Molecular phylogeny and taxonomy of colorless, filamentous sulfur bacteria of the genus Thiothrix

Comprehensive investigation combining molecular genetic techniques and comparative studies of morphological and physiological properties made it possible to resolve the disputed issue of the taxonomic status of the groups ??T. nivea?? and ??Eikelboom type 021N?? of the genus Thiothrix. The phylogenetic trees constructed on the basis of 16S rRNA and gyrB gene sequences demonstrated that members of the genus Thiothrix formed a cluster within the order Thiotrichales. According to the ??ribosomal?? tree, the cluster of the genus Thiothrix was divided into two main groups, I and II, corresponding to the groups ??T. nivea?? and ??Eikelboom type 021N??. The levels of similarity between the 16S rRNA gene sequences of Thiothrix species reached 88.9?C100%. On the contrary, in the ??gyrase?? tree, these species were not divided into ??T. nivea?? and ??Eikelboom type 021N?? groups. The levels of similarity between the amino acid sequences of the gyrB gene fragments of Thiothrix species varied from 74.5 to 99.2%. Importantly, members of the groups ??T. nivea?? and ??Eikelboom type 021N?? formed very similar 16S rRNA secondary structures in the variable region V3, where a 30-nucleotide deletion characteristic of all Thiothrix species was detected. Phenotypic analysis of the studied bacteria revealed some morphological and physiological properties shared by the groups ??T. nivea?? and ??Eikelboom type 021N??. The data obtained indicate that members of the groups ??T. nivea?? and ??Eikelboom type 021N?? are phenotypically and genetically heterogeneous species within the single monophyletic genus Thiothrix..     

Evidence for Inbreeding and Genetic Differentiation among Geographic Populations of the Saprophytic Mushroom Trogia venenata from Southwestern China

During the past 40 years, more than 400 Sudden Unexplained Deaths (SUDs) have occurred in Yunnan, southwestern China. Epidemiological and toxicological analyses suggested that a newly discovered mushroom called Trogia venenata was the leading culprit for SUDs. At present, relatively little is known about the genetics and natural history of this mushroom. In this study, we analyzed the sequence variation at four DNA fragments among 232 fruiting bodies of T. venenata collected from seven locations. Our ITS sequence analyses confirmed that all the isolates belonged to the same species. The widespread presence of sequence heterozygosity within many strains at each of three protein-coding genes suggested that the fruiting bodies were diploid, dikaryotic or heterokaryotic. Within individual geographic populations, we found significant deviations of genotype frequencies from Hardy-Weinberg expectations, with the overall observed heterozygosity lower than that expected under random mating, consistent with prevalent inbreeding within local populations. The geographic populations were overall genetically differentiated. Interestingly, while a positive correlation was found between population genetic distance and geographic distance, there was little correlation between genetic distance and barium concentration difference for the geographic populations. Our results suggest frequent inbreeding, geographic structuring, and limited gene flow among geographic populations of T. venenata from southwestern China.     

Evidence against Barium in the Mushroom Trogia venenata as a Cause of Sudden Unexpected Deaths in Yunnan,China

This study examined barium concentrations in the mushroom Trogia venenata, the leading culprit for sudden unexpected deaths in Yunnan, southwest China. We found that barium concentrations in T. venenata from Yunnan were low and comparable to other foods, inconsistent with barium concentrations in this mushroom as a significant contributor to these deaths.     

A novel xylan degrading β-d-xylosidase: purification and biochemical characterization

Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular ??-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. ??-xylosidase is a glycoprotein (39?% carbohydrate content) and has a molecular mass of 137?kDa by SDS-PAGE, with optimal temperature and pH at 70?°C and 3.0?C5.5, respectively. ??-xylosidase was stable in acidic pH (3.0?C6.0) and 70?°C for 1?h. The enzyme was activated by 5?mM MnCl₂ (28?%) and MgCl₂ (20?%) salts. The ??-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-??-d-xylopyranoside, exhibiting apparent K_m and V_max values of 0.66?mM and 39?U (mg protein)^?1 respectively, and to a lesser extent p-nitrophenyl-??-d-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel ??-d-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by ??-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose.     

云南横坑切梢小蠹生物学研究

横坑切梢小蠹Tomicus minor (Hartig)是云南松Pinus yunnanensis Franchet的主要次期性害虫之一。1980年以来,该虫与纵坑切梢小蠹T. piniperda（L.）一起在中国西南部大量发生,导致数十万公顷云南松林受害。本文报道了横坑切梢小蠹在云南地区的生活史、生长、发育、繁殖等生物学特征。横坑切梢小蠹年生活史为一代,前后两代在冬春季有部分重叠。成虫羽化于4月下旬开始陆续,5 月下旬结束。成虫羽化后即飞到树冠上蛀食枝梢,直到11月发育成熟,开始繁殖。在此期间,每头成虫可以蛀食4～6个枝梢。横坑切梢小蠹在云南没有越冬习性。繁殖期从11月至次年3月。成虫主要在已经受到纵坑切梢小蠹危害的树木的中、下部产卵。繁殖期较纵坑切梢小蠹约迟1周。由于横坑切梢小蠹从枝梢到树干对云南松持续危害,对树木的危害性较在其它地区更为严重。横坑切梢小蠹利用受到纵坑切梢小蠹蛀害的树木繁殖产卵,加强了蠹虫对云南松树的危害,加速了受害树木的死亡进程。横坑切梢小蠹的上述生物生态学特征是该虫对云南松造成严重危害的重要原因。从横坑切梢小蠹虫体和虫坑中检测到伴生真菌云南半帚孢Leptographium yunnanensis。横坑切梢小蠹对该菌的带菌率在蛀梢期为11.5%;在蛀干中期约为10%～26%。     

Origin and Expansion of the Yunnan Shoot Borer,Tomicus yunnanensis (Coleoptera: Scolytinae): A Mixture of Historical Natural Expansion and Contemporary Human-Mediated Relocation

The Yunnan shoot borer, Tomicus yunnanensis, is a recently-discovered, aggressive pest of the Yunnan pine stands in southwestern China. Despite many bionomics studies and massive controlling efforts, research on its population genetics is extremely limited. The present study, aimed at investigating the origin and dispersal of this important forestry pest, analyzed the population genetic structure and demographic history using a mitochondrial cox1 gene fragment. Our results showed that T. yunnanensis most likely originated from the Central-Yunnan Altiplano, and the divergence time analysis placed the origin approximately 0.72 million-years ago. Host separation and specialization might have caused the speciation of T. yunnanensis. Genetic structure analyses identified two population groups, with six populations near the origin area forming one group and the remaining six populations from western and eastern Yunnan and southwestern Sichuan comprising the other. Divergence time analysis placed the split of the two groups at approximately 0.60 million-years ago, and haplotype phylogenetic tree, network, as well as migration rate suggested that populations of the latter group were established via a small number of individuals from the former one. Migration analysis also showed a certain degree of recent expansion from southwestern Sichuan to eastern Yunnan. Our findings implied that T. yunnanensis underwent both historical expansion and recent dispersal. The historical expansion may relate to the oscillation of regional climate due to glacial and interglacial periods in the Pleistocene, while human-mediated transportation of pine-wood material might have assisted the relocation and establishment of this pest in novel habitats.     

Influence of Ficus carica and Olea europaea leaves extracts on the mycelial growth of mushrooms in vitro

The use of 20% plant leaves extracts included fig (Ficus carica) and olive (Olea europaea) and their mixture 1:1 as an amendment in the solid agar medium (PDA) is beneficial to promote the growth of four mycelial mushrooms. These are Pleurotus ostreatus (Grey oyster mushroom), Pleurotus cornucopiae (Yellow oyster mushroom), Coriolus versicolor (Turkey Tail mushroom), and Ganoderma lucidum (Reishi mushroom). C. versicolor showed better growth reached 67?mm significantly (p?<?0.05) on OC medium after five days. While, P. cornucopiae recorded the lowest growth on FC medium reached 35.3?mm. Induction percentage of mycelial growth is changing according to the type of medium and species of fungus. In general, FOH medium exhibited the best percentage of induction was 14.89%, followed 12.48% and 9.43% by OH and OC media, while the lower percentages were 5.02% and 5.12% on FH and FC media, respectively. FC medium did not induce growth of P. cornucopiae and C. versicolor. The sterilization by Autoclave and Millipore filter showed different induction percentages. Finally, the extracts of fig and olive were useful to add in the culture media to improve the growth of mycelial mushroom in vitro.     

Genetic identification of Thunnus orientalis, T. thynnus, and T. maccoyii by a cytochrome b gene analysis

The three species of bluefin tunas, Thunnus orientalis, T. maccoyii, and T. thynnus, are morphologically similar, which can pose problems for fisheries management and marketing. We examined intraspecific genetic diversity and interspecific genetic boundaries among these three species by analyzing the cytochrome (Cyt) b gene. The full lengths of the nucleotide sequences were 1,141 bp in T. orientalis and T. thynnus and ranged 1,138?~?1,141 bp in T. maccoyii. Mean nucleotide diversities were 0.0019?±?0.0002 in T. thynnus (n?=?8), 0.0063?±?0.0005 in T. orientalis (n?=?22), and 0.0059?±?0.0007 in T. maccoyii (n?=?24). Average numbers of nucleotide differences and nucleotide substitutions per site among the three species were 18.748?±?2.879 and 0.017?±?0.003, respectively. The Neighbor-joining and minimum-evolution trees showed distinct clades with high bootstrapping value support, and the high Fst value indicated significant differentiation among the three species. T. thynnus, T. orientalis, and T. maccoyii could be individually distinguished from each other Thunnus tunas by the 132nd, 375th, and 1,023rd sites of the Cyt b sequences. In the mismatch analysis, Fu??s and Tajima??s tests of sequences from T. orientalis and T. maccoyii provided evidence of their population expansion dating to the middle Pleistocene.     

Recombinant antimicrobial peptide hPAB-�� expressed in Pichia pastoris, a potential agent active against methicillin-resistant Staphylococcus aureus

As a potential therapeutic agent, antimicrobial peptide has received increased attention in recent years. However, high-level expression of a small peptide with antimicrobial activity is still a challenging task. In this study, the coding sequence of antimicrobial peptide hPAB-??, a variant derived from human beta-defensin 2, was cloned into pPIC9K vector and transformed into Pichia pastoris. P. pastoris transformants harbored with multi-copy plasmids were screened by G418 selection. When the transformed cells were induced by methanol, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and matrix-assisted laser desorption ionization-time of flight mass spectrometry revealed recombinant hPAB-?? products consisting of three protein species of 4,680.4, 4,485.3, and 4,881.9?Da at proportions of 58%, 36%, and 6%, respectively, which may be due to the incomplete processing of the fusion signal peptide of ??-factor by the STE13 protease. Expressed hPAB-?? was secreted into the culture medium at a level of 241.2?±?29.5?mg/L. Purified hPAB-?? with 95% homogeneity was obtained by 10?kDa membrane filtration followed by cation ion-exchange chromatography with a SP-Sepharose? XL column. The two major protein species separated through a SOURCE? 30RPC reverse phase chromatography column showed definite antimicrobial activities against Staphylococcus aureus. All 22 methicillin-resistant S. aureus (MRSA) isolates with multidrug resistance phenotype were sensitive to the recombinant hPAB-?? with minimal inhibitory concentrations of 8?C64???g/ml. Our results show that the methylotrophic yeast-inducible system is suitable for high-level expression of active hPAB-??, and that expressed hPAB-?? in P. pastoris may be a potential antimicrobial agent against MRSA infection.     

A Miocene leaf fossil record of Rosa (R. fortuita n. sp.) from its modern diversity center in SW China

Roses (Rosa, Rosaceae) are arguably the most admired ornamental plants in the world. Southwestern China is the center of diversity for many extant native species of Rosa and fossils found in this region are critical for understanding the evolution of this genus. Herein, we report a leaf fossil record with good preservation from the late Miocene of Yunnan Province. The opposite and odd-pinnate leaf is composed of seven elliptical leaflets, with close, crenulate, and regular marginal teeth. The stipules are lanceolate and adnate to the petiole. Additionally, the secondary veins are semicraspedodromous, showing the same venation pattern as most living Rosa species in southwestern China. On the basis of the extensive morphological comparisons, we propose a new species, R. fortuita T. Su et Z.K. Zhou n. sp. This is the first confirmed Rosa leaf fossil record in China, and the discovery of R. fortuita n. sp. indicates that Rosa existed in southwestern China by the late Miocene. It suggests that Rosa was distributed in subtropical or temperate forests and shared a similar ecological niche as Rosa in Europe during the Oligocene and Miocene. The modern diversification of Rosa in southwestern China is thought to have been closely associated with the continuous uplift of the Qinghai-Tibet Plateau since the late Miocene, creating complex topography and a variety of climate conditions.     

Molecular characterization of intergenic spacer region of 5S ribosomal RNA genes in subgenus Vigna: extensive hybridization among V. unguiculata subspecies

We report the molecular analysis of the 5S ribosomal RNA intergenic spacer (IGS) region from 57 Vigna species of subgenus Vigna. Sequence analysis revealed that the 5S IGS was highly variable in length (189?C237?bp) and sequence (58% polymorphic sites). Most of the Vigna species analysed harboured a single type of 5S rRNA repeat unit, except V. unguiculata and V. reticulata that showed multiple ??intragenomic?? 5S types. The intragenomic 5S types among the six V. unguiculata subspecies were characterized by PCR-RFLP, genomic RFLP and sequencing. The 5S IGS was phylogenetically informative (comparable to ITS-1 and ITS-2 spacers) in inferring species relationships among the Vigna species analysed. However, due to the presence of multiple intragenomic 5S types and their incomplete homogenization among V. unguiculata subspecies the relationships in section Catiang could not be resolved below species level. The results presented indicate that intraspecies hybridization might have resulted in the ??horizontal transfer?? of 5S types among the V. unguiculata subspecies, while their maintenance could be due to a slow molecular drive.     

Chemical composition of essential oils of four Eucalyptus species and their phytotoxicity on silverleaf nightshade (Solanum elaeagnifolium Cav.) in Australia

Phytotoxicity and chemical composition of essential oils from four selected Eucalyptus species in Australia were investigated. Essential oils had stronger inhibitory effects on germination and seedling growth of silverleaf nightshade (Solanum elaeagnifolium Cav.) when compared with a commercial eucalyptus oil and with 1,8-cineole. E. salubris oil had the highest inhibition index for silverleaf nightshade germination, root growth and shoot growth, while E. spathulata had the lowest inhibitory effect except root growth. Gas chromatography-mass spectrometry analysis revealed 56 compounds present in E. salubris oil, with 1,8-cineole (57.6?%), ??-pinene (10.9?%) and p-cymene (8.3?%) predominant. E. dundasii oil contained 55 identified compounds with 1,8-cineole (65.5?%) and ??-pinene (19.9?%) being the richest fractions. There were 56 compounds identified from E. brockwayii oil with ??-pinene (31.1?%), isopentyl isovalerate (20.2?%) and 1,8-cineole (16.9?%) as the most abundant components. E. spathulata oil contained 60 compounds, predominantly 1,8-cineole (52.9?%) and ??-pinene (31.0?%). Further study is required to determine the phytoxicity of the individual identified compounds on silverleaf nightshade and whether the observed phytotoxicity is attributable to a single compound or to the synergistic effects of several compounds.     

Residue Tyr224 is critical for the thermostability of Geobacillus sp. RD-2 lipase

A thermophilic lipase (lipGRD) from Geobacillus sp. RD-2, isolated from a hot spring in Yunnan, China, was cloned and over-expressed in Escherichia coli. The function of the conserved residue, Tyr224, near the presumed temperature switch site was analyzed by site-directed saturation mutagenesis. The activity of the wild type lipGRD was optimal at 55??C and pH?7.5, but that from mutant Y224C was optimally active at 35??C, whereas Y224P lipase was optimally active at 65??C. Furthermore, the latter lipase retained 60% of its activity after incubation at 65??C for 5?h. The conserved residue Tyr224, which is close to the lid helix, is the key amino acid residue determining the thermostability of the thermostable lipase.     

中国毛茛科翠雀属的分类学研究(五):秋翠雀花二新异名

通过标本室和野外观察,发现根据四川西南部越西标本描述的毛茛科凉山翠雀花(Delphinium liangshanense W. T. Wang)和根据四川西南部木里标本描述的光轴翠雀花(D. leiostachyum W. T. Wang)只是此前记载分布于四川木里、盐源和云南西北部宁蒗一带的秋翠雀花(D. autumnale Hand.-Mazz.)植株较高的类型,故将二者均处理为秋翠雀花的异名.     

CW EPR and 9 GHz EPR imaging investigation of stable paramagnetic species and their antioxidant activities in dry shiitake mushroom (Lentinus edodes)

We investigated the antioxidant activities and locations of stable paramagnetic species in dry (or drying) shiitake mushroom (Lentinus edodes) using continuous wave (CW) electron paramagnetic resonance (EPR) and 9?GHz EPR imaging. CW 9?GHz EPR detected paramagnetic species (peak-to-peak linewidth (ΔH_pp)?=?0.57?mT) in the mushroom. Two-dimensional imaging of the sharp line using a 9?GHz EPR imager showed that the species were located in the cap and shortened stem portions of the mushroom. No other location of the species was found in the mushroom. However, radical locations and concentrations varied along the cap of the mushroom. The 9?GHz EPR imaging determined the exact location of stable paramagnetic species in the shiitake mushroom. Distilled water extracts of the pigmented cap surface and the inner cap of the mushroom showed similar antioxidant activities that reduced an aqueous solution of 0.1?mM 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl. The present results suggest that the antioxidant activities of the edible mushroom extracts are much weaker than those of ascorbic acid. Thus, CW EPR and EPR imaging revealed the location and distribution of stable paramagnetic species and the antioxidant activities in the shiitake mushroom for the first time.     

Application of MALDI-TOF mass spectrometry for differentiation of closely related species of the “Arthrobacter crystallopoietes” phylogenetic group

MALDI mass spectra were generated for the type strain of Arthrobacter crystallopoietes VKM Ac-1107^T and for closely related (99.6?C100% 16S rRNA gene similarity) halotolerant Arthrobacter strains, as well as for some other Arthrobacter species. Results of the cluster analysis of the spectra were in agreement with the genotypic characteristics of bacteria (DNA-DNA hybridization and BOX-PCR). The data obtained in this study indicate that the halotolerant strains belong to two new Arthrobacter species. Specific peaks which can serve as chemotaxonomic markers of the species composing the phylogenetic group ??Arthrobacter crystallopoietes?? were revealed.     

The Chloroplast Genome Sequence of Date Palm (Phoenix dactylifera L. cv. ‘Aseel’)

Date palm (Phoenix dactylifera L.) is an economically important and widely cultivated palm of the family Arecaceae. We sequenced the complete date palm chloroplast genome (cpDNA) from Pakistani cv. ??Aseel??, using a combination of Sanger-based and next-generation sequencing technologies. Being very similar to a sequence from a Saudi Arabian date palm cultivar ??Khalas?? published recently, the size of the genome was 158,458?bp with a pair of inverted repeat (IR) regions of 27,276?bp that were separated by a large single-copy (LSC) region of 86,195?bp and a small single-copy (SSC) region of 17,711?bp. Genome annotation demonstrated a total of 138 genes, of which 89 were protein coding, 39 were tRNA, and eight were rRNA genes. Comparison of cpDNA sequences of cultivars ??Aseel?? and ??Khalas?? showed following intervarietal variations in the LSC region; (a) two SNPs in intergenic spacers and one SNP in the rpoc1 gene, (b) polymorphism in two mono-nucleotide simple sequence repeats (SSR), and (c) a 4-bp indel in the accD-psaI intergenic spacer. The SSC region has a polymorphic site in the mono-nucleotide SSR located at position 120,710. We also compared cv. ??Aseel?? cpDNA sequence with partial P. dactylifera cpDNA sequence entries deposited in Genbank and identified a number of potentially useful polymorphisms in this species. Analysis of date palm cpDNA sequences revealed a close relationship with Typha latifolia. Occurrence of small numbers of forward and inverted repeats in date palm cpDNA indicated conserved genome arrangement.     

Assessment of the role of meta-topolins on in vitro produced phenolics and acclimatization competence of micropropagated ‘Williams’ banana

The effects of five topolins (meta-Topolin, mT; meta-Topolin riboside, mTR; meta-Methoxy topolin, MemT; meta-Methoxy topolin riboside, MemTR and meta-Methoxy topolin 9-tetrahydropyran-2-yl, MemTTHP) on the phenolic content and subsequent acclimatization potential of micropropagated ??Williams?? bananas were compared to benzyladenine (BA). Sterile shoot-tip explants were cultured on modified Murashige and Skoog (MS) media containing 10, 20 or 30 ??M of the above aromatic cytokinins (CKs) for 42?days. Phenolic contents were quantified spectrophotometrically. Growth parameters and photosynthetic pigments of the greenhouse-acclimatized plants were determined after 5?months. Total phenolic levels were highest in 10 ??M mT-treated plantlets within the aerial parts and 30 ??M MemTTHP for the underground parts. In the underground parts, 10 ??M mT resulted in the production of the highest amount of proanthocyanidins which was approximately five-fold higher than in the control plants. Furthermore, 10 ??M MemTTHP-treated plantlets had significantly higher total flavonoids (30.1?±?0.24 mg CE/g DW) within the aerial parts. Plantlets regenerated using MemT, MemTR and MemTTHP had significantly longer roots and better shoot/root ratios compared to the control and BA-treated plants. In terms of root fresh weight, it was significantly higher in MemT-treated plantlets than in the control and BA treatments. Chlorophyll a/b ratio was significantly improved with the use of MemT, mTR and mT compared to control. Current findings indicate the potential of topolins in stimulating the accumulation of phenolic compounds in micropropagated plantlets. In view of the importance of plant secondary metabolites, their substantial accumulation probably enhanced the acclimatization and subsequent ex vitro survival of the micropropagated plantlets. Topolins, particularly, the new derivative MemTTHP could be an alternative CK for the micropropagation of plant species based on their stimulatory effect on ex vitro rooting that inevitably enhances acclimatization competence. Furthermore, topolins are demonstrated as potential elicitors in micropropagation.     

Redescription of a rare deepwater cardinalfish, Epigonus ctenolepis Mochizuki and Shirakihara 1983, and comparison with related species (Perciformes: Epigonidae)

A rare epigonid fish, Epigonus ctenolepis Mochizuki and Shirakihara 1983, is redescribed in detail on the basis of two type specimens (90.0??8.0?mm standard length, SL) and an additional specimen (128.8?mm SL) collected from Suruga Bay, Japan, and compared with related species of Epigonus. The species is characterized by the following combination of characters: first dorsal-fin rays VII, second dorsal-fin rays I, 9??0, vertebrae 10?+?15, pored lateral-line scales 52??3 (hypural end 48??9?+?caudal fin 4), pyloric caeca 9??1, total gill rakers 24??5, presence of a pungent opercular spine, lateral-line scales ctenoid, absence of a maxillary mustache-like process, and absence of a rib on the last abdominal vertebra. A key to the Epigonus species characterized by a pungent opercular spine is provided.