Gene Phylogenetic Tree Research Articles

Genomics and Phylogeny of Rhodotorula glutinis and Rhodotorula kratochvilovae Isolated from the Northern Peruvian Andes

Genomes of oleaginous yeast strains Rhodotorula glutinis CON-5 and Rhodotorula kratochvilovae POR-3, isolated from areas in the northern Peruvian Andes using SPAdes, were sequenced and assembled applying Illumina and de novo. Genomes of 20,515,696 and 20,738,185 bp, respectively, were determined. From the structural and functional annotations, the Basidiomycota phylum showed a similarity of 76.8% and 86.5% with 6,976 and 8,124 pairs of proteins in both yeasts respectively, with homologues in the UniProt data bank. Using OrthoVenn, a relationship between both yeasts was obtained from 450 orthologous groups. Likewise, the above-mentioned yeasts and R. toruloides (oleaginous Basidiomycota) showed 1,574 orthologous groups, indicating a good relationship. Construction of phylogenetic trees of genes encoding metabolic enzymes was also carried out, based on the ITS sequences which showed that CON-5 and POR-3 have a greater relationship with R. graminis. Their phylogenetic relationship was ascertained and determined that the enzymes involved in the metabolism of CON-5 and POR-3 are related to each other. It was also found that the protein sequences of the Basidiomycota phylum differ from Ascomycota. The study showed functional evidence regarding the lipid accumulation phenotype, an important aspect in the context of obtaining lipids or oleochemicals.

Species delineation and hybrid identification using diagnostic nuclear markers for Mediterranean groupers Epinephelus marginatus and Mycteroperca rubra

Globally, groupers (Serranidae) are commercially important fish species. Hybridization within this family has been reported in captivity and natural environments, usually among congeneric species. However, intergeneric hybridization has been reported only occasionally. Given the commercial interest in these fish, artisanal or recreational fishermen easily notice “weird-looking” individuals. This is the case of an unidentified grouper noticed by a spearfisher in Lebanese Mediterranean waters. Visual comparisons raised the possibility that the observed specimen was a hybrid. Molecular analysis based on two mitochondrial and three nuclear markers was performed to investigate this potential hybrid’s affinity. The discordance of the phylogenetic gene trees of the mitochondrial and nuclear markers indicates that E. marginatus and M. rubra are potential parent species of a hybrid individual in our samples. The subsequent haploweb analysis based on the S7 nuclear marker reveals the affinity of the hybrid individual to both E. marginatus and M. rubra. In this specific hybridization case, the nuclear marker S7 emerges as a valuable diagnostic tool for species delineation and hybrid detection.

Phylogenetic and Expression Analysis of the Sucrose Synthase and Sucrose Phosphate Synthase Gene Family in Potatoes.

Sucrose synthase (SUS) and sucrose phosphate synthase (SPS) are essential in plant sucrose metabolism. The potato is an important crop worldwide, but systematic analyses of the StSUS and StSPS gene families in potatoes are still lacking. Ten sucrose metabolism-related genes were identified in this study. The SUSs and SPSs could each be split into three subgroups through phylogenetic analysis. StSUSIc was the most highly expressed gene in different developmental tissues. Ka/Ks analysis showed that StSUSIb and StSUSIc were subjected to more-significant homozygous selection pressure. Our cis-acting element analysis of the StSUS and StSPS promoter sequences showed four elements: defense- and stress-responsive, hormone-responsive, light-responsive, and transcription factor elements. The expression of StSUS and StSPS genes was found to be regulated by circadian rhythm. In the treatments of 1% to 5% sucrose, glucose, and fructose, the expression of StSUS and StSPS family genes was enhanced by sucrose, but inhibited at high-glucose and fructose concentrations. This study identified six StSUS and four StSPS genes and analyzed their gene structure, conserved motifs, chromosome position, promoter elements, phylogenetic tree, and tissue-specific expression patterns. Our results will motivate more research into the biological process underlying the genes of sucrose metabolism in potatoes.

Comamonas endophytica sp. nov., a novel indole acetic acid producing endophyte isolated from bamboo in China.

Two novel indole acetic acid-producing strains, 5MLIRT and D4N7, were isolated from Indosasa shibataeoides in Yongzhou, Hunan province, and Phyllostachys edulis in Hangzhou, Zhejiang province, respectively. Based on their 16S rRNA sequences, strains 5MLIRT and D4N7 were closely related to Comamonas antarcticus 16-35-5T (98.4 % sequence similarity), and the results of 92-core gene phylogenetic trees showed that strains 5MLIRT and D4N7 formed a phylogenetic lineage within the clade comprising Comamonas species. The complete genome size of strain 5MLIRT was 4.49 Mb including two plasmids, and the DNA G+C content was 66.5 mol%. The draft genome of strain D4N7 was 4.26 Mb with 66.7 mol% G+C content. The average nucleotide identity and digital DNA-DNA hybridization values among strain 5MLIRT and species in the genus Comamonas were all below the species delineation threshold. The colonies of strain 5MLIRT and D4N7 were circular with regular margins, convex, pale yellow and 1.0-2.0 mm in diameter when incubated at 30 °C for 3 days. Strains 5MLIRT and D4N7 grew optimally at 30 °C, pH 7.0 and 1.0 % NaCl. The respiratory isoprenoid quinone was ubiquinone-8. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Polyphasic analyses indicated that strains 5MLIRT and D4N7 could be distinguished from related validly named Comamonas species and represent a novel species of the genus Comamonas, for which the name Comamonas endophytica sp. nov. is proposed. The type strain is 5MLIRT (=ACCC 62069T=GDMCC 1.2958T=JCM 35331T).

Serological, Morphological and Molecular Diagnosis of Mycobacterium bovis in Cattle at Sulaimani Province of Iraq

Background:Mycobacterium bovis (M. bovis) causes bovine tuberculosis (BTB), is an endemic disease in cattle and poses a high risk of spreading to humans.Objective:This study aimed to determine M. bovis in cattle and assess the similarities between cattle and humans through molecular methods and histopathological examinations.Methodology:Randomly, blood samples from 411 healthy appearance cows (1% of the target population) in five districts in Sulaimani province, Iraq, were collected from January to March 2022. Sera were obtained immediately and used for ELISA test to determine M. bovis. Additionally, the disease prevalence was confirmed by gross lesions at the slaughterhouse and histopathological examination of collected lymph nodes. Moreover, a PCR assay was used to detect M. bovis in suspected cow samples and previously diagnosed human samples. Gene sequencing and phylogenetic tree analysis were also done to determine the molecular differences between animal and human M. bovis.Results:Using an ELISA test, 46 (11.11%) of 414 samples were positive, while 368 (88.89%) were negative without significant differences between the districts (p>0.05). According to postmortem lesions at the slaughterhouse, only three cows were infected with TB, and typical gross lesions were calcified necrotic and multiple well-demarcated granulomas. The molecular test using two primers (CSB2 and oxyR gene) revealed that M. bovis was found in animal and human extra-pulmonary lymph nodes with no molecular change.Conclusion:Healthy cows harbored M. bovis, the causative agent of a contagious disease that spreads and causes a persistent health problem in humans.

New sand fly (Diptera, Psychodidae) records and COI DNA barcodes in the state of Maranhão, Eastern Amazon, Brazil

The sand fly fauna and the usefulness of the DNA barcoding fragment of the cytochrome c oxidase subunit I (COI) gene were accessed in a forest fragment in the municipality of Governador Newton Bello, state of Maranhão, Brazil. We performed entomological collections in three independent campaigns in May and October 2021, and January 2023. Sand flies were morphologically-identified and then DNA barcoded. Sequences were deposited and analyzed in the BOLD System Database, and various species delimitation algorithms, to assess whether DNA sequences merge into taxonomic units in accordance with nominal species. In total, 1,524 sand flies were collected, comprising 32 nominal species. Nyssomyia antunesi was the most abundant species (31.5 %), followed by Psychodopygus davisi (27 %). We reported for the first time in the state of Maranhão, the presence of Lutzomyia evangelistai, Lutzomyia sherlocki, Pressatia equatorialis, and Psathyromyia barrettoi. We amplified and analyzed 67 COI barcodes of 23 species, which were merged with conspecific sequences extracted from GenBank. The maximum intraspecific p distances ranged from 0.0 % to 14.74 %, while the distances to the nearest neighbor varied from 1.67 % to 13.64 %. The phylogenetic gene tree and species delimitation tools clustered sequences into well-supported clades/clusters for each nominal species, except for Pressatia choti/Pr. equatorialis, which have the lowest interspecific genetic distance (1.67 %). We sequenced for the first time COI barcodes of Brumptomyia brumpti, Evandromyia monstruosa, Micropygomyia rorotaensis, Micropygomyia pilosa, Pintomyia christenseni, Pintomyia pacae, Pr. equatorialis, Pa. barrettoi, and Psathyromyia hermanlenti, which will be useful for further molecular identification and classification proposals of Neotropical species. This study updated the current list of the sand fly fauna for the state of Maranhão to 97, and demonstrated that COI barcodes are useful for specific identification.

Functional Identification of Olfactory Receptors of Cnaphalocrocis medinalis (Lepidoptera: Crambidae) for Plant Odor.

Cnaphalocrocis medinalis (Lepidoptera: Crambidae) is a migratory insect pest on rice crops. The migratory C. medinalis population in a particular location may be immigrants, local populations, emigrants, or a mix of these. Immigrants are strongly attracted to plant odor. We conducted research to identify the olfactory receptors in a floral scent mixture that is strongly attractive to C. medinalis. Through gene cloning, 12 olfactory receptor (OR) genes were amplified and expressed in Xenopus oocytes in vitro, and three of them were found to be responsive to plant foliar and floral volatiles. These were CmedOR31, a specific receptor for geraniol; CmedOR32, a broad-spectrum OR gene that responded to both foliar and floral odors; and CmedOR1, which strongly responded to 10-4 M phenylacetaldehyde. The electrophysiological response to phenylacetaldehyde was extremely high, with a current of 3200 ± 86 nA and an extremely high sensitivity. We compared the phylogenetic tree and sequence similarity of CmedOR genes and found that CmedOR1 belonged to a uniquely conserved OR pedigree in the evolution of Glossata species, and the ORs of this pedigree strongly responded to phenylacetaldehyde. The expression of OR1 was significantly higher in the females than in the males. Localization of CmedOR1 in the antennae of C. medinalis by fluorescence in situ hybridization showed that CmedOR1 was expressed in both males and females. CmedOR1 may be an odor receptor used by females to locate food sources. The function of these ORs and their role in pest monitoring were discussed.

First record of Ixodes (Scaphixodes) caledonicus in the Carpathian Basin and first time molecular-phylogenetic analysis of this tick species with updated host records and geographical range

Four Ixodes species represent the subgenus Scaphixodes Schulze, 1941 in Europe, but none of them were reported to be compared in a molecular-phylogenetic context. This study compensates for this lack of data. A tick larva, morphologically identified as Ixodes (Scaphixodes) caledonicus Nuttall, 1910, was collected from an Alpine swift (Tachymarptis melba) during its nesting period in Transylvania, Romania. Following DNA extraction, PCR analyses and sequencing in part with newly designed primers, three genetic markers of this specimen were amplified and compared to GenBank data, and two were analyzed phylogenetically. Based on sequence comparisons of its mitochondrial cytochrome c oxidase subunit I (cox1) and nuclear 28S rRNA genes I. caledonicus appeared to be closely related to members of the subgenus Pholeoixodes. However, the topology of the concatenated cox1 and 16S rRNA gene phylogenetic tree clearly showed its clustering with Ixodes (Scaphixodes) philipi. In conclusion, I. caledonicus is part of the tick fauna of Romania and is expected to occur also in other countries of the Carpathian Basin where rocky cliffs are available for nesting of swifts and other birds. This is the first species of the subgenus Scaphixodes in Europe, for which the traditional (morphology-based) taxonomic assignment is confirmed by molecular-phylogenetic analyses.

Genome-wide identification of CaARR-Bs transcription factor gene family in pepper and their expression patterns under salinity stress

In plants, ARRs-B transcription factors play a crucial role in regulating cytokinin signal transduction, abiotic stress resistance, and plant development. A number of adverse environmental conditions have caused severe losses for the pepper (Capsicum annuum L.)—a significant and economically important vegetable. Among the transcription factors of the type B-ARRs family, multiple members have different functions. In pepper, only a few members of the ARRs-B family have been reported and characterized. The current study aimed to characterize ARRs-B transcription factors in C. annuum, including phylogenetic relationships, gene structures, protein motif arrangement, and RT-qPCR expression analyses and their role in salinity stress. In total, ten genes encode CaARRs-B transcription factors (CaARR1 to CaARR10) from the largest subfamily of type-B ARRs were identified in C. annum. The genome-wide analyses of the CaARRs-B family in C. annuum were performed based on the reported ARRs-B genes in Arabidopsis. An analysis of homologous alignments of candidate genes, including their phylogenetic relationships, gene structures, conserved domains, and qPCR expression profiles, was conducted. In comparison with other plant ARRs-B proteins, CaARRs-B proteins showed gene conservation and potentially specialized functions. In addition, tissue-specific expression profiles showed that CaARRs-B genes were differentially expressed, suggesting functionally divergent. CaARRs-B proteins had a typical conserved domain, including AAR-like (pfam: PF00072) and Myb DNA binding (pfam: PF00249) domains. Ten of the CaARRs-B genes were asymmetrically mapped on seven chromosomes in Pepper. Additionally, the phylogenetic tree of CaARRs-B genes from C. annuum and other plant species revealed that CaARRs-B genes were classified into four clusters, which may have evolved conservatively. Further, using quantitative real-time qRT-PCR, the study assessed the expression patterns of CaARRs-B genes in Capsicum annuum seedlings subjected to salt stress. The study used quantitative real-time qRT-PCR to examine CaARRs-B gene expression in Capsicum annuum seedlings under salt stress. Roots exhibited elevated expression of CaARR2 and CaARR9, while leaves showed decreased expression for CaARR3, CaARR4, CaARR7, and CaARR8. Notably, no amplification was observed for CaARR10. This research sheds light on the roles of CaARRs-B genes in pepper’s response to salinity stress. These findings enrich our comprehension of the functional implications of CaARRs-B genes in pepper, especially in responding to salinity stress, laying a solid groundwork for subsequent in-depth studies and applications in the growth and development of Capsicum annuum.

Transcriptome analysis and identification of genes related to environmental adaptation of Grylloprimevala jilina Zhou & Ren 2023.

Grylloprimevala jilina is a true cave insect living in the dark areas of caves. It has the characteristics of sparse skin pigmentation, degeneration of the compound eyes and monocular eyes, and obvious preference for high-humidity and low-temperature environments. Given the highly specialized, rare, and limited distribution, G. jilina is considered an endangered species and also a first-level national protected insect in China. Cave creatures often undergo dramatic morphological changes in their sensory systems to adapt to the cave environment. Most previous studies mainly focused on morphological adaptive changes in cave insects, and only a few studied the changes at the gene level. In this study, we performed transcriptome analysis of G. jilina and constructed phylogenetic trees of genes that are related to environmental adaptation, including chemosensory, visual-related, reproduction-related, temperature adaptation-related, and winged morph differentiation-related genes. Besides, the expression levels of environmental adaption-related genes in different tissues, including antennae, heads, thoraxes, abdomens, legs, and tails, were analyzed. The results showed the loss of chemosensory genes and vision-related genes, the conservation of reproduction-related genes and temperature adaptation-related genes, and the conservation of wing-related genes despite the loss of wings, and the results were consistent with other cave insects. The identification and expression study of genes possibly related to the environmental adaptability in G. jilina provided basic data for the protection of this endangered species and increased knowledge about insect evolution in general.

Structural divergence and phylogenetic relationships of Ajania (Asteraceae) from plastomes and ETS

BackgroundAjania Poljakov, an Asteraceae family member, grows mostly in Asia’s arid and semi-desert areas and is a significant commercial and decorative plant. Nevertheless, the genus’ classification has been disputed, and the evolutionary connections within the genus have not been thoroughly defined. Hence, we sequenced and analyzed Ajania’s plastid genomes and combined them with ETS data to assess their phylogenetic relationships.ResultsWe obtained a total of six new Ajania plastid genomes and nine ETS sequences. The whole plastome lengths of the six species sampled ranged from 151,002 bp to 151,115 bp, showing conserved structures. Combined with publicly available data from GenBank, we constructed six datasets to reconstruct the phylogenetic relationships, detecting nucleoplasmic clashes. Our results reveal the affinities of Artemisia, Chrysanthemum and Stilpnolepis to Ajania and validate the early taxonomy reclassification. Some of the plastid genes with low phylogenetic information and gene trees with topological differences may have contributed to the ambiguous phylogenetic results of Ajania. There is extensive evolutionary rate heterogeneity in plastid genes. The psbH and ycf2 genes, which are involved in photosynthesis and ATP transport, are under selective pressure. Plastomes from Ajania species diverged, and structural aspects of plastomes may indicate some of the real evolutionary connections. We suggest the ycf1 gene as a viable plastid DNA barcode because it has significant nucleotide diversity and better reflects evolutionary connections.ConclusionOur findings validate the early Ajania taxonomy reclassification and show evolutionary rate heterogeneity, genetic variety, and phylogenetic heterogeneity of plastid genes. This research might provide new insights into the taxonomy and evolution of Ajania, as well as provide useful information for germplasm innovation and genetic enhancement in horticultural species.

MSSD: An Efficient Method for Constructing Accurate and Stable Phylogenetic Networks by Merging Subtrees of Equal Depth

Background:: Systematic phylogenetic networks are essential for studying the evolutionary relationships and diversity among species. These networks are particularly important for capturing non-tree-like processes resulting from reticulate evolutionary events. However, existing methods for constructing phylogenetic networks are influenced by the order of inputs. The different orders can lead to inconsistent experimental results. Moreover, constructing a network for large datasets is time-consuming and the network often does not include all of the input tree nodes. Aims: This paper aims to propose a novel method, called as MSSD, which can construct a phylogenetic network from gene trees by Merging Subtrees with the Same Depth in a bottom-up way. background: Phylogenetic trees can represent the evolutionary history of genes vertically. There is a difference between phylogenetic trees of different genes due to the reticulate evolution events of species. Phylogenetic networks can represent reticulate evolutionary processes and show the difference between rooted gene trees. Methods:: The MSSD first decomposes trees into subtrees based on depth. Then it merges subtrees with the same depth from 0 to the maximum depth. For all subtrees of one depth, it inserts each subtree into the current networks by means of identical subtrees. Results:: We test the MSSD on the simulated data and real data. The experimental results show that the networks constructed by the MSSD can represent all input trees and the MSSD is more stable than other methods. The MSSD can construct networks faster and the constructed networks have more similar information with the input trees than other methods. Conclusion:: MSSD is a powerful tool for studying the evolutionary relationships among species in biologyand is free available at https://github.com/xingjiajie2023/MSSD. conclusion: The MSSD can construct networks faster and the constructed networks have more similar information with the input trees than other methods.

Exploring the Efficacy of Bacillus oceanisediminis Ba9 from Asian Seabass Cage Sediment in Saline Wastewater Treatment

Preventing toxicity in aquaculture systems from ammonia and nitrite is important. This study isolated the salt-tolerant Bacillus sp. strain Ba9 from bottom sediment under an Asian Seabass (Lates calcarifer) cage cultivating at Koh Yor, Songkhla, Thailand. Morphological characteristics showed that strain Ba9 was rod-shaped, endospore-forming, and Gram-positive. Strain Ba9 grew well at a salinity of 1.5 to 4.0% NaCl. The catalase test of the isolate was positive, while the oxidase test was negative. Based on 16S rRNA gene sequencing data and phylogenetic tree analysis, strain Ba9 was identified as B. oceanisediminis with 97% similarity (strain HQB337T). The result showed that the ammonium removal efficiency of Ba9 in a high ammonium medium was 64.24%. The nitrite and nitrate production were 0.10% and 0.08%, respectively. Consequently, sucrose had been the optimal carbon source for Ba9, which showed ammonium removal was 61.05%. Ammonium sulfate is the most suitable for ammonium oxidation, with 50.53% for the nitrogen source. The optimal C/N ratio of strain Ba9 was 8, with 71.15% ammonia removal. For wastewater improvement, strain Ba9 was inoculated into artificial wastewater for 14 days. The result showed that the ammonium removal efficiency of Ba9 was 96.87%. In addition, the biochemical oxygen demand (BOD) removal efficiency of Ba9 was 90.86%. From this result, the salt-tolerant B. oceanisediminis Ba9 has a high potential as a microbial product for water quality management in marine aquaculture.

Trichophoromyia auraensis: evidence for cryptic species and first record in the state of Maranhão, Brazil.

Trichophoromyia auraensis (Mangabeira, 1942) (Diptera, Psychodidae, Phlebotominae) has a wide geographic distribution in the western region of the Amazon biome, where it is a putative Leishmania vector. Here, we reported for the first time a population of this species in the Brazilian state of Maranhão, in the eastern Amazon, from which we DNA-barcoded and compared with previously processed specimens from Acre State, in the western Amazon. For this, we analyzed the DNA barcoding fragment (658 bp) of the mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear internal transcribed spacer 2 (ITS2) of Trichophoromyia species using phylogeneticgene trees, and species delimitation algorithms. The analyses of COI barcodes showed high values of genetic distance (mean K2P = 5.17) and well-supported clades/MOTUs for the eastern and western populations of T. auraensis, which may indicate a possible complex of cryptic species. The western population of this taxon merged with the close-related sand fly Trichophoromyia velezbernali Posada-López, Galvis and Galati, 2018 from Colombia, which may be associated with the recent speciation history and introgression between these populations. These evidences should be evaluated with a more comprehensive sampling in terms of analyzed populations and molecular markers.

RNA-seq provides insights into potato deubiquitinase responses to drought stress in seedling stage.

Ubiquitination is a specific protein degradation and reversible post-translational modification process that can be reversed by deubiquitinase (DUBs). DUBs can hydrolyze and release ubiquitin in the substrate protein so that the substrate can avoid degradation or change its activity, and it has an impact on plant growth and development, cell cycle, abiotic stress response, and other biological processes. Transcript sequences of potato varieties "DM1-3", "Atlantic" and "Cooperation-88" downloaded from Potato Genome Resources were used for genome-wide identification of the DUB gene family using Hidden Markov Models and verified in the NCBI CD-Search tool. The characteristics of DUB genes from different potato varieties were analyzed including subcellular localization, gene structural motifs, phylogenetic tree, and sequence homology. Polyethylene glycol 6000 (PEG6000) induced drought stress transcriptome analysis was performed on the "Atlantic", and differentially expressed genes were screened, with emphasis on the characterization of deubiquitinase. DUB genes have a complex gene structure, often with a large number of exons and alternative splicing. Their promoters contain abundant abiotic stress-responsive elements, such as 425 MYC, 325 ABRE, and 320 MYB. There are also a large number of orthologous genes in the DUBs of the three potato varieties, and these genes are often clustered in similar regions on the genome. We performed transcriptome sequencing of the potato under PEG-induced drought stress and analyzed it for the first time using the Atlantic as a reference genome. We identified a total of 6067 down-regulated differentially expressed genes (DEGs) and 4950 up-regulated DEGs under PEG-induced drought stress. We screened the expression of DUBs and observed that 120 DUBs were up-regulated where most of them functioned in the nucleus, and the interacting proteins of DUBs were also localized in the nucleus. We have comprehensively identified and analyzed potato DUBs, and the accurately aligned transcriptome data which will further deepen the understanding of DUBs involved in the regulation of osmotic stress.

Phylogenetic analysis reveals multiple origins of Chinese gamecocks

Cockfighting is popular worldwide, dating back to 2,800 BC. Primarily, 5 modern Chinese gamecock breeds exist, located in the northeast (Luxi and Henan), west (Turpan), south (Xishuangbanna), and southeast (Zhangzhou) of China. However, whether Chinese gamecocks were derived from a single origin or multiple origins remains controversial. Therefore, this study used next-generation resequencing data to elucidate the origin of Chinese gamecocks by constructing genome-wide and SRY-box transcription factor 5 (SOX5) gene phylogenetic trees. Data from 161 chickens from 27 breeds, including 9 gamecock breeds, were included. Before constructing the SOX5 gene tree, we validated that the pea-comb phenotype mutation in all gamecock breeds was attributed to copy number variation in intron 1 of the SOX5 gene, as previously reported. The specific region was chr1: 65,838,000 to 65,846,000. The phylogenetic tree results suggested that Zhangzhou and Xishuangbanna gamecocks have a monophyletic origin, while Luxi, Henan, and Turpan gamecocks have a common ancestor. Our study provides genome-wide evidence that Chinese gamecocks have multiple origins and advances the understanding of the genetic mechanisms of the pea-comb characteristic.

Validation of 16S rRNA and Complete rpoB Gene Sequence Analysis for the Identification of Elizabethkingia Species.

Bacteria in the genus Elizabethkingia have emerged as a cause of life-threatening infections in humans. However, accurate species identification of these pathogens relies on molecular techniques. We aimed to evaluate the accuracy of 16S rRNA and complete RNA polymerase β-subunit (rpoB) gene sequences in identifying Elizabethkingia species. A total of 173 Elizabethkingia strains with whole-genome sequences in GenBank were included. The 16S rRNA gene and rpoB gene sequences from the same Elizabethkingia strains were examined. Of the 41 E. meningoseptica strains, all exhibited >99.5% 16S rRNA similarity to its type strain. Only 83% of the 99 E. anophelis strains shared >99.5% 16S rRNA gene similarity with its type strain. All strains of E. meningoseptica and E. anophelis formed a cluster distinct from the other Elizabethkingia species in the 16S rRNA and rpoB gene phylogenetic trees. The polymorphisms of 16S rRNA gene sequences are not sufficient for constructing a phylogenetic tree to discriminate species in the E. miricola cluster (E. miricola, E. bruuniana, E. occulta, and E. ursingii). The complete rpoB gene phylogenetic tree clearly delineates all strains of Elizabethkingia species. The complete rpoB gene sequencing could be a useful complementary phylogenetic marker for the accurate identification of Elizabethkingia species.

Molecular cloning and sequence analysis of Annexin A2a and Annexin A2b coding sequences from grass carp (Ctenopharyngodon idella)

Annexin A2, a Ca2+-regulating and phospholipid-binding protein, distributing in most of cells which plays roles in the inflammatory regulation and pathogenic resistance. It performs membrane binding function in various physiological processes such as membrane repair, vesicular transportation and autophagy. In the present study, Annexin A2a and Annexin A2b gene were cloned from Ctenopharyngodon Idella (grass carp). The open reading frame (ORF) of Annexin A2a and Annexin A2b encoded 337 and 338 amino acid residues, respectively. The gene phylogenetic tree showed that Annexin A2a and Annexin A2b gene in grass carp had a great evolutionary conservation compared with their orthologues in danio rerio. The predicted 3D models presented that Annexin A2a equipped calcium ion binding sites, and its structure identity was 66.96% compared with human Annexin A2. However, Annexin A2b possessed no calcium ion binding sites, but it still had 64.79% identity compared with human Annexin A2.

Philodulcilactobacillus myokoensis gen. nov., sp. nov., a fructophilic, acidophilic, and agar-phobic lactic acid bacterium isolated from fermented vegetable extracts.

Lactic acid bacteria are commonly in the fermentation industry and pose potential positive effects on health. In this study, a new lactic acid bacterium was isolated from fermented vegetable extracts in Myoko, Niigata, Japan. This bacterium is fructophilic, acidophilic, and hard to grow on agar medium. The isolate is Gram-stain-positive, non-spore-forming, non-motile, rod-shaped, and catalase-negative. Growth occurred at pH 3.5-5.5, with optimal growth at pH 4.5-5.0. The cells formed colonies on a solid MRS medium with 20% (w/v) sucrose and 0.8% (w/v) gellan gum under anaerobic conditions. The bacterium was able to grow on up to 50% (w/v) sucrose but not on d-glucose. Moreover, 16S rRNA gene sequence analysis revealed that the strain was most closely related to Apilactobacillus ozensis (93.1% sequence similarity). The values of average nucleotide identity, digital DNA-DNA hybridization, average amino acid sequence identity, and amino acid identity of conserved genes were calculated between the isolated strain (type strain is WR16-4T = NBRC 115064T = DSM 112857T) and its phylogenetically closest type strains. The average nucleotide identity values (73.36-78.28%) and DNA-DNA hybridization values (16.3-32.9%) were significantly lower than the threshold values for species boundaries. The average amino acid sequence identity values (53.96-60.88%) were significantly below the threshold boundary of genus demarcation (68%). The amino acid identity of conserved genes values compared to strain WR16-4T were the genera Apilactobacillus, Nicoliella spurrieriana SGEP1_A5T, Acetilactobacillus jinshanensis HSLZ-75T, and Fructilactobacillus were 62.51-63.79%, 62.87%, 62.03%, and 58.00-61.04%, respectively. The 16S rRNA gene and core genome phylogenetic trees suggested that this novel strain was most closely related to the type strain of A. jinshanensis HSLZ-75T. Based on the physiological, morphological, and phenotypical characteristics of strain WR16-4T, we propose its classification as a novel genus, Philodulcilactobacillus myokoensis gen. nov., sp. nov.

First Report of Yellow Rot Caused by Physarum galbeum in Grifola frondosa in China.

Grifola frondosa (Dicks.) Gray, named "Maitake" in Japan, is mainly cultivated in China, Japan and Korea as a rare delicacy (Park et al. 2015). G. frondosa is a medicinal and edible mushroom that can enhance the human immunology system. In recent years, the production of G. frondosa has increased in China due to its high economic value and as a source of livelihood for small scale farmers. From August to September 2017, a serious slime mold disease was observed on G. frondosa under greenhouse conditions in Qingyuan County, Lishui city, Zhejiang Province, China. Incidence was 10 to 30% in most surveyed mushroom greenhouses, sometimes more than 80% in mushroom greenhouses without proper management. The disease reduced G. frondosa production by 10% on average, and over 80% in severe cases. Slime mold disease usually appeared after irrigation, the kelly plasmodia migrate firstly from the root of fruiting body to stem and finally to pileus, then the infected parts became soft and putrid with slime on the surface. Additionally, many other organisms grow on decayed fruiting bodies, such as bacteria, fungi, and insects. The disease can spread rapidly through soil to adjacent fruiting bodies resulting in yield reduction. Samples were collected and cultures were isolated by transferring diseased fruiting bodies with yellow green plasmodia onto 2% water agar medium. Plasmodia were purified through aseptically transferring their edge segment to a new sterile 2% water agar medium, and this procedure was repeated three or four times to free the isolate from contaminating organisms. Purified plasmodia were then placed on the solid bacteriological test medium (SGM), containing glucose, peptone, yeast extract, mineral salts and hematin used in the axenic culture of Physarum polycephalum (Daniel et al. 1964), to verify bacteria presence. Plasmodia were also induced to form sporocarps. Voucher specimens were deposited in the Fungarium of Jiangxi Academy of Agricultural Sciences (FJAAS-M0001) and the Herbarium of the Mycology, Engineering Research Center of Edible and Medicinal Fungi, Chinese Ministry of Education, Jilin Agricultural University (HMJAU-M1561). Sporocarps were stalked, globose to discoid, golden-yellow, 0.9-1.8 mm in height, 0.28-0.55 mm in diameter. Hypothallus was small, thin, orange. Stalks were subulated, about twice to thrice the diameter of the sporotheca, bright orange below, yellow above, furrowed. Peridium was weak, thin, and plated with yellow calcareous flakes. Capillitium was a small meshed, persistent net of tubules with small and yellow angular lime nodes. Spores were globose, free, dark brown to black in mass, purplish brown in transmitted light, 8-10 μm in diameter, smooth under light microscopy. Irregular spinulose spores showed clusters of small warts that are conspicuous under scanning electron microscopy. Plasmodia were yellow green. The 18S ribosomal RNA gene was amplified with primer SMNUR101/NS4 (Rusk et al. 1995; White et al. 1990). The 18S rRNA gene sequence was submitted to GenBank (OP373728) and an 18S rRNA gene phylogenetic tree of Physarum obtained by maximum likelihood analysis (ML) and Bayesian inferences (BI) of 23 taxa and 1,608 aligned positions was produced. Based on sporocarps morphological characteristics, plasmodial cultural traits, and the sequence of 18S rRNA, the slime mold was identified as Physarum galbeum. A pathogenicity test was performed by gently inoculating a 12 mm diameter circinal patch of SGM with plasmodia on three healthy fruiting bodies of G. frondosa. All treatments were cultured in a mushroom-growing room with temperature 24 to 29 ℃ and relative humidity of 87 to 96%. Three fruiting bodies inoculated with a 12 mm diameter SGM served as controls. All fruiting bodies inoculated with plasmodia showed the same symptom. No symptoms developed on the controls. The pathogen was consistently reisolated from the symptomatic fruiting bodies of G. frondosa and confirmed to be P. galbeum based on cultural, morphological and molecular characteristics, thus fulfilling Kock's postulates. This is the first report of P. galbeum causing yellow rot disease on cultivated G. frondosa.