Genetic similarity analysis within Pyropia yezoensis blades developed from both conchospores and blade archeospores using AFLP1

Pyropia yezoensis (Ueda) M. S. Hwang et H. G. Choi (previously called Porphyra yezoensis) is an economically important alga. The blades generated from conchospores are genetic chimeras, which are not suitable for genetic similarity analysis. In this study, two types of blades from a single filament of P. yezoensis sporophyte filament were obtained. One type, ConB, consisted of 40 blades that had germinated from conchospores. The other type, ArcB, consisted of 88 blades that had germinated from archeospores released from ConB. Both of them were analyzed by amplified fragment length polymorphism. The low genetic similarity levels for both conchospore‐germinated and archeospore‐germinated blades demonstrated that the conchcelis we used was cross‐fertilized. Furthermore, a higher polymorphic loci ratio (98.6%) was detected in ArcB than in ConB (80.7%), and the average genetic similarity of ArcB (average 0.61) was lower than that of ConB (average 0.71). These differences indicated that genetic analysis using ArcB gives more accurate results.     

Co‐suppression in Pyropia yezoensis (Rhodophyta) Reveals the Role of PyLHCI in Light Harvesting and Generation Switch

The red macroalga Pyropia yezoensis is an economically important seaweed widely cultured in Asian countries and is a model organism for molecular biological and commercial research. This species is unique in that it utilizes both phycobilisomes and transmembrane light‐harvesting proteins as its antenna system. Here, one of the genes of P. yezoensis (PyLHCI) was selected for introduction into its genome to overexpress PyLHCI. However, the co‐suppression phenomenon occurred. This is the first documentation of co‐suppression in algae, in which it exhibits a different mechanism from that in higher plants. The transformant (T1) was demonstrated to have higher phycobilisomes and lower LHC binding pigments, resulting in a redder color, higher sensitivity to salt stress, smaller in size, and slower growth rate than the wildtype (WT). The photosynthetic performances of T1 and WT showed similar characteristics; however, P700 reduction was slower in T1. Most importantly, T1 could release a high percentage of carpospores in young blades to switch generation during its life cycle, which was rarely seen in WT. The co‐suppression of PyLHCI revealed its key roles in light harvesting, stress resistance, and generation alternation (generation switch from gametophytes to sporophytes, and reproduction from asexual to sexual).     

NADPH from the oxidative pentose phosphate pathway drives the operation of cyclic electron flow around photosystem I in high‐intertidal macroalgae under severe salt stress

Pyropia yezoensis (Bangiales, Rhodophyta) is a representative species of high‐intertidal macroalgae, whose blades can tolerate extreme stresses, such as salt stress and desiccation. In this study, the photosystem (PS) responses of P. yezoensis blades under salt stress were studied. Our results showed that when the effective photochemical quantum yield of PS (Y) II decreased to almost zero under high salt stress, YI still had a relatively high activity rate. PSII was therefore more sensitive to salt stress than PSI. Furthermore, in the presence of 3‐(3′, 4′‐dichlorophenyl)‐1,1‐dimethylurea (DCMU), YI rose as salinity increased. The YI values for DCMU‐treated thalli decreased in the presence of glucose‐6‐phosphate dehydrogenase (EC 1.1.1.49, G6PDH) inhibitor (glucosamine, Glucm). The YI values were ～0.09 in the presence of methyl viologen (MV) and almost zero in the presence of dibromothymoquinone (DBMIB). These results demonstrated that under severe salt stress (120‰ salinity) PSI activity was driven from a source other than PSII, and that stromal reductants probably supported the operation of PSI. Under salt stress, the starch content decreased and soluble sugar levels increased. The G6PDH and 6‐phosphogluconate dehydrogenase (EC 1.1.1.44) activities increased, but cytosolic glyceraldehyde 3‐phosphate dehydrogenase (EC 1.2.1.12) activity decreased. Furthermore, the NADPH content increased, but NADH decreased, which suggested that soluble sugar entered the oxidative pentose phosphate pathway (OPPP). All these results suggested that NADPH from OPPP increases the cyclic electron flow around PSI in high‐intertidal macroalgae under severe salt stress.     

Genetic toolkits of the red alga Pyropia tenera against the three most common diseases in Pyropia farms

Disease outbreaks devastate Pyropia aquaculture farms every year. The three most common and serious diseases are Olpidiopsis‐blight and red‐rot disease caused by oomycete pathogens and green‐spot disease caused by the PyroV1 virus. We hypothesized that a basic genetic profile of molecular defenses will be revealed by comparing and analyzing the genetic response of Pyropia tenera against the above three pathogens. RNAs isolated from infected thalli were hybridized onto an oligochip containing 15,115 primers designed from P. tenera expressed sequence tags (EST)s. Microarray profiles of the three diseases were compared and interpreted together with histochemical observation. Massive amounts of reactive oxygen species accumulated in P. tenera cells exposed to oomycete pathogens. Heat shock genes and serine proteases were the most highly up‐regulated genes in all infection experiments. Genes involved in RNA metabolism, ribosomal proteins and antioxidant metabolism were also highly up‐regulated. Genetic profiles of P. tenera in response to pathogens were most similar between the two biotrophic pathogens, Olpidiopsis pyropiae and PyroV1 virus. A group of plant resistance genes were specifically regulated against each pathogen. Our results suggested that disease response in P. tenera consists of a general constitutive defense and a genetic toolkit against specific pathogens.     

Neuraminidase Inhibitors from marine‐derived actinomycete Streptomyces seoulensis

Aims

This work was performed to characterize new secondary metabolites with neuraminidase (NA) inhibitory activity from marine actinomycete strains.

Methods and Results

An actinomycete strain IFB‐A01, capable of producing new NA inhibitors, was isolated from the gut of shrimp Penasus orientalis and identified as Streptomyces seoulensis according to its 16S rRNA sequence (over 99% homology with that of the standard strain). Repeated chromatography of the methanol extract of the solid‐substrate culture of S. seoulensis IFB‐A01 led to the isolation of streptoseolactone ( 1 ), and limazepines G ( 2 ) and H ( 3 ). The structures of 1 – 3 were determined by a combination of IR, ESI‐MS, 1D (¹H and ¹³C NMR, and DEPT) and 2D NMR experiments (HMQC, HMBC, ¹H‐¹H COSY and NOESY). Compounds 1 – 3 showed significant inhibition on NA in a dose‐dependent manner with IC₅₀ values of 3·92, 7·50 and 7·37 μmol l^?1, respectively.

Conclusions

This is the first report of two new ( 1 and 2 ) and known ( 3 , recovered as a natural product for the first time in the work) NA inhibitors from the marine‐derived actinomycete S. seoulensis IFB‐A01.

Significance and Impact of the Study

The three natural NA inhibitors maybe of value for the development of drug(s) necessitated for the treatment of viral infections.     

Genetic diversity of Chinese cattle revealed by Y‐SNP and Y‐STR markers

With its vast territory and complex natural environment, China boasts rich cattle genetic resources. To gain the further insight into the genetic diversity and paternal origins of Chinese cattle, we analyzed the polymorphism of Y‐SNPs (UTY19 and ZFY10) and Y‐STRs (INRA189 and BM861) in 34 Chinese cattle breeds/populations, including 606 males representative of 24 cattle breeds/populations collected in this study as well as previously published data for 302 bulls. Combined genotypic data identified 14 Y‐chromosome haplotypes that represented three haplogroups. Y2‐104‐158 and Y2‐102‐158 were the most common taurine haplotypes detected mainly in northern and central China, whereas the indicine haplotype Y3‐88‐156 predominates in southern China. Haplotypes Y2‐108‐158, Y2‐110‐158, Y2‐112‐158 and Y3‐92‐156 were private to Chinese cattle. The population structure revealed by multidimensional scaling analysis differentiated Tibetan cattle from the other three groups of cattle. Analysis of molecular variance showed that the majority of the genetic variation was explained by the genetic differences among groups. Overall, our study indicates that Chinese cattle retain high paternal diversity (H = 0.607 ± 0.016) and probably much of the original lineages that derived from the domestication center in the Near East without strong admixture from commercial cattle carrying Y1 haplotypes.     

Diversity of bladed Bangiales (Rhodophyta) in western Mediterranean: recognition of the genus Themis and descriptions of T. ballesterosii sp. nov., T. iberica sp. nov., and Pyropia parva sp. nov.

The diversity of the bladed species of the red algal order Bangiales from the Iberian Mediterranean shores has been reassessed after a detailed study of this region. Prior to this study, 11 bladed species of Bangiales had been reported from Mediterranean waters: Porphyra atropurpurea, P. cordata, P. coriacea, P. dioica, P. linearis, P. purpurea, P. umbilicalis, Pyropia leucosticta, Pyropia koreana (as P. olivii), Py. elongata (as P. rosengurttii) and Py. suborbiculata. A combined analysis of the nuclear nSSU and the plastid rbcL genes together with detailed morphological studies has confirmed the presence of species within the genera Porphyra and Pyropia and also revealed a third, undescribed genus, Themis gen. nov. Porphyra linearis, Pyropia elongata and the introduced Pyropia koreana had been previously listed for the Mediterranean and were recorded in this study. An additional four species, including the introduced Pyropia suborbiculata and three new species: Pyropia parva sp. nov., Themis ballesterosii sp. nov., and Themis iberica sp. nov. were also observed. Hence, most of the Porphyra species traditionally reported along these shores were not reported in this survey. This new floristic Bangiales composition confirms the importance of the Mediterranean basin as a hotspot for biodiversity, possible endemics of ancient origin and high proportion of introductions. Our data also continue to confirm the extent of Bangiales diversity at regional and worldwide levels.     

ICAM‐1 and MKL‐1 polymorphisms impose considerable impacts on coronary heart disease occurrence

This study was aimed to explore the correlation of intercellular adhesion molecule‐1 (ICAM‐1) K469E and megakaryoblastic leukaemia factor‐1 (MKL‐1) ?184C/T polymorphisms with the susceptibility to coronary heart disease (CHD) in the Chinese Han population. 100 CHD patients and 91 healthy people that had no blood connection with each other were enrolled in this case‐control study. ICAM‐1 and MKL‐1 polymorphisms were genotyped by polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) approach. Multiple logistic regression was used to analyse the correlation between polymorphisms of ICAM‐1 and MKL‐1 and CHD susceptibility. Differences of genotype and allele frequencies of the two SNPs between case and control groups were analysed by chi‐square test. Odds ratios (ORs) and 95% confidence intervals (CIs) were indicated relative susceptibility of CHD. The distributions of ICAM‐1 and MKL‐1 polymorphisms in each group conformed to Hardy‐Weinberg equilibrium (HWE). After adjusting for traditional risk factors, the TT genotype frequency of MKL‐1 ?184C/T polymorphism was found significantly higher in case group than in control group (P < .05). Meanwhile, T allele frequency increased in case group compared with control group, and the differences had statistical significance (P = .04, OR = 2.34, 95% CI = 1.34‐5.26). Logistic regression analysis in this study proved that smoking, hypertension, diabetes and triglyceride (TG) were all risk factors for CHD ICAM‐1 K469E polymorphism has no association with the onset of CHD. But MKL‐1 ?184C/T polymorphism is associated with the risk of CHD and T allele might be a susceptibility factor for CHD.     

Comparative study of wild and cultivated <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Bangiales,Rhodophyta) based on molecular and morphological data

We compared the wild Porphyra strain OGATSU from northeastern Japan with cultivated Porphyra yezoensis f. narawaensis using the RuBisCO spacer, rbcL, and ITS-1 DNA sequences as well as early gametophyte development. Based on the molecular analyses and detailed morphological observations, OGATSU was identified as P. yezoensis, but also revealed important differences from the cultivated form. Under the same culture conditions, gametophytic blades of OGATSU produced more archeospores than P. yezoensis f. narawaensis strain HG-4. The length of blades and their length-to-width ratios were significantly lower in OGATSU than in HG-4, and the color of OGATSU blades was darker than that of HG-4. The first lateral cell division in conchospore germlings occurred significantly earlier in the OGATSU strain than in the HG-4 strain, resulting in the rounder shape of the OGATSU blade compared to that of P. yezoensis f. narawaensis. These results suggested that wild strains such as OGATSU can provide useful characters that could enhance cultivated varieties in a careful breeding program.     

Molecular Identification and Diversity of ‘Candidatus Phytoplasma solani’ Associated with Red‐leaf Disease of Salvia miltiorrhiza in China

Reddening disease has recently been threatening Salvia miltiorrhiza in China, ranging from 30 to 50%. The main symptoms observed, such as plant stunting, inflorescence malformation, leaf reddening, fibrous roots browning, skin blackening and eventually root rot, are typically associated with phytoplasma infection. The presence of phytoplasmas was demonstrated through phytoplasma‐specific PCR, with the expected amplification (1.8 kb) from symptomatic S. miltiorrhiza plants from Shangluo, Shangzhou and Luonan fields in Shaanxi Province of China. The sequences of 16S rRNA, tuf, secY and vmp1 genes amplified from LN‐1 phytoplasma shared the closest homologies of 99%, 100%, 99% and 98% with those of the reference strain Candidatus Phytoplasma solani (subgroup 16SrXII‐A), respectively. The phylogenetic trees showed that LN‐1 phytoplasma clustered with the members of 16SrXII‐A group, including Ca. P. solani. Computer‐simulated restriction fragment length polymorphism analysis further supported this classification. Diversity analysis showed that all ‘Ca. P. solani’ strains identified from the three different regions examined shared 100% identical 16S rRNA, tuf, secY and vmp1 nucleotide sequences. To the best of our knowledge, this is the first report of phytoplasma infecting the medicinal plant of S. miltiorrhiza. The results demonstrate that ‘Ca. P. solani’ is the presumptive aetiological agent of S. miltiorrhiza reddening disease in China.     

Non‐host plant essential oil volatiles with potential for a ‘push‐pull’ strategy to control the tea green leafhopper,Empoasca vitis

The tea green leafhopper, Empoasca vitis Göthe (Hemiptera: Cicadellidae), is an economically important pest of tea crops, Camellia sinensis (L.) O. Kuntze (Theaceae), in China. The use of non‐host plant essential oils for manipulation of E. vitis was investigated for potential incorporation into a ‘push‐pull’ control strategy for this pest. The effectiveness of 14 plant essential oils in repelling E. vitis was investigated in laboratory assays. Rosemary oil, geranium oil, lavender oil, cinnamon oil, and basil oil repelled leafhoppers in a Y‐shaped olfactometer. We also compared the efficacy of these five plant essential oils to repel E. vitis in the presence of a host plant volatile‐based leafhopper attractant, (Z)‐3‐hexenyl acetate, in a tea plantation. In the treatment combination, four plates (north, south, east, and west) treated with an essential oil surrounded a central sticky plate treated with (Z)‐3‐hexenyl acetate. Fewer E. vitis were found on the plates treated with rosemary oil (12.5% reduction) than on the four water‐sprayed control treatment plates surrounding a central plate with (Z)‐3‐hexenyl acetate. We compared the distribution of E. vitis on the plates, and the relative numbers of E. vitis on each plate were compared with similar plates in the control treatment. When four plates treated with rosemary oil surrounded a central (Z)‐3‐hexenyl acetate‐treated plate, the distribution of E. vitis on the different plates changed significantly compared with that of the control. Relatively fewer E. vitis were found on the east (13.0% reduction) rosemary oil‐treated plates and more E. vitis (11.3% increase) were found on the central attractant‐treated plate. Our findings indicate that rosemary oil is a promising leafhopper repellent that should be tested further in a ‘push‐pull’ strategy for control of E. vitis.     

Herd prevalence of Enterobacteriaceae producing CTX‐M‐type and CMY‐2 β‐lactamases among Japanese dairy farms

Aims

To determine the herd prevalence of Enterobacteriaceae producing CTX‐M‐type extended‐spectrum β‐lactamases (ESBLs) among 381 dairy farms in Japan.

Methods and Results

Between 2007 and 2009, we screened 897 faecal samples using BTB lactose agar plates containing cefotaxime (2 μg ml^?1). Positive isolates were tested using ESBL confirmatory tests, PCR and sequencing for CTX‐M, AmpC, TEM and SHV. The incidence of Enterobacteriaceae producing CTX‐M‐15 (n = 7), CTX‐M‐2 (n = 12), CTX‐M‐14 (n = 3), CMY‐2 (n = 2) or CTX‐M‐15/2/14 and CMY‐2 (n = 4) in bovine faeces was 28/897 (3·1%) faecal samples. These genes had spread to Escherichia coli (n = 23) and three genera of Enterobacteriaceae (n = 5). Herd prevalence was found to be 20/381 (5·2%) dairy farms. The 23 E. coli isolates showed clonal diversity, as assessed by multilocus sequence typing and pulsed‐field gel electrophoresis. The pandemic E. coli strain ST131 producing CTX‐M‐15 or CTX‐M‐27 was not detected.

Conclusions

Three clusters of CTX‐M (CTX‐M‐15, CTX‐M‐2, CTX‐M‐14) had spread among Japanese dairy farms.

Significance and Impact of the Study

This is the first report on the prevalence of multidrug‐resistant CTX‐M‐15–producing E. coli among Japanese dairy farms.     

Selection and characterization of Bacillus thuringiensis strains toxic against pyralid stored‐product pests

In this study, we present the selection and the characterization of Bacillus thuringiensis strains toxic against pyralid pests of stored products. Among 201 new B. thuringiensis strains isolated from different countries, two strains (BLB249 and BLB384) showed greater toxicity than the commercialized strain B. thuringiensis kurstaki HD1 against Ephestia kuehniella larvae. Morphological, molecular and biochemical investigations revealed that these strains were similar to HD1 but presented different cry gene content. Additional bioassays revealed that only strain BLB249 displayed higher toxicity than HD1 against Plodia interpunctella larvae. The study of Cry protoxin activation by midgut proteases of E. kuehniella and P. interpunctella larvae supported that higher toxicity of BLB249 and BLB384 strains compared to HD1 was not due to differential protoxin activation. Moreover, the toxic strains produced δ‐endotoxins and spores in similar amounts to HD1. Interestingly, the δ‐endotoxin production and the yield of BUPM26 strain were 32.87% and 35.12% greater than that of HD1. The potent insecticidal activities of BLB249 and BLB384 strains and the high level of δ‐endotoxin production by BUPM26 strain make them excellent candidates for use against E. kuehniella and P. interpunctella larvae.     

High‐yield lipid production from lignocellulosic biomass using engineered xylose‐utilizing Yarrowia lipolytica

Lignocellulosic biomass shows high potential as a renewable feedstock for use in biodiesel production via microbial fermentation. Yarrowia lipolytica, an emerging oleaginous yeast, has been engineered to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into lipids for lignocellulosic biodiesel production. Yet, the lipid yield from xylose or lignocellulosic biomass remains far lower than that from glucose. Here we developed an efficient xylose‐utilizing Y. lipolytica strain, expressing an isomerase‐based pathway, to achieve high‐yield lipid production from lignocellulosic biomass. The newly developed xylose‐utilizing Y. lipolytica, YSXID, produced 12.01 g/L lipids with a maximum yield of 0.16 g/g, the highest ever reported, from lignocellulosic hydrolysates. Consequently, this study shows the potential of isomerase‐based xylose‐utilizing Y. lipolytica for economical and sustainable production of biodiesel and oleochemicals from lignocellulosic biomass.