were picked and purified separately by successive streaking. A pinprick method was used for pathogenicity tests with an inoculum concentration of 108 CFU/ml (Singh et al. 2013). Among 10 isolates, only strain YKX exhibited soft rot symptoms on detached mustard leaves. In addition, ten two-month-old leaf mustard plants grown in the greenhouse were used for in vivo pathogenicity tests. Briefly, sterilized pins were dipped in the bacterial suspension, and then leaf mustard petioles were pricked with these pins. After inoculation, each plant was kept in a plastic bag for 12 hours to maintain high humidity. As expected, strain YKX caused obvious rot symptoms on eight plants at 1-2 days post-inoculation while the control group including two plants treated with sterile water showed no symptoms. The colonies of strain YKX on NA were white, roughly circular, and convex. For a preliminary identification, total DNA was extracted and used as the template in PCR amplification of 16S rDNA with the universal PCR primer pair 27F/1492R (Weisburg et al. 1991). The quality-filtered DNA sequence (871 bp) showed 100% query coverage and 99.47% identity to the 16S rDNA sequences of type strain Pectobacterium aroidearum SCRI 109T (GenBank: NR_159926) found in the NCBI rRNA/ITS database. Whole-genome sequencing of strain YKX was then performed using the Illumina and Nanopore sequencing platforms by Tsingke Biotechnology Co., Ltd. (Beijing, China). A single contig (GenBank: CP129239) with a length of approximately 4.9 Mb was obtained by de novo hybrid assembly using Unicycler v0.5.0 (Wick et al. 2017). The quality of the genomic data was evaluated by BUSCO v5.4.7 (Manni et al. 2021) against the gammaproteobacteria_odb10 dataset. A BUSCO complete score of 99.5% indicated high assembly quality. The genome sequence of strain YKX was uploaded to the Type Strain Genome Server for a genome-based taxonomic analysis (Meier-Kolthoff et al. 2022). The distance-based phylogeny showed that strain YKX and P. aroidearum L6 (GenBank: CP065044) and P. aroidearum PC1 (GenBank: NC_012917) form a clade. When comparing strain YKX with L6 or PC1, the digital DNA-DNA hybridization value (83.5-83.8%) was above the species delineation threshold (70% for DDH), clearly indicating that strain YKX should be classified as P. aroidearum. Additionally, P. aroidearum was reisolated from inoculated leaves and identified based on morphological similarities and 16S rDNA sequencing, thus fulfilling Koch's postulates. It is worth noting that a previous study reported occurrences of soft rot disease on leaf mustard attributed to Rhizopus microsporus var. chinensis (Wang et al. 2020). To our knowledge, this is the first report of P. aroidearum causing soft rot on leaf mustard in China, which expands the known host range of this pathogen and benefits the control of this disease.

A prospective study of nanopore-targeted sequencing in the diagnosis of central nervous system infections.

This study aims to prospectively evaluate the ability of nanopore-targeted sequencing (NTS) in the detection of pathogens in cerebrospinal fluid (CSF). It was the first time combining mNGS and microbial culture to verify the NTS-positive results also using 16S rDNA amplification with Sanger sequencing. Although microbial culture was thought to be the gold standard for pathogens detection and diagnosis of infectious diseases, this study suggested that microbial culture of CSF is not the most appropriate way for diagnosing central nervous system (CNS) infection. NTS should be recommended to be used in CSF for diagnosing CNS infection. When evaluating the value of NTS for diagnosis of CNS infections, the results of CSF TNC should be combined, and NTS-positive result is observed to be more reliable in patients with CSF TNC level >10 cells/μL.

Isolation, Identification and Screening of Potential Marine Actinomycete for Lipase Production under Submerged Fermentation by using Tributyrin substrate

Ninety-six isolates were collected at various depths and sea shores of the Bay of Bengal near Visakhapatnam Coastal of Andhra Pradesh. These isolates were screened for the isolation of lipase activity by tributyrin agar clearing method and cultured under submerged fermentation and assayed for extra cellular lipase production by using tributyrin as substrate. Cultural characteristics of the selected strain was done by incorporating different media. Various parameters namely morphological, biochemical, physiological and molecular were used for the characterization and identification of actinomycetes isolates. Results indicated that all the isolates showed lipolytic activity after primary screening. The marine isolate T9 was the most active one thus the same was selected for production of lipase. The 16S rDNA amplification for phylogenetic study revealed that the isolate was highly related to Nocardiopsis dassonvillei. In conclusion, these results increased the scope of finding industrially important marine actinomycetes from Visakhapatnam Coastal of Andhra Pradesh.

Verminous bronchitis and pneumonia by nasal trematodes in Greater Caribbean manatees from Puerto Rico.

Five adult Greater Caribbean manatees Trichechus manatus manatus were found stranded on various coasts of Puerto Rico; 2 stranded alive and 3 stranded dead. Clinical signs observed in live-stranded manatees included emaciation, weakness, bradypnea, arrhythmia, and nasal mucus discharge. Postmortem examinations revealed serosanguinous, mucohemorrhagic, or suppurative exudate in bronchi associated with luminal adult Pulmonicola cochleotrema (range: 18-182 trematodes), accompanied by pulmonary abscesses in 2 cases. Histologically, we observed eosinophilic bronchopneumonia of varying severity (n = 4) and chronic erosive to eosinophilic tracheobronchitis (n = 4) with squamous metaplasia (n = 3) and intralesional trematodes and eggs. The trematode identity was confirmed and compared through molecular analysis for the amplified 18S rDNA fragment. Comorbidities included enteric chiorchosis (n = 5), gastric heterocheilosis (n = 4), malnutrition (n = 4), trauma related to watercraft collision (n = 3), systemic toxoplasmosis (n = 1), acute bacterial peritonitis (n = 1), and interstitial nephritis (n = 1), suggesting that immunosuppression was a predisposing factor for lower respiratory tract pulmonicolosis. Based on lesion severity, clinical signs, and the presence and absence of other findings to explain death, this condition was considered the primary cause of death in 1 manatee, a contributory cause of death in 3 manatees, and an incidental finding in 1 individual. These clinicopathological descriptions will facilitate the diagnosis and clinical management of pulmonicolosis in T. manatus, a species endangered with extinction.

Salivary microbial diversity – an investigation on possible biomarkers for polycystic ovarian syndrome from eastern India

Introduction and aim. Polycystic ovarian syndrome (PCOS) is one of the major reproductive health issues, thought to be multifactorial, needs serious attention as a dual burden (health and economic) mainly for developing countries like India, due to its rapid rise (30%) in the last couple of years. Therefore, widespread and liberal screening for this disorder towards prognosis, diagnosis and intervention seems to be an urgent area of research. In this background, the present study attempts to unravel the association of salivary microbial diversity and PCOS. Material and methods. To achieve the purpose 100 clinically diagnosed PCOS individuals and 110 age matched non-PCOS participants from Bengalee Hindu caste population, West Bengal, India was considered. Obtained salivary samples were identified with 16S rDNA amplification and microbial diversity were determined by Alu I restriction enzyme digestion. Results. The present study revealed an explicit pattern of DNA fragment lengths varied between 200 bp and 225 bp in PCOs in comparison to the non-PCOS group. Conclusion. The cardinal feature of the present study as the first attempt from India envisaged, utilization of salivary microbial diversity as an additional potential and economizing biomarker for PCOS that stimulate new horizon of research in 21st century’s anthropology – the anthropology of microbes.

Desulfurization of coal using heterogeneous microbial species isolated from acid mine drainage, Talabira Coal Mines, Sambalpur, Odisha

Aim: The present study aims to assess the coal desulfurizing efficiency of heterogenous microbial consortia isolated from drainage water of Talabira Coal Mines, Sambalpur, Odisha. Methodology: Microbial species in the mine drainage water were isolated and cultured using 9K+ medium. Identification of microbial species was performed by the amplification of 16S rDNA. The heterogeneous microbial consortia were incubated with pulverized coal to assess their desulphurizing efficiency. Results: The proximate analysis of the coal sample revealed 34.92% fixed carbon, 9.01% moisture, 33.38% ash and 25.44% volatile matter. After incubation of the pulverized coal with isolated and cultured heterogeneous microbial species from MDW water of Talabira coal mines area, Odisha for 24 days in aerobic condition, the total sulphur content was reduced from 2.86% to 1.61%, while significant removal of pyritic Sulphur (48%) was obtained from 1.72% to 0.91%. However, no remarkable reduction was seen in the case of organic Sulphur. Interpretation: The results showed that drainage water of Talabira Mines inhabits numerous potential desulfurizing bacterial species that need further isolation, characterization, and process optimization to achieve optimal sulphur removal efficiency. Key words: Autotrophic bacteria, Coal, Microbial desulfurization

First Report of Pectobacterium versatile Causing Diseases on Potato in Xinjiang Uygur Autonomous Region and Heilongjiang Province, China.

Pectobacterium spp. and Dickeya spp. cause aerial stem rot on potatoes worldwide (Charkowski, 2018). Potato plants (cv. Xisen6# or Youjin) with aerial stem rot or blackleg symptoms (Fig. S1) were observed in the commercial fields in Changji, Xinjiang Province in September 2021 and Harbin, Heilongjiang Province in August 2021, in China. The field disease incidences were 45-50% and 15-20% in Changji (2 ha) and Harbin (1 ha), respectively. Five diseased plants from each field site were collected to isolate the pathogen. Symptomatic stems were soaked in 75% ethanol for 2 min, rinsed, and ground in sterile distilled water (Handique et al. 2022). The suspension was plated onto a crystal violet pectate agar (CVP) plate (Ge et al. 2018). Three days after incubation at 28°C, bacterial colonies that developed pits on CVP plates were purified and sequenced for identification using the universal 16S rRNA gene primer set 27F/1492R (Monciardini et al. 2002). Amplified 16S rDNA sequences from two isolates designated as ZRIMU1267 and ZRIMU1366 showed more than 99% sequence identity to P. versatile CFBP6051T type strain and the sequences were submitted to GenBank (accession numbers: OP476349, OP476350). Additionally, six housekeeping genes sequences were uploaded to GenBank: proA (OP487826, OP487832), gyrA (OP487828, OP487834), icdA (OP487823, OP487829), mdh (OP487825, OP487831), gapA (OP487824, OP487830), and rpoS (OP487827, OP487833) (Ma et al. 2007; Waleron et al. 2008). A phylogenetic tree based on concatenated sequences (MLSA) of the housekeeping genes (Fig. S2) of the two isolates was constructed using MEGA X (Tamura et al. 2013).The phylogenetic tree of MLSA sequences shows that the sequences from isolates ZRIMU1267 and ZRIMU1366 clustered with P. versatile CFBP6051T indicating that these isolates are P. versatile at the species level. Koch's postulate were performed on 3-week-old potato seedlings (cv. Favorita) and tubers. The bacterial suspension (100 μl, 105 CFU/ml) or sterile phosphate-buffered solution was injected into the crown area of the seedlings for the development of aerial stem rot or drenched in the potting mix for the development of blackleg, and the plants were covered with polybags to keep 100% humidity at 25° for 2 days. Five seedlings were inoculated for each strain and the experiment was repeated twice. Seven days after stem injection, the infected area of the inoculated seedlings was rotten, turned black, or even lodged, while the controls were symptomless (Fig. S3a). Four days after drench application, the seedlings were wilted and lodged, while the controls were symptomless (Fig. S3 c). Tuber slice assay for soft rot development was performed by adding bacterial suspension (100 μl, 105 CFU/ml). One day after inoculation, the infected tubers rotted, while the controls were symptomless (Fig. S3 b). ZRIMU1267 and ZRIMU1366 were reisolated from infected tissues on CVP plates and identified by 16S rRNA sequences to complete Koch's postulate. Diseases on potato has been reported to be caused by P. atrosepticum, P. carotovorum, P. brasiliense, P. parmentieri, P. polaris, and P. punjabense in China (Zhao et al. 2018; Cao et al. 2021; Wang et al. 2021; Handique et al. 2022). P. versatile causing aerial stem rot on potatoes have been reported in Hebei province (Han et al. 2022), while our study reports P. versatile strains that are able to cause multiple diseases on potatoes in Xinjiang Uygur Autonomous Region and Heilongjiang provinces of China. The results indicate that P. versatile might be widely distributed in northern China, and it is necessary to include cropping season and post-harvest strategies to control diseases caused by P. versatile.

First report of 'Candidatus Phytoplasma asteris' associated with yellowing, scorching and decline of almond trees in India.

The almond, a commercially important tree nut crop worldwide, is native to the Mediterranean region. Stone fruit trees are affected by at least 14 'Candidatus Phytoplasma' species globally, among which 'Candidatus Phytoplasma asteris' is one of the most widespread phytoplasma infecting Prunus dulcis, causing aster yellows disease. Recently, almond plantations of Nauni region were consistently affected by phytoplasma, as evidenced by visible symptoms, fluorescent microscopic studies and molecular characterization. During several surveys from May to September 2020-2022, almond aster yellows phytoplasma disease showing symptoms such as chlorosis, inward rolling, reddening, scorching and decline with an incidence as high as 40%. Leaf samples were collected from symptomatic almond trees and the presence of phytoplasma was confirmed through fluorescent microscopic studies by employing DAPI (4, 6-diamino-2-phenylindole) that showed distinctive light blue flourescent phytoplasma bodies in phloem sieve tube elements. The presence of phytoplasma in symptomatic almond trees was further confirmed using nested PCR with specific primer pairs followed by amplification of 16S rDNA and 16S-23S rDNA intergenic spacer (IS) fragments. Sequencing and BLAST analysis of expected amplicon of the 16S rDNA gene confirmed that the almond phytoplasma in Himachal Pradesh was identical to the aster yellows group phytoplasma. Phylogenetic analysis of 16S rDNA almond phytoplasma also grouped 'Prunus dulcis' aster yellows phytoplasma within 16SrI-B subgroup showed 94% nucleotide identity with 'Prunus dulcis' phytoplasma PAEs3 and 'Prunus dulcis' phytoplasma PAE28 from Iran. This research presents the first host report of 'Candidatus Phytoplasma asteris' infecting almonds in India, expanding the knowledge of the diversity and distribution of phytoplasma strains affecting almond trees globally.

Dynamics of Infections in Cattle and Rhipicephalus microplus: A Preliminary Study.

Tick-borne pathogens (TBPs) pose a significant threat to livestock, including bovine species. This study aimed to investigate TBPs in cattle and ticks across four sampling points, utilizing real-time microfluidic PCR. The results revealed that Rhipicephalus microplus ticks were found infesting all animals. Among the detected TBPs in cattle, Anaplasma marginale was the most frequently identified, often as a single infection, although mixed infections involving Rickettsia felis, uncharacterized Rickettsia sp., and Anaplasma sp. were also observed. In ticks, A. marginale was predominant, along with R. felis, Rickettsia sp., and Ehrlichia sp. It is noteworthy that although A. marginale consistently infected all cattle during various sampling times, this pathogen was not detected in all ticks. This suggests a complex dynamic of pathogen acquisition by ticks. A phylogenetic analysis focused on the identification of Anaplasma species using amplified 16S rDNA gene fragments revealed the presence of A. marginale and Anaplasma platys strains in bovines. These findings underscore the presence of multiple TBPs in both cattle and ticks, with A. marginale being the most prevalent. Understanding the dynamics and phylogenetics of TBPs is crucial for developing effective control strategies to mitigate tick-borne diseases in livestock.

Exploring the Milk Microbiota of Healthy and Mastitic Nili Ravi Buffalo Using 16S rRNA Gene Base Metagenomic Analysis.

The Nili Ravi, a buffalo breed from Pakistan, significantly contributes to the dairy industry. Mastitis is one of the major challenges affecting milk production in this breed. The objective of the current study was to identify the bacterial communities and diversity in healthy and mastitic milk of this breed. Milk samples (n = 14) were collected from Nili Ravi buffaloes with different udder health statuses, i.e., healthy (5), subclinical mastitis (4), and clinical mastitis (5). The DNAs were extracted, subjected to partial amplification of 16S rDNA (V3 and V4 regions), and sequenced using the Illumina platform. The results revealed variations in the bacterial communities in the milk of animals with different udder health statuses. Proteobacteria was the predominant phylum in the healthy group, while clinical and subclinical mastitis milk had a higher abundance of Firmicutes. Dominant bacterial genera in the healthy group were Streptococcus (11.60%), Herbaspirillum (7.65%), and Staphylococcus (4.70%), whereas the clinical mastitis group was dominated by Streptococcus (33.96%), Staphylococcus (7.87%), and Corynebacterium (2.68%), and the subclinical mastitis group was dominated by Bacillus (15.70%), Corynebacterium (6.70%), and Staphylococcus (6.58%). Assignment of operational taxonomic units at the species level resulted in most species being assigned to uncultured or unknown bacteria or remaining unassigned. Alpha diversity indices indicated lower microbial diversity in the clinical mastitis group, while beta diversity indices showed a scattered pattern of sample clustering in PCA plots among different groups. It is concluded that bacterial diversity in the milk of Nili Ravi buffaloes suffering from clinical mastitis is lower compared to healthy and subclinical mastitis cases. It is concluded that the variations in the microbiota of healthy and mastitic milk may be further investigated and exploited as signature microbes associated with the udder health status of Nili Ravi buffalo.

Germicidal effect of hydrogen peroxide nano-silver ion composite disinfectant and its effect on the microbial community of shrimp intestine and rearing water

In this study, the inhibitory effect of different doses of hydrogen peroxide nano-silver ion composite disinfectant (HPS) on DIV1 and bacteria was analyzed, and the antibacterial activity of three disinfectants, HPS, potassium monopersulfate (KMPS) and calcium hypochlorite (Ca(ClO)2), was compared. In addition, 16S rDNA amplification sequencing technology was used to analyze the effects of these three disinfectants on the intestinal microflora of Litopenaeus vannamei and the structure and composition of water microflora. The results showed that HPS did not positively affect the survival rate of prawns infected with DIV1, which needs to be verified in future studies. In the experimental design range, the higher the dosage of HPS, the more obvious the killing effect on the number of Vibrio and total bacteria in rearing water, and the two showed a negative correlation. 5,104 OTUs were obtained based on 16S rDNA high-throughput sequencing, of which 3,012 (59.01%) and 1,475 (28.90%) OUTs were annotated at the phylum and genus levels, respectively. Proteobacteria, Firmicutes, Actinobacteriota, Bacteroidetes, and Cyanobacteria dominated the water samples at the phylum level. At the genus level, the dominant bacterial genera in the intestinal bacterial community of shrimp were Photobacterium, Vibrio, and Ruegeria. The most dominant bacteria genera in water samples were Vibrio, Ruegeria, Pseudoalteromonas, and Nautella. In the water samples, the composition and structure of the Ca(ClO)2 microbial community were relatively simple, and the species richness and diversity of Ca(ClO)2 were significantly lower than those of HPS and KMPS disinfectant groups at 12 h and 24 h (p < 0.05). In terms of inhibiting the diversity and richness of the microbial community, Ca(ClO)2 had a significant effect (p < 0.05), but the intestinal microbial community diversity of shrimp treated with HPS was higher than that of the other two groups. It is worth noting that, compared with the three disinfectants, HPS has the strongest killing effect on Vibrio and total bacteria, and has a certain positive significance for maintaining the stable state of the microbial community. This study provides a scientific basis for applying HPS in aquaculture and broadens the application range of HPS.

Genetic and Physiological Diversity of Marine Actinobacteria from the Okha Coast, Gujarat, India

Saline and alkaline habitats of the Okha coastline, Gujarat, India, were explored for the diversity of actinobacteria. Based on the phenotypic and genotypic heterogeneity, nine haloalkaliphilic actinobacterial strains were characterized and identified. The PCR amplification of 16S rDNA using universal and genus-specific primers corroborated the trends that emerged through cultural and physiological characterization. Streptomyces was the most abundant genus amplified with the Streptomyces-specific primers (StreptB/E, StreptB/F), whereas other actinobacterial strains were amplified by the universal primers U1 and U2. Further molecular diversity was investigated by the gradient PCR-DGGE as a fingerprinting tool that generated group-specific DGGE patterns. Based on the nucleotide homology and phylogenetic analysis, strains OK-1 and OK-2 were identified as Streptomyces somaliensis. At the same time, OK-3 and OK-7 were detected as Streptomyces sp., while OK-5, OK-6, OK-8, OK-9, and OK-10 belonged to Nocardiopsis alba. Further, the cluster analysis using the UPGMA method generated 3 clusters based on biochemical characterization, sugar utilization, and enzyme production. The dendrogram based on the DGGE band pattern created with Jaccard-distance revealed two major clades with 33.33% similarity. Further, the study of alpha diversity calculation using phenotypic characteristics discloses highly diverse sugar utilization abilities. Moreover, a stress value of 0.1236 was obtained based on the NMDS analysis of the plots using Bary-Curties dissimilarity. Overall, the distinct phenotypic, metabolic, and molecular profiling illustrated the diversity among marine actinobacteria.

Screening of antibiotics to obtain axenic cell cultures of a marine microalga Chrysotila roscoffensis.

Due to high growth rate, outstanding abiotic stress tolerance, and rich value-added substances, Chrysotila roscoffensis, belonging to the phylum of Haptophyta, can be considered as a versatile resource for industrial exploitation of bioactive compounds. However, the application potential of C. roscoffensis has drawn attention until just recently, and the understanding related to the biological properties of this species is still scarce. For example, the sensitivities of C. roscoffensis to antibiotics, which is essential for the verification of heterotrophic capacity and the establishment of efficient genetic manipulation system is still unavailable. Aiming to provide fundamental information for future exploitation, the sensitivities of C. roscoffensis to nine types of antibiotics were tested in this study. The results demonstrated that C. roscoffensis exhibited relatively high resistances to ampicillin, kanamycin, streptomycin, gentamicin, and geneticin, while was sensitive to bleomycin, hygromycin B, paromomycin, and chloramphenicol. Using the former five types of antibiotics, a bacteria removal strategy was established tentatively. Finally, the axenicity of treated C. roscoffensis was confirmed based on a multi-strategy method including solid plate, 16S rDNA amplification, and nuclear acid staining. This report can provide valuable information for the development of optimal selection markers, which are meaningful for more extensive transgenic studies in C. roscoffensis. Moreover, our study also paves the way for the establishment of heterotrophic/mixotrophic cultivation modes of C. roscoffensis.