Culturable soil microbial biodiversity in post-mining areas of the Konin lignite mine and its potential for ecosystem restoration

Traditional morphology-based ciliate classification is often time-consuming and inaccurate, necessitating molecular approaches. Although 18S rRNA gene sequencing is widely used for taxonomic analyses of ciliates, its high degree of conservation makes it challenging to achieve species-level resolution. This study explores the potential of internal transcribed spacers (ITS1 and ITS2) and the 28S rRNA gene to improve taxonomic resolution beyond that offered by 18S rRNA gene in free-living and host-associated ciliates. A comparative analysis of ITS, the 18S, and 28S rRNA gene sequences retrieved from public databases indicated that ITS regions exhibit greater inter- and intra-specific sequence dissimilarity compared to 18S rRNA gene, supporting existing literature. We then designed universal primers targeting the ITS and 28S rRNA gene for freshwater and rumen ciliates. These primers were rigorously evaluated for their inclusiveness, specificity, and amplification efficiency using in-silico PCR, experimental PCR, followed by sequencing and metataxonomic analyses of the ciliate communities. In-silico analyses revealed inclusiveness exceeding 80%, while experimental analyses validated their specificity. Metataxonomic analyses of ciliates demonstrated that the ITS and 28S rRNA gene captured significantly greater taxonomic diversity than 18S rRNA gene. Also, ITS1 offered superior taxonomic resolution by detecting the most ciliate species that went unnoticed by the 18S rRNA gene. These findings underscore the superiority of ITS1, and to a lesser extent ITS2, as taxonomic markers for enhancing the resolution of freshwater and rumen ciliate communities. We recommend ITS1 as an alternative marker to overcome the limitations of 18S rRNA gene-based approaches in free-living and host-associated ciliate taxonomy.

ABSTRACTThe present study aimed to investigate microbial communities in seven Indian composts and their potential for biocontrol of Fusarium oxysporum f. sp. lycopersici. In addition, identification of bioactive substances in disease suppressive composts was also attempted. Composts were chosen based on disease suppressiveness and subjected to molecular microbial analyses. Total genomic DNA from the composts was extracted and amplified with polymerase chain reaction using primers targeting the 18S rRNA and 16S rRNA genes of fungi and bacteria, respectively. Denaturing gradient gel electrophoresis (DGGE) fingerprinting and DNA sequencing were used to identify the fungal and bacterial targets. Phylogenetic analysis of the fungal 18S rRNA ITS gene sequences showed that phylum Ascomycota was dominant in all composts, while in the bacterial 16S rRNA gene sequences, the phylum Proteobacteria was dominant. Some fungi in disease suppressive composts grouped phylogenetically close to F. oxysporum. Bacterial sequences with close similarity (>95% identity) with Actinobacterium showed a strong presence only in disease suppressive composts. Disease suppressive composts formed a separate group in the cluster analysis of 18S rRNA ITS and 16S rRNA gene sequences. Gas chromatography-time of flight-mass spectrometry was performed with compost extracts to determine if bioactive substances were present in disease suppressive composts. The analysis of compost organic matter showed a negative association of disease suppressiveness with phloroglucinol, sitosterol, and monoenoic fatty acid, while cholesterol and certain organic acids were positively associated with suppressiveness.

Ginseng (Panax ginseng) is an economically valuable medicinal herb mainly planted in Jilin Province, China. In September 2013, during harvest, suspected bacterial rots were observed on ginseng roots with about 10% incidence in Fusong County, Jilin Province, China (127°29.48' N, 42°11.12' E). Rotted roots completely lost their economic value. Symptoms on roots began as water-soaked lesions, and developed rapidly into a soft, watery, decayed mass within 3 to 5 days. Three diseased root tissues were surface-sterilized in 70% ethanol for 30 s, rinsed 3 times in sterilized water and cut into small pieces (2 to 3 mm). Tissues were then macerated for 5 min in sterilized water, streaked onto nutrient agar (NA) medium, and incubated at 28°C for 2 days. Representative colonies were selected from each plate and further purified by sub-culturing onto NA medium. Five strains of the bacteria were gram-negative, short straight rods, 0.5 to 1.0 × 1.5 to 3.0 μm with a single, polar flagellum. Colonies were round, smooth, translucent, and yellowish green on NA medium. The bacteria were identified based on physiological and biochemical tests as follows (3): They were levan and potato rot negative, oxidase, aerobic, and arginine dihydrolase positive, converted nitrate to N2, hydrolyzed gelatin, produced nitrites from nitrates, produced pyocyanin, and grew at 41°C. Bacterial identity was further confirmed by amplifying the 16S rRNA (1,461 bp), gyrB (1,134 bp), and 16S-23S ITS genes (523 bp) with 27F/1492R, UP1/UP2, and L1/L2 primer sets, respectively. The 16S rRNA gene sequence (NCBI Accession No. KJ156527), gyrB gene sequence (KJ748373), and 16S-23S ITS gene sequence (KJ748374) had 99% identity to that of Pseudomons aeruginosa strain BS01 (JQ229778), ATCC25011 (FJ652721), and ATCC15522 (AB547908), respectively. The strains were also identified by using BD Phoenix-100 Automated Microbiology System (BD Ltd., New Jersey) as P. aeruginosa with 99% confidence. A pathogenicity test was conducted by spraying a suspension of five strains individually (108 CFU/ml) onto 4-year-old ginseng roots (cv. Damaya) wounded with a sterilized needle. Five ginseng roots were inoculated with each strain and five ginseng roots were inoculated with sterilized water as controls. All inoculated plants were maintained at 28°C with 80 to 85% relative humidity. Soft, watery tissue rot symptoms developed 3 to 5 days after inoculation, and were similar to those observed on the diseased plants under natural conditions. In contrast, no symptoms developed on control plants. The bacteria were readily re-isolated from inoculated plants and identified as P. aeruginosa using bacterial colony morphology, physiological and biochemical tests, as well as sequence analysis of the 16S rRNA gene, fulfiling Koch's postulates. The bacterium was not isolated from control plants. P. aeruginosa has been reported to cause diseases in a variety of plants including onion (1,2), arabidopsis, and sweet basil (4). To our knowledge, this is the first report of P. aeruginosa causing ginseng root rot in China.

Raillietina echinobothrida (R. echinobothrida) is one of the most pathogenic and prevalent tapeworms threat to the commercial chickens in China. However, there is a lack of research on their molecular identification and morphological characteristics. This study explored the molecular identification markers for R. echinobothrida in North China based on 18s ribosomal RNA (18s rRNA) gene and the ribosomal DNA second internal transcribed spacer (ITS-2) gene. The BLAST results of 18s rRNA (1643 bp) and ITS-2 (564 bp) gene sequences showed that the isolated intestinal tapeworms were R. echinobothrida. Phylogenetic trees obtained by maximum likelihood (ML) or neighbor-joining (NJ) method revealed that the R. echinobothrida in North China had the closest evolutionary relationship with the species found on the Qinghai-Tibet plateau, China. Morphological observations by hematoxylin staining and scanning electron microscope showed four round suckers and a retractable rostellum on the spherical scolex of R. echinobothrida. Two rows of alternately arranged hooks distributed around the rostellum. There were 30-40 testes in each mature segment. A well-developed cirrus pouch lied outside the excretory duct of mature segment. The gravid segment contained 200-400 eggs and there was a well-developed oncosphere in each egg. In addition, abundant ultrastructural features in mature proglottid of R. echinobothrida in North China were identified by transmission electron microscopy. In conclusion, the present study established ways of molecular phylogenetic identification for R. echinobothrida based on 18s rRNA and ITS-2 gene, and identified the morphological and ultrastructural characteristics of R. echinobothrida in North China.

Genetic Diversity and Phylogenetic Relationships between Birches and Alders Using ITS, 18S rRNA, and rbcL Gene Sequences

Rapid and reasonable molecular identification of bacteria and fungi in microbiological diagnostics using rapid real-time PCR and Sanger sequencing

ObjectiveThe purpose of this systematic bioinformatics analysis was to describe the compositions and differences in submucosal microbial profiles of peri-implants’ diseases and healthy implant.Material and methodsPubMed, Embase, ETH Z, Scopus, CNKI, and Wanfang databases were searched to screen relevant literature on the analysis of peri-implant microflora based on the sequencing analysis technique of 16S ribosomal RNA (16S rRNA) gene. High-throughput sequencing of the 16S rRNA gene of microorganisms from healthy implants, peri-implant mucositis, and peri-implantitis was downloaded from the screened articles. EasyAmplicon and Usearch global algorithm were used to match the reads from each dataset to a full length of 16S rRNA or ITS gene sequence. The microorganisms based on the Human Oral Microbiome Database (HOMD) were re-classified, and the microbial diversity, flora composition, and differential species of the samples were re-analyzed, including taxonomic classification and alpha and beta diversity calculations. The co-occurrence network was also re-analyzed.ResultsA total of seven articles with 240 implants were included. Among them, 51 were healthy implants (HI), 43 were in the peri-implant mucositis (PM) group, and 146 were in the peri-implantitis (PI) group. A total of 26,483 OTUs were obtained, and 877 microorganisms were annotated. The alpha diversity including Chao1 (healthy implants, 121.04 ± 92.76; peri-implant mucositis, 128.21 ± 66.77; peri-implantitis, 131.15 ± 84.69) and Shannon (healthy implants, 3.25 ± 0.65; peri-implant mucositis, 3.73 ± 0.61; peri-implantitis, 3.53 ± 0.67) of the samples from the three groups showed a significant difference. The beta diversity of the three samples was statistically different among groups. The genera of Treponema and Fretibacterium were significantly more abundant in the PI group than in the other two groups, and the genus of Streptococcus was more abundant in the HI group. The relative abundance of Porphyromonas in the peri-implantitis group was 6.1%. The results of the co-occurrence network showed differences in the network topology among the three groups of samples. The most connected three genera in the healthy implants were Halomonas, Fusobacterium, and Fretibacterium. The most connected three genera in peri-implant mucositis were Alistipes, Clostridia UCG-014, and Candidatus Saccharimonas. The most connected three genera in the peri-implantitis group were Lachnoanaerobaculum, Fusobacterium, and Atopobium. The betweenness of Porphvromonas gingivalis (red complex) in the PI group (7,900) was higher than in the HI group (23).ConclusionsThe community compositions of peri-implant submucosal microorganisms were significantly different in healthy implants, peri-implant mucositis, and peri-implantitis. The submucosal microbial communities in peri-implantitis were characterized by high species richness and diversity compared with the healthy implants; the relative abundance of red complex, some members of the yellow complex, and some novel periodontal pathogens was higher in the peri-implantitis and peri-implant mucositis groups than in the healthy implant group. The core flora of the co-occurrence network of healthy implants, peri-implant mucositis, and peri-implantitis varied considerably. The peri-implantitis site presented a relative disequilibrium microbial community, and Porphyromonas may play an important role in the co-occurrence network.

To identify the species of trematodes isolated from laying ducks in Nanchang City using morphological and molecular approaches. Trematodes were isolated from the hepatobiliary duct, gallbladder and large intestine of market-sold laying ducks in Nanchang City. Following morphological characterization, total DNA was extracted from all trematode specimens, and internal transcribed spacer region (ITS) and cytochrome C oxidase subunit 1 (Cox1) genes were amplified using PCR assay and sequenced. Sequence alignment was performed using the Blast software, and homology and phylogenetic analyses were done in the trematode isolates based on ITS and Cox1 gene sequences. The morphological characteristics of two trematode isolates from the large intestine of laying ducks were similar to those of Echinostoma revolutum and E. miyagawai, and the morphological characteristics of eight trematode samples isolated from the hepatobiliary duct and gallbladder of laying ducks were similar to those of Amphimerus anatis. The ITS and Cox1 gene sequences of the two trematode isolates from the large intestine of laying ducks had 99.3% and 98.9%-99.4% homology with E. miyagawai, and the phylogenetic analysis showed that two trematode isolates had the closest genetic relationship with E. miyagawai based on ITS and Cox1 gene sequences. The ITS gene sequences of eight trematode isolates from the hepatobiliary duct and gallbladder of laying ducks shared 95.1%-95.5% with Opisthorchis sudarikovi and Clonorchis sinensis, while the Cox1 gene sequences of eight trematode isolates from the hepatobiliary duct and gallbladder of laying ducks shared 86.3%-86.4% and 85.5%-85.7% with O. viverrini and O. sudarikovi. ITS gene sequence-based phylogenetic analysis showed that the duck-derived trematode isolates had the closest genetic relationship with C. sinensis, and Cox1 gene sequence-based phylogenetic analysis showed that the duck-derived trematode isolates had the closest genetic relationship with Metorchis orientalis and O. viverrini. The trematode isolates from the large intestine of laying ducts in Nanchang City may be E. miyagawai, and the trematode isolates from the hepatobiliary duct and gallbladder may be an unidentified trematode species of the family Opisthorchiidae.

First Report in China on the Identification and Drug Sensitivity of Mycobacterium elephantis Isolated from the Milk of a Cow with Mastitis

In this study, we attempted species-level identification of frog specimen collected from Faridpur district of Bangladesh beyond it’s location outside Orissa, India. Specimen was identified morphologically at genus level as Fejervarya sp. belonging to the family Dicroglossidae using finger formula F3>F4>F1=F1 where F denote as toe finger. From the study two nucleotide sequences of 16S and 12S rRNA genes were obtained which contained 508bp and 408bp respectively. The Sequences were submitted to Gene Bank database with the accession number OQ231604 and OQ240197 for 16S and 12S rRNA gene sequences. Furthermore, the 16S rRNA gene sequence was used as molecular bar-code for the identified Orissa frog F. orissaensis species from Bangladesh. GC content of partial 12S and 16S rRNA genes have been calculated as 44% and 45% respectively. For 16S rRNA gene sequence there was no intra specific divergence. Whereas the inter specific polymorphic divergence were calculated 4.13% and 6.3% when the collected Orissa frog F. orissaensis was compared with that of F. iskandari and F. kupitzi, respectively. In case of 12S rRNA gene intra specific divergence was found 2.45% where the inter specific divergence were 4.41% and 6.86% when the collected Orissa frog F. orissaensis was compared with that of F. iskandari and F. limnocaris, respectively. Maximum likelihood tree also indicates that our sample Orissa frog formed a monophyletic group with F. orissaensis in both the cases of 16S and 12S rRNA genes and thus can be concluded as closely related. Therefore, the collected specimen was identified to be belonging to Fejervarya orissaensis which would be first report from Bangladesh outside Orissa, India. Bangladesh J. Zool. 51(3): 301-313, 2023

Kyoung-Bo Kim, M.D., Sung-Gyun Park, M.D., Jae-Seok Park, M.D., Wonmok Lee, M.D., Jung-Sook Ha, M.D., Nam-Hee Ryoo, M.D., Dong-Seok Jeon, M.D., and Jae-Ryong Kim, M.D.. Ann Lab Med 2015;35:379-81. https://doi.org/10.3343/alm.2015.35.3.379

Cycads have developed a complex root system categorized either as normal or coralloid roots. Past literatures revealed that a great diversity of key microbes is associated with these roots. This recent study aims to comprehensively determine the diversity and community structure of bacteria and fungi associated with the roots of two Cycas spp. endemic to China, Cycas debaoensis Zhong & Chen and Cycas fairylakea D.Y. Wang using high-throughput amplicon sequencing of the full-length 16S rRNA (V1-V9 hypervariable) and short fragment ITS region. The total DNA from 12 root samples were extracted, amplified, sequenced, and analyzed. Resulting sequences were clustered into 61 bacteria and 2128 fungal OTUs. Analysis of community structure revealed that the coralloid roots were dominated mostly by the nitrogen-fixer Nostocaceae but also contain other non-diazotrophic bacteria. The sequencing of entire 16S rRNA gene identified four different strains of cyanobacteria under the heterocystous genera Nostoc and Desmonostoc. Meanwhile, the top bacterial families in normal roots were Xanthobacteraceae, Burkholderiaceae, and Bacillaceae. Moreover, a diverse fungal community was also found in the roots of cycads and the predominating families were Ophiocordycipitaceae, Nectriaceae, Bionectriaceae, and Trichocomaceae. Our results demonstrated that bacterial diversity in normal roots of C. fairylakea is higher in richness and abundance than C. debaoensis. On the other hand, a slight difference, albeit insignificant, was noted for the diversity of fungi among root types and host species as the number of shared taxa is relatively high (67%). Our results suggested that diverse microbes are present in roots of cycads which potentially interact together to support cycads survival. Our study provided additional knowledge on the microbial diversity and composition in cycads and thus expanding our current knowledge on cycad-microbe association. Our study also considered the possible impact of ex situ conservation on cyanobiont community of cycads.

ObjectivesThis study examined feline haemoplasmas (Mycoplasma haemofelis, 'Candidatus Mycoplasma haemominutum' [CMhm] and 'Candidatus Mycoplasma turicensis') infecting Thai domestic cats, using the 16S and 23S rRNA genes as genetic markers.MethodsBlood samples from 20 cats were obtained from a diagnostic laboratory and nucleic acids were extracted from each sample using a commercial kit. PCR targeting the 16S rRNA gene was used to screen haemoplasmas in the samples. Positive PCR samples were further sequenced using the 16S and 23S rRNA genes. The sequences from each genetic marker were analysed using Nucleotide BLAST, phylogeny and genetic network analyses.ResultsAmong the 20 samples, five were infected with haemoplasmas. In the 16S rRNA gene sequencing, four sequences were assigned to CMhm and the remaining sequence was likely to be a closely related species of CMhm. In the 23S rRNA gene sequencing, four sequences from the same samples used for 16S rRNA gene sequencing were identified as CMhm and one sequence could be a putative novel haemoplasma species closely related to CMhm.Conclusions and relevanceOnly CMhm and its closely related species were identified in this study. Although CMhm has been recognised as a low-virulence parasite, cases of severe anaemia in cats infected with CMhm have been found. Thus, such cases could be confirmed via the analysis of 16S and 23S rRNA genes. Furthermore, molecular detection and genetic analyses of feline haemoplasmas in additional cat blood samples should be conducted using PCR assay and DNA sequencing based on universal primers of 16S rRNA and 23S rRNA genes to enable more specific identification.

The current classification of non-pigmented and late-pigmenting rapidly growing mycobacteria (RGM) capable of producing disease in humans and animals consists primarily of three groups, the Mycobacterium fortuitum group, the Mycobacterium chelonae-abscessus group and the Mycobacterium smegmatis group. Since 1995, eight emerging species have been tentatively assigned to these groups on the basis of their phenotypic characters and 16S rRNA gene sequence, resulting in confusing taxonomy. In order to assess further taxonomic relationships among RGM, complete sequences of the 16S rRNA gene (1483-1489 bp), rpoB (3486-3495 bp) and recA (1041-1056 bp) and partial sequences of hsp65 (420 bp) and sodA (441 bp) were determined in 19 species of RGM. Phylogenetic trees based upon each gene sequence, those based on the combined dataset of the five gene sequences and one based on the combined dataset of the rpoB and recA gene sequences were then compared using the neighbour-joining, maximum-parsimony and maximum-likelihood methods after using the incongruence length difference test. Combined datasets of the five gene sequences comprising nearly 7000 bp and of the rpoB+recA gene sequences comprising nearly 4600 bp distinguished six phylogenetic groups, the M. chelonae-abscessus group, the Mycobacterium mucogenicum group, the M. fortuitum group, the Mycobacterium mageritense group, the Mycobacterium wolinskyi group and the M. smegmatis group, respectively comprising four, three, eight, one, one and two species. The two protein-encoding genes rpoB and recA improved meaningfully the bootstrap values at the nodes of the different groups. The species M. mucogenicum, M. mageritense and M. wolinskyi formed new groups separated from the M. chelonae-abscessus, M. fortuitum and M. smegmatis groups, respectively. The M. mucogenicum group was well delineated, in contrast to the M. mageritense and M. wolinskyi groups. For phylogenetic organizations derived from the hsp65 and sodA gene sequences, the bootstrap values at the nodes of a few clusters were <70 %. In contrast, phylogenetic organizations obtained from the 16S rRNA, rpoB and recA genes were globally similar to that inferred from combined datasets, indicating that the rpoB and recA genes appeared to be useful tools in addition to the 16S rRNA gene for the investigation of evolutionary relationships among RGM species. Moreover, rpoB gene sequence analysis yielded bootstrap values higher than those observed with recA and 16S rRNA genes. Also, molecular signatures in the rpoB and 16S rRNA genes of the M. mucogenicum group showed that it was a sister group of the M. chelonae-abscessus group. In this group, M. mucogenicum ATCC 49650(T) was clearly distinguished from M. mucogenicum ATCC 49649 with regard to analysis of the five gene sequences. This was in agreement with phenotypic and biochemical characteristics and suggested that these strains are representatives of two closely related, albeit distinct species.

BackgroundApples are important for human nutrition because these provide vital nutrients, including vitamins and minerals, that are needed for a balanced diet. A suitable environment for the growth and survival of various microorganisms is also provided by multiple nutrients, such as carbohydrates, minerals, vitamins, and amino acids. Penicillium spp. are the cause of blue mold, one of the most common postharvest apple fruit diseases. Using morphological description and ITS gene sequencing, the current study aimed to identify Penicillium isolates associated with infected apple. High-performance liquid chromatography (HPLC) was used to demonstrate these isolates' capability for producing mycotoxins.ResultsThe initial identification of fungi was based on micromorphological characteristics, growth texture, and colony patterns. Penicillium (35.01%) and Talaromyces (15.62%) were the most prevalent fungal genera. These isolates' morphological identity was validated by ITS gene amplification and sequencing. The generated fragments' ITS gene sequencing values were 477, 480, 510, 478, and 478 bps for Penicillium expansum strain AP1, Penicillium crustosum strain AP2, Talaromyces atroroseus strain AP3, Penicillium expansum strain AP4, and Penicillium expansum strain AP5, respectively. It was examined whether the five isolates could produce citrinin and patulin. The highest value of toxins was recorded for patulin (24180 ppb), that produced by P. expansum AP5, followed by P. crustosum AP2 (19360 ppb). However, the highest value for citrinin was 24890 ppb detected for P. expansum AP4, followed by P. expansum AP5 (19320 ppb).ConclusionBlue mold caused by Penicillium expansum, Penicillium crustosum, and Talaromyces atroroseus. These isolates have the ability to produce citrinin and patulin with different degrees. So blue mold is one of the most harmful diseases in post-harvest apple fruits.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-025-07205-2.